RECEPTEURS COUPLES PAR DES PROTEINES G EXPRIMES DANS LE CERVEAU

G PROTEIN-COUPLED RECEPTORS EXPRESSED IN HUMAN BRAIN

Abrégé français

La présente invention concerne des gènes codant des récepteurs couplés par des protéines G jusqu'ici inconnus, des constructions et des cellules hôtes recombinées incorporant les gènes; les polypeptides GPCR codés par les gènes; des anticorps dirigés contre les polypeptides; et des méthodes de production et d'utilisation de tout ce qui est précité.

Abrégé anglais

The present invention provides genes encoding heretofore unknown G protein-
coupled receptors, constructs and recombinant host cells incorporating the
genes; the GPCR polypeptides encoded by the genes; antibodies to the
polypeptides; and methods of making and using all of the foregoing.

Revendications

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CLAIMS
What is claimed is:
1. A purified and isolated seven transmembrane receptor polypeptide
comprising an amino acid sequence at least 90% identical to an amino acid
sequence
set forth in any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, l8 or 20, or a
fragment
thereof comprising an epitope specific to said seven transmembrane receptor
polypeptide.
2. A purified and isolated seven transmembrane receptor polypeptide
according to claim 1 comprising an amino acid sequence at least 90% identical
to the
amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof
comprising an
epitope specific to said seven transmembrane receptor polypeptide.
3. A purified and isolated seven transmembrane receptor polypeptide
according to claim 1 comprising an amino acid sequence at least 90% identical
to the
amino acid sequence set forth in SEQ ID NO: 4, or a fragment thereof
comprising an
epitope specific to said seven transmembrane receptor polypeptide.
4. A purified and isolated seven transmembrane receptor polypeptide
according to claim 1 comprising an amino acid sequence at least 90% identical
to the
amino acid sequence set forth in SEQ ID NO: 6, or a fragment thereof
comprising an
epitope specific to said seven transmembrane receptor polypeptide.
5. A purified and isolated seven transmembrane receptor polypeptide
according to claim 1 comprising an amino acid sequence at least 90% identical
to the
amino acid sequence set forth in SEQ ID NO: 8, or a fragment thereof
comprising an
epitope specific to said seven transmembrane receptor polypeptide.

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6. A purified and isolated seven transmembrane receptor polypeptide
according to claim 1 comprising an amino acid sequence at least 90% identical
to the
amino acid sequence set forth in SEQ ID NO: 10, or a fragment thereof
comprising an
epitope specific to said seven transmembrane receptor polypeptide.
7. A purified and isolated seven transmembrane receptor polypeptide
according to claim 1 comprising an amino acid sequence at least 90% identical
to the
amino acid sequence set forth in SEQ ID NO: 12, or a fragment thereof
comprising an
epitope specific to said seven transmembrane receptor polypeptide.
8. A purified and isolated seven transmembrane receptor polypeptide
according to claim 1 comprising an amino acid sequence at least 90% identical
to the
amino acid sequence set forth in SEQ ID NO: 14, or a fragment thereof
comprising an
epitope specific to said seven transmembrane receptor polypeptide.
9. A purified and isolated seven transmembrane receptor polypeptide
according to claim 1 comprising an amino acid sequence at least 90% identical
to the
amino acid sequence set forth in SEQ ID NO: 16, or a fragment thereof
comprising an
epitope specific to said seven transmembrane receptor polypeptide.
10. A purified and isolated seven transmembrane receptor polypeptide
according to claim 1 comprising an amino acid sequence at least 90% identical
to the
amino acid sequence set forth in SEQ ID NO: 18, or a fragment thereof
comprising an
epitope specific to said seven transmembrane receptor polypeptide.
11. A purified and isolated seven transmembrane receptor polypeptide
according to claim 1 comprising an amino acid sequence at least 90% identical
to the
amino acid sequence set forth in SEQ ID NO: 20, or a fragment thereof
comprising an
epitope specific to said seven transmembrane receptor polypeptide.

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12. A purified and isolated seven transmembrane receptor polypeptide
according to any one of claims 1-11.
13. A purified and isolated polypeptide according to any one of claims
1-11 comprising at least one extracellular domain of the seven transmembrane
receptor polypeptide.
14. A purified and isolated polypeptide according to any one of claims
1-11 comprising the N-terminal extracellular domain of the seven transmembrane
receptor polypeptide.
15. A purified and isolated polypeptide according to any one of claims
1-11 comprising a seven transmembrane receptor fragment selected from the
group
consisting of an N-terminal extracellular domain transmembrane domains,
extracellular loops connecting transmembrane domains, intracellular loops
connecting
transmembrane domains, a C-terminal cytoplasmic domain, and fusions thereof.
16. A polypeptide according to any one of claims 1-15, wherein the
polypeptide further includes a heterologous tag amino acid sequence.
17. A purified and isolated polynucleotide comprising a nucleotide
sequence that encodes the polypeptide of claim 16.
18. A purified and isolated polynucleotide comprising a nucleotide
sequence that encodes a polypeptide according to any one of claims 2, 3, 4, 8
or 9.
19. A purified and isolated polynucleotide comprising a heterologous
expression control sequence operatively linked to a nucleotide sequence that
encodes
a polypeptide according to any one of claims 1-16.

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20. The polynucleotide according to claim 19, wherein the expression
control sequence is a promoter sequence that promotes expression of said
polynucleotide in an eukaryotic cell.
21. The polynucleotide according to claim 19, wherein the promoter is
a heterologous promoter that promotes expression of the polynucleotide in a
human
cell.
22. A purified and isolated polynucleotide comprising a nucleotide
sequence that encodes a mammalian seven transmembrane receptor, wherein said
polynucleotide hybridizes to any one of the nucleotide sequences set forth in
SEQ ID
NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 or the non-coding strand
complementary
thereto, under the following hybridization conditions:
(a) hybridization for 16 hours at 42°C in a hybridization solution
comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and
(b) washing 2 times for 30 minutes at 60°C in a wash solution
comprising 0.1× SSC and 1% SDS,
with the proviso that the nucleotide sequence of the polynucleotide differs
from the
coding sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15,
17, or
19 and from its complementary strand by at least one nucleotide.
23. A polynucleotide according to claim 22 that encodes a human
seven transmembrane receptor.
24. A vector comprising a polynucleotide according to any one of
claims 17-23.
25. A vector according to claim 24 that is an expression vector for
expressing the polynucleotide in a mammalian cell.

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26. A host cell stably transformed or transfected with a polynucleotide
according to any one of claims 17-23 in a manner allowing the expression in
said host
cell of the polypeptide or fragment thereof encoded by the polynucleotide.
27. A host cell stably transformed or transfected with a vector
according to claim 24 or 25 in a manner allowing the expression in said host
cell of
the polypeptide or fragment thereof encoded by the polynucleotide.
28. A method for producing a seven transmembrane receptor
polypeptide comprising the steps of growing a host cell according to claim 26
or 27 in
a nutrient medium under conditions in which the host cell expresses a seven
transmembrane receptor encoded by the polynucleotide.
29. A method according to claim 28, further comprising a step of
isolating said polypeptide from said cell or said medium.
30. A method according to claim 29, further comprising a step of
isolating cell membranes from the host cell, wherein the cell membrane
comprises the
seven transmembrane receptor.
31. An antibody specific for a polypeptide according to any one of
claims 1-15.
32. The antibody of claim 31 which is a monoclonal antibody.
33. A hybridoma that produces an antibody according to claim 32.
34. An antibody according to claim 31 that is a humanized antibody.

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35. An antibody according to claim 31 that specifically binds an
extracellular epitope of a seven transmembrane receptor having an amino acid
sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12,
14,
16, 18 or 20.
36. An antibody according to claim 35 that specifically binds to the
amino-terminal extracellular domain of the seven transmembrane receptors.
37. A cell-free composition comprising polyclonal antibodies, wherein
at least one of said antibodies is an antibody according to claim 31.
38. An anti-idiotypic antibody specific for an antibody according to
claim 31.
39. A polypeptide comprising a fragment of an antibody according to
claim 31, wherein said fragment and said polypeptide specifically bind to a
seven
transmembrane receptor having an amino acid sequence selected from the group
consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20.
40. A polypeptide according to claim 39 that is selected from the
group consisting of single chain antibodies and CDR-grafted antibodies.
41. A composition comprising a polypeptide according to any one of
claims 1-16 in a pharmaceutically acceptable carrier.
42. A composition comprising an antibody according to any one of
claims 31, 32, 34, 35, or 36, or a polypeptide according to claim 39 or 40, in
a
pharmaceutically acceptable carrier.

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43. A method for modulating ligand binding of a seven
transmembrane receptor polypeptide according to any one of claims 1-15,
comprising
the step of contacting said seven transmembrane receptor polypeptide with an
antibody specific for said seven transmembrane receptor, under conditions
wherein
the antibody binds the receptor.
44. A method for modulating ligand binding of a seven
transmembrane receptor polypeptide comprising the step of contacting said
seven
transmembrane receptor polypeptide with a polypeptide according to claim 39 or
40.
45. An assay to identify compounds that bind a seven transmembrane
receptor polypeptide, said assay comprising the steps of:
(a) contacting a composition comprising a seven transmembrane
receptor polypeptide according to any of claims 1-15 with a compound suspected
of
binding the seven transmembrane receptor polypeptide; and
(b) measuring binding between the compound and the seven
transmembrane receptor polypeptide.
46. A method for identifying a modulator of binding between a seven
transmembrane receptor polypeptide and a binding partner of the seven
transmembrane receptor polypeptide, comprising the steps of:
(a) contacting the binding partner and a composition comprising
the seven transmembrane receptor polypeptide in the presence and in the
absence of a
putative modulator compound, where the seven transmembrane receptor
polypeptide
is a polypeptide according to any one of claims 1-15;
(b) measuring binding between the binding partner and said seven
transmembrane receptor polypeptide; and
(c) identifying a putative modulator compound in view of
decreased or increased binding between the binding partner and seven
transmembrane
receptor polypeptide in the presence of the putative modulator, as compared to
binding in the absence of the putative modulator.

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47. An assay according to claim 45 or 46 wherein the composition
comprises a cell expressing the seven transmembrane receptor polypeptide on
its
surface.
48. An assay according to claim 47 wherein the measuring step
comprises measuring intracellular signaling of the seven transmembrane
receptor
polypeptide induced by the compound.
49. A method for treating a neurological disorder comprising the step
of administering to a mammal in need of such treatment a pharmaceutical
composition comprising a compound in an amount effective to modulate
biological
activity of a seven transmembrane receptor in neurons of said mammal, wherein
the
compound is selected from the group consisting of:
(a) an antibody according to any one of claims 31, 32, 34, 35, or 36;
(b) an anti-idiotypic antibody according to claim 38;
(c) a polypeptide according to claim 39 or 40;
(d) a compound identified according to the method of claim 45; and
(e) a modulator identified according to claim 46.
50. The method of claim 49 wherein the neurological disorder is
schizophrenia.
51. A method according to claim 50, wherein the seven
transmembrane receptor comprises a polypeptide according to claim 8.
52. A method of treating schizophrenia comprising the step of
administering to a human diagnosed with schizophrenia an amount of a modulator
of
CON202 receptor activity sufficient to modulate CON202 receptor activity or
CON202 ligand binding in said human.

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53. A method of diagnosing schizophrenia or a susceptibility to
schizophrenia comprising the steps of:
(a) measuring the presence or amount of expression or activity of a
polypeptide according to claim 8 in a cell of a human patient; and
(b) comparing the measurement of step (a) to a measurement of expression
or activity of the polypeptide in a cell from a normal subject or the patient
at an earlier
time, wherein the diagnosis of schizophrenia or susceptibility to
schizophrenia is
based on the presence or amount of CON202 polypeptide expression or activity.
54. A method of screening a human subject to diagnose a disorder
affecting the brain or genetic predisposition therefor, comprising the steps
of:
(a) assaying nucleic acid of a human subject to determine a presence or an
absence of a mutation altering the amino acid sequence, expression, or
biological
activity of at least one seven transmembrane receptor that is expressed in the
brain,
wherein the seven transmembrane receptor comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16,
18, and
20, or an allelic variant thereof, and wherein the nucleic acid corresponds to
the gene
encoding the seven transmembrane receptor; and
(b) diagnosing the disorder or predisposition from the presence or absence of
said mutation, wherein the presence of a mutation altering the amino acid
sequence,
expression, or biological activity of allele in the nucleic acid correlates
with an
increased risk of developing the disorder.
55. A method according to claim 54, wherein the seven
transmembrane receptor is CON202 comprising an amino acid sequence set forth
in
SEQ ID NO: 14, or an allelic variant thereof.
56. A method according to claim 55, wherein the disease is
schizophrenia.

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57. A method according to claim 56, wherein the assaying step
comprises at least one procedure selected from the group consisting of:
(a) determining a nucleotide sequence of at least one codon of at least one
CON202 allele of the human subject;
(b) performing a hybridization assay to determine whether nucleic acid
from the human subject has a nucleotide sequence identical to or different
from one or
more reference sequences;
(c) performing a polynucleotide migration assay to determine whether
nucleic acid from the human subject has a nucleotide sequence identical to or
different
from one or more reference sequences; and
(d) performing a restriction endonuclease digestion to determine whether
nucleic acid from the human subject has a nucleotide sequence identical to or
different
from one or more reference sequences.
58. A method according to claim 56 wherein the assaying step
comprises: performing a polymerase chain reaction (PCR) to amplify nucleic
acid
comprising CON202 coding sequence, and determining nucleotide sequence of the
amplified nucleic acid.
59. A method of screening for a CON202 hereditary schizophrenia
genotype in a human patient, comprising the steps of:
(a) providing a biological sample comprising nucleic acid from
said patient, said nucleic acid including sequences corresponding to said
patient's
CON202 alleles;
(b) analyzing said nucleic acid for the presence of a mutation or
mutations;
(c) determining a CON202 genotype from said analyzing step; and
(d) correlating the presence of a mutation in a CON202 allele with
a hereditary schizophrenia genotype.

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60. The method according to claim 59 wherein said biological
sample is a cell sample.
61. The method according to claim 59 wherein said analyzing
comprises sequencing a portion of said nucleic acid, said portion comprising
at least
one codon of said CON202 alleles.
62. The method according to claim 59 wherein said nucleic acid is
DNA.
63. The method according to claim 59 wherein said nucleic acid is
RNA.
64. A kit for screening a human subject to diagnose schizophrenia
or a genetic predisposition therefor, comprising, in association:
(a) an oligonucleotide useful as a probe for identifying polymorphisms in a
human CON202 seven transmembrane receptor gene, the oligonucleotide comprising
6-50 nucleotides that have a sequence that is identical or exactly
complementary to a
portion of a wild type human CON202 gene sequence or CON202 coding sequence,
except for one sequence difference selected from the group consisting of a
nucleotide
addition, a nucleotide deletion, or nucleotide substitution; and
(b) a media packaged with the oligonucleotide containing information
identifying polymorphisms identifyable with the probe that correlate with
schizophrenia or a genetic predisposition therefor.

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65. A method of identifying a seven transmembrane allelic variant
that correlates with a mental disorder, comprising steps of:
(a) providing a biological sample comprising nucleic acid from a
human patient diagnosed with a mental disorder, or from the patient's genetic
progenitors or progeny;
(b) analyzing said nucleic acid for the presence of a mutation or
mutations in at least one seven transmembrane receptor that is expressed in
the brain,
wherein the at least one seven transmembrane receptor comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12,
14, 16,
18, and 20, or an allelic variant thereof, and wherein the nucleic acid
includes
sequence corresponding to the gene or genes encoding the at least one seven
transmembrane receptor;
(c) determining a genotype for the patient for the at least one seven
transmembrane receptor from said analyzing step; and
(d) identifying an allelic variant that correlates with the mental
disorder from the determining step.
66. A method according to claim 65, wherein the disorder is
schizophrenia, and wherein the at least one seven transmembrane receptor
comprises
CON202 having an amino acid sequence set forth in SEQ ID NO: 14, or an allelic
variant thereof.
67. A purified and isolated polynucleotide comprising a nucleotide
sequence encoding a CON202 receptor allelic variant identified according to
claim 66.
68. A host cell transformed or transfected with a polynucleotide
according to claim 67 or with a vector comprising the polyncleotide.

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69. A purified polynucleotide comprising a nucleotide sequence
encoding a CON202 seven transmembrane receptor protein of a human that is
affected
with schizophrenia;
wherein said polynucleotide hybridizes to the complement of SEQ ID
NO: 13 under the following hybridization conditions:
(a) hybridization for 16 hours at 42°C in a hybridization solution
comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and
(b) washing 2 times for 30 minutes at 60°C in a wash solution
comprising 0.1x SSC and 1% SDS; and
wherein the polynucleotide encodes a CON202 amino acid sequence
that differs from SEQ ID NO: 14 at at least one residue.
70. A vector comprising a polynucleotide according to claim 69.
71. A host cell that has been transformed or transfected with a
polynucleotide according to claim 70 and that expresses the CON202 protein
encoded
by the polynucleotide.
72. A host cell according to claim 71 that has been co-transfected
with a polynucleotide encoding the CON202 amino acid sequence set forth in SEQ
ID
NO: 14 and that expresses the con202 protein having the amino acid sequence
set
forth in SEQ ID NO: 14.
73. A method for identifying a modulator of CON202 biological
activity, comprising the steps of:
a) contacting a cell according to claim 71 in the presence and in
the absence of a putative modulator compound;
b) measuring CON202 biological activity in the cell; and
c) identifying a putative modulator compound in view of
decreased or increased CON202 biological activity in the presence versus
absence of
the putative modulator.

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74. An assay to identify compounds useful for the treatment of
schizophrenia, said assay comprising steps of:
(a) contacting a composition comprising a seven transmembrane
receptor polypeptide according to claim 8 with a compound suspected of binding
the
seven transmembrane receptor polypeptide;
(b) measuring binding between the compound and the seven
transmembrane receptor polypeptide; and
(c) identifying molecules that bind the seven transmembrane receptor
as candidate compounds useful for the treatment of schizophrenia.
75. A method for identifying compound useful for a modulator of
binding between a seven transmembrane receptor polypeptide and a binding
partner of
the seven transmembrane receptor polypeptide, which modulator is useful for
treatment of schizophrenia, comprising the steps of:
(a) contacting the binding partner and a composition comprising
the seven transmembrane receptor polypeptide in the presence and in the
absence of a
putative modulator compound, where the seven transmembrane receptor
polypeptide
is a polypeptide according to claim 8;
(b) measuring binding between the binding partner and the seven
transmembrane receptor polypeptide;
(c) identifying a modulator compound useful for the treatment of
schizophrenia in view of decreased or increased binding between the binding
partner
and seven transmembrane receptor polypeptide in the presence of the putative
modulator, as compared to binding in the absence of the putative modulator.
76. An assay according to claim 74 or 75 wherein the composition
comprises a cell expressing the seven transmembrane receptor polypeptide on
its
surface.

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77. An assay according to claim 76 wherein the composition
comprises a cell transformed or transfected with a polynucleotide encoding the
seven
transmembrane polypeptide and expressing the seven transmembrane receptor
polypeptide on its surface.

Description

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
G PROTEIN--COUPLED RECEPTORS EXPRESSED IN BRAIN
RELATED APPLICATIONS
This patent application is a continuation-in-part of the following U.S.
patent applications: Serial No. 09/481,794 filed January 12, 2000; Serial No.
09/454,399 filed December 3, 1999; Serial Nos. 09/429,517, 09/429,555,
09/429,676,
09/429, G9S filed October 28, 1999; and Serial Nos. 09/428,114, 09/428,020,
091427,859 and 09/427,653 filed October 27, 1999. All these application are
incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates generally to the fields of genetics and
cellular and molecular biology. More particularly, the invention relates to a
novel G
protein-coupled seven transmembrane receptor polynucleotide and polypeptide
sequences that are expressed in the brain.
DESCRIPTION OF RELATED ART
Humans and other life forms are comprised of living cells. Among the
mechanisms through which the cells of an organism communicate with each other
and
obtain information and stimuli from their environment is through cell membrane
receptor molecules expressed on the cell surface. Many such receptors have
been
identified, characterized, and sometimes classified into major receptor
superfamilies
based on structural motifs and signal transduction features. Such families
include (but
are not limited to) ligand-gated ion channel receptors, voltage-dependent ion
channel
receptors, receptor tyrosine kinases, receptor protein tyrosine phosphatases,
arid G
protein-coupled receptors. The receptors are a first essential link for
translating an
extracellular signal into a cellular physiological response.
The G protein-coupled receptors (GPCR) form a vast superfamily of
cell surface receptors which are characterized by an amino-terminal
extracellular
domain, a carboxyl-terminal intracellular domain, and a serpentine structure
that
passes through the cell membrane seven times. Hence, such receptors are
sometimes
also referred to as seven transmembrane (7TM) receptors. These seven

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transmembrane domains define three extracellular loops and three intracellular
loops,
in addition to the amino- and carboxyl-terminal domains. The extracellular
portions
of the receptor have a role in recognizing and binding one or more
extracellular
binding partners (ligands), whereas the intracellular portions have a role in
recognizing and communicating with downstream effector molecules.
The G protein-coupled receptors bind a variety of ligands including
calcium ions, hormones, chemokines, neuropeptides, neurotransmitters,
nucleotides,
lipids, odorants, and even photons, and are important in the normal (and
sometimes
the aberrant) function of many cell types. (See generally A.D. Strosberg, Eur.
J.
Biochern., 19G: 1-10 (1991) and S. K. Bolun et ccl., Biochem J., 322: 1-18
(1997).]
When a specific ligand binds to its corresponding receptor, the ligand
stimulates the
receptor to activate a specific heterotrimeric guanine-nucleotide-binding
regulatory
protein (G-protein) that is coupled to the intracellular portion of the
receptor. The G
protein in tum transmits a signal to an effector molecule within the cell, by
either
stimulating or inhibiting the activity of that effector molecule. These
effector
molecules include adenylate cyclase, phospholipases, and ion channels.
Adenylate
cyclase and phospholipases are enzymes that are involved in the production of
the
second messenger molecules cAMP, inositol triphosphate and diacyglycerol. It
is
through this sequence of events that an extracellular ligand stimuli exerts
intracellular
changes through a G protein-coupled receptor. Each such receptor has its own
characteristic primary structure, expression pattern, ligand-binding profile,
and
intracellular effector system.
Because of the vital role of G protein-coupled receptors in the
communication between cells and their environment, such receptors are
attractive
targets for therapeutic intervention, and many drugs have been registered
which are
directed towards activating or antagonizing such receptors. For receptors
having a
known ligand, the identification of agonists or antagonists may be sought
specifically
for enhancing or inhibiting the action of the ligand. Some G protein-coupled
receptors have roles in disease pathogenesis (e.g., certain chemokine
receptors that act
as HIV co-receptors and may have a role in AIDS pathogenesis), and are
attractive
targets for therapeutic intervention even in the absence of knowledge of the
natural

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ligand of the receptor. Other receptors are attractive targets for therapeutic
intervention by virtue of their expression pattern in tissues or cell types
that are
attractive targets for therapeutic intervention. Examples of this latter
category of
receptors include receptors expressed in immune cells, for targeting to
enhance
immune responses to f ght pathogens or cancer or inhibit autoimmune responses;
and
receptors expressed in the brain or other neurons, for targeting to treat
schizophrenia,
depression, bipolar disease, or other neurological disorders. This latter
category of
receptor is also useful as a marker for identifying and/or purifying (e.g.,
via
fluorescence activated cell sorting) cellular subtypes that express the
receptor.
Unfortunately, only a limited number of G protein receptors from the central
nervous
system (CNS) are known. A need exists for identifying the existence and
structure of
such G protein-coupled receptors.
SUMMARY OF THE INVENTION
The present invention addresses one or more of the needs identified
above in that it provides purified polynucleotides encoding heretofore unknown
G
protein-coupled receptors (GPCR); constructs and recombinant host cells
incorporating the polynucleotides; GPCR polypeptides encoded by the
polynucleotides; antibodies to the polypeptides; and methods of making and
using all
of the foregoing. As set forth in detail herein, the GPCR polypeptides
described
herein are expressed in the brain, providing a therapeutic indication for GPCR
polypeptides and binding partners to treat diseases associated with this
tissue.
The invention provides purified and isolated GPCR seven
transmembrane receptor polypeptides comprising any one of the amino acid
sequences set forth in SEQ >D NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20, or a
fragment
thereof comprising an epitope specific to the seven transmembrane receptor. By
"epitope specific lo" is meant a portion of the receptor that is recognizable
by an
antibody that is specific for that seven transmembrane receptor, as defined in
detail
below.
One preferred embodiment comprises a purified and isolated
polypeptide designated CON193, comprising the complete amino acid sequence set

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forth in SEQ ID NO: 2. 'this amino acid sequence was deduced from a
polynucleotide CON193 (SEQ NO:I),
sequence LD as
encoding set
forth
below:
ntggttgttg gaccattaaa aagttggggg agagggagac60
atgcattatg
gaatttttaa
agtaaaaata acctatattt tttttttttt aactctagga
aagcccagac120
tctcttgttt
aaattttgag ctatttcata cttatc atgcta acactgaat aaa 174
acctaccaga
Me t Thr u Lys
Leu Le Asn
1 5
aca gac ctaatacca gettcattt attctgaatgga gtcccagga ctg 222
Thr Asp LeuIlePro AlaSerPhe IleLeuAsnGly ValProGly Leu
10 15 zo
gaa gac acacaactc tggatttcc ttcccattctgc tctatgtat gtt 270
Glu Asp ThrGlnLeu TrpIleSer PheProPheCys SerMetTyr Val
25 30 35
gtg get atggtaggg aattgtgga ctcctctacetc attcactat gag 318
I$ Val Ala MetValGly AsnCysGly LeuLeuTyrLeu IleHisTyr Glu
40 45 50
gat gcc ctgcacaaa cccatgtac tacttcttggcc atgctttcc ttt 366
Asp Ala LeuHisLys ProMetTyr TyrPheLeuAla MetLeuSer Phe
55 60 65 70
act gac cttgttatg tgctctagt acaatccctaaa gccctctgc atc 414
Thr Asp LeuValMet CysSerSer ThrIleProLys AlaLeuCys Ile
75 80 85
ttc tgg tttcatctc aaggacatt ggatttgatgaa tgccttgtc cag 462
Phe Trp PheHisLeu LysAspIle GlyPheAspGlu CysLeuVal Gln
2$ 90 95 100
atg ttc ttcatccac accttcaca gggatggagtct ggggtgctt atg 510
Met Phe PheIleHis ThrPheThr GlyMetGluSer GlyValLeu Met
105 110 115
ctt atg gccctggat cgctatgtg gccatctgctac cccttacgc tat 558
Leu Met AlaLeuAsp ArgTyrVal AlaIleCysTyr ProLeuArg Tyr
120 125 130
tca act atcctcacc aatcctgta attgcaaaggtt gggactgcc acc 606
Ser Thr IleLeuThr AsnProVal IleAlaLysVal GlyThrAla Thr
135 140 145 150
ttc ctg agaggggta ttactcatt attccctttact ttcctcacc aag 654
Phe Leu ArgGlyVal LeuLeuIle IleProPheThr PheLeuThr Lys
155 160 165
cgc ctg ccctcctgc agaggcaat atacttccccat acctactgt gac 702
Arg Leu ProSerCys ArgGlyAsn IleLeuProHis ThrTyrCys Asp
170 175 180

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cac atg tct gta gcc aaa ttg tcc tgt ggt aat gtc aag gtc aat gcc 750
His Met SerValAla LysLeuSer CysGlyAsn ValLysValAsn Ala
185 190 195
atc tat ggtctgatg gttgccctc ctgattggg ggctttgacata ctg 798
Ile Tyr GlyLeuMet ValAlaLeu LeuIleGly GlyPheAspIle Leu
200 205 210
tgt atc accatctcc tataccatg attctccgg gcagtggtcagc ctc 846
Cys Ile ThrIleSer TyrThrMet IleLeuArg AlaValValSer Leu
215 220 225 230
tec tca gcagatget cggcagaag gectttaat acetgcactgce eac 894
Ser Ser AlaAspAla ArgGlnLys AlaPheAsn ThrCysThrAla His
235 240 245
att tgt gccattgtt ttctcetat actecaget ttcttctcctte ttt 942
Ile Cys AlaIleVal PheSerTyr ThrProAla PhePheSerPhe Phe
1$ 250 255 260
tcc cac cgctttggg gaacacata atcccccct tcttgccacatc att 990
Ser His ArgPheGly GluHisIle IleProPro SerCysHisIle Ile
265 270 275
gta gcc aatatttat ctgctccta ccacccact atgaaccctatt gtc 1038
Val Ala AsnIleTyr LeuLeuLeu ProProThr MetAsnProIle Val
280 285 290
tat ggg gtgaaaacc aaacagata cgagactgt gtcataaggatc ctt 1086
Tyr Gly ValLysThr LysGlnIle ArgAspCys ValIleArgIle Leu
295 300 305 310
tca ggt tctaaggat accaaatcc tacagcatg tgaatgaaca ctt 1132
Ser Gly SerLysAsp ThrLysSer TyrSerMet
315 320
gccaggagtg agaagagaag cttctatttg cctcttatgc
aggagttcat1192
gaaagaatta
aaaatctttc tggaagtact caaaatggag tttgntgact
ggtgcattc 1252
gtattgatca
caataagtac cttgggaatc ggaaggccca ccacatttct
ataaat 1308
tnacatcact
Another preferred embodiment fiedand
comprises isolated
a
puri
polypeptide designated CON166, comprising the complete amino acid sequence set
forth in SEQ ID NO: 4. This amino acid sequence was deduced from a
polynucleotide
sequence
encoding
CON166
(SEQ
ID NO:
3), as
set forth
below:
atg gat gaaacagga aatctgaca tcttct acatgc catgac
gta gcc 48
Met Asp GluThrGly AsnLeuThr SerSer ThrCys HisAsp
Val Ala
1 5 10 15
act att gatgacttc cgcaatcaa tattcc ttgtac tctatg
gtg acc 96
Thr Ile AspAspPhe ArgAsnGln TyrSer LeuTyr SerMet
Val Thr
20 25 30

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atc tct gtt gta ggc ttc ttt ggc aat ggc ttt gtg ctc tat gtc ctc 144
Ile Ser Val Val Gly Phe Phe Gly Asn Gly Phe Val Leu Tyr Val Leu
35 40 45
ata aaa acc tat cac aag aag tca gcc ttc caa gta tac atg att aat 192
S Ile Lys ThrTyrHisLys LysSer AlaPheGln ValTyrMet IleAsn
50 55 60
tta gca gtagcagatcta ctttgt gtgtgcaca ctgcctctc cgtgtg 240
Leu Ala ValAlaAspLeu LeuCys ValCysThr LeuProLeu ArgVal
65 70 75 80
gtc tat tatgttcacaaa ggcatt tggctcttt ggtgacttc ttgtgc 288
Val Tyr TyrValHisLys GlyIle TrpLeuPhe GlyAspPhe LeuCys
85 90 95
cgc ctc agcacctatget ttgtat gtcaacctc tattgtagc atcttc 336
Arg Leu SerThrTyrAla LeuTyr ValAsnLeu TyrCysSer IlePhe
100 1os 110
ttt atg acagccatgagc tttttc cggtgcatt gcaattgtt tttcca 384
Phe Met ThrAlaMetSer PhePhe ArgCysIle AlaIleVal PhePro
115 120 125
gtc cag aac att aat ttg gtt aca cag aaa aaa gcc agg ttt gtg tgt 432
Val Gln AsnIleAsn LeuValThr GlnLysLys AlaArgPhe ValCys
130 135 140
gta ggt atttggatt tttgtgatt ttgaccagt tctccattt ctaatg 480
Val Gly IleTrpIle PheValIle LeuThrSer SerProPhe LeuMet
145 150 155 160
2$ gcc aaa ccacaaaaa gatgagaaa aataat-acc aagtgcttt gagccc 528
Ala Lys ProGlnLys AspGluLys AsnAsnThr LysCysPhe GluPro
165 170 175
cca caa gacaatcaa actaaaaat catgttttg gtcttgcat tatgtg 576
Pro Gln AspAsnGln ThrLysAsn HisValLeu ValLeuHis TyrVal
180 185 190
tca ttg tttgttggc tttatcatc ccttttgtt attataatt gtctgt 624
Ser Leu PheValGly PheIleIle ProPheVal IleIleIle ValCys
195 200 205
tac aca atgatcatt ttgacctta ctaaaaaaa tcaatgaaa aaaaat 672
35 Tyr Thr MetIleIle LeuThrLeu LeuLysLys SerMetLys LysAsn
210 215 220
ctg tca agtcataaa aaggetata ggaatgatc atggtcgtg accget 720
Leu Ser SerHisLys LysAlaIle GlyMetIle MetValVal ThrAla
225 230 235 240
40 gcc ttt ttagtcagt ttcatgcca tatcatatt caacgtacc attcac 768
Ala Phe LeuValSer PheMetPro TyrHisIle GlnArgThr IleHis
245 250 255

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_7_
ctt cat ttt tta cac aat gaa act aaa ccc tgt gat tct gtc ctt aga 816
Leu His Phe Leu His Asn Glu Thr Lys Pro Cys Asp Ser Val Leu Arg
260 265 270
atg cag aag tcc gtg gtc ata acc ttg tct ctg get gca tcc aat tgt 864
$ Met Gln Lys Ser Val Val Ile Thr Leu Ser Leu Ala Ala Ser Asn Cys
275 280 285
tgc ttt gac cct ctc cta tat ttc ttt tct ggg ggt aac ttt agg aaa 912
Cys Phe Asp Pro Leu Leu Tyr Phe Phe Ser Gly Gly Asn Phe Arg Lys
290 295 300
agg ctg tct aca ttt aga aag cat tct ttg tcc agc gtg act tat gta 960
Arg Leu Ser Thr Phe Arg Lys His Ser Leu Ser Ser Val Thr Tyr Val
305 310 315 320
ccc aga aag aag gcc tct ttg cca gaa aaa gga gaa gaa ata tgt aaa 1008
Pro Arg Lys Lys Ala Ser Leu Pro Glu Lys Gly Glu Glu Ile Cys Lys
325 330 335
gta tag 1014
Val
Still another preferred embodiment comprises a purified and isolated
polypeptide designated CON103, comprising the complete amino acid sequence set
forth in SEQ ID NO: 6. This amino acid sequence was deduced from a
polynucleotide sequence encoding CON103 (SEQ ID NO: 5), as set forth below:
ggggcctact tcaccgtgta cccggacttg ggaccatcac agacttcaga accatcagga 60
acctgggagc aactgaaagc tgaactacag tgggctttca gacacacagc aggctgcgga 120
gcacaaatag gactggttcc ctccaggcca ccagcagggc ggtggaggtc ttcactgact 180
2S ccctgcctac ctctcaggac aatgtccttt tggctccaca gtccctgaag ccagagctgg 240
tgggggcagg gaggcagcca ccagcctcta tatgtagtgg aggagggggt gtccagggag 300
ggctgcatga tcctgagagc ccccacctca cccggctgga ctatcctccc acttcagggt 360
ttctctgggc ttccatcttg cccctgctga gccctgcttc ctcctctacc agcagcacaa 420
cccccaggct gggctcagag acctcatgtg gtgggatcac tcagtacccc gaggcggagg 480
gaaggaggga gggctgcagg gttccccttg gcctgcaaac aggaacacag ggtgtttctc 540
agtggctgcg agaatgctga tgaaaacccc aggatgttgt gtcaccgtgg tggccagctg 600
atagtgccaa tcatcccact ttgccctgag cactcctgca ggggtagaag actccagaac 660
cttctctcag gcccatggcc caagcagccc atg gaa ctt cat aac ctg agc tct 714
Met Glu Leu His Asn Leu Ser Ser
1 5
cca tct ccc tct ctc tcc tcc tct gtt Ctc cct ccc tcc ttc tct ccc 762
Pro Ser Pro Ser Leu Ser Ser Ser Val Leu Pro Pro Ser Phe Ser Pro
10 15 20
tca ccc tec tct get cce tct gcc ttt ace act gtg ggg ggg tce tct 810
Ser Pro Ser Ser Ala Pro Ser Ala Phe Thr Thr Val Gly Gly Ser Ser
25 30 35 40

CA 02386509 2002-04-16
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_g_
gga ggg ccc tgc cac ccc acc tct tcc tcg ctg gtg tct gcc ttc ctg 858
Gly Gly ProCysHis ProThrSer SerSerLeu ValSer AlaPheLeu
45 50 55
gca cca atcctggcc ctggagttt gtcctgggc ctggtg gggaacagt 906
$ Ala Pro IleLeuAla LeuGluPhe ValLeuGly LeuVal GlyAsnSer
60 65 70
ttg gcc ctcttcatc ttctgcatc cacacgcgg ccctgg acctccaac 954
Leu Ala LeuPheIle PheCysIle HisThrArg ProTrp ThrSerAsn
75 80 85
acg gtg ttcctggtc agcctggtg gccgetgac ttcctc ctgatcagc 1002
Thr Val PheLeuVal SerLeuVal AlaAlaAsp PheLeu LeuIleSer
90 95 100
aac ctg cccctccgc gtggactac tacctcctc catgag acctggcgc 1050
Asn Leu ProLeuArg ValAspTyr LeuLeuHis GluThr TrpArg
105 110 115 120
ttt ggg getgetgcc tgcaaagtc aacctcttc atgctg tccaccaac 1098
Phe Gly AlaAlaAla CysLysVal AsnLeuPhe MetLeu SerThrAsn
125 130 135
cgc acg gccagcgtt gtcttcctc acagccatc gcactc aaccgctac 1146
Arg Thr AlaSerVal ValPheLeu ThrAlaIle AlaLeu AsnArgTyr
140 145 150
ctg aag gtggtgcag ccccaccac gtgctgagc cgtget tccgtgggg 1194
Leu Lys ValValGln ProHisHis ValLeuSer ArgAla SerValGly
155 160 165
gca get gcccgggtg gccggggga ctctgggtg ggcatc ctgctcctc 1242
Ala Ala AlaArgVal AlaGlyGly LeuTrpVal GlyIle LeuLeuLeu
170 175 180
aac ggg cacctgctc ctgagcacc ttctccggc ccctcc tgcctcagc 1290
Asn Gly HisLeuLeu LeuSerThr PheSerGly ProSer CysLeuSer
185 190 195 200
tac agg gtgggcacg aagccctcg gcctcgctc cgctgg caccaggca 1338
Tyr Arg ValGlyThr LysProSer AlaSerLeu ArgTrp HisGlnAla
205 210 215
ctg tac ctgctggag ttcttcctg ccactggcg ctcatc ctctttget 1386
Leu Tyr LeuLeuGlu PhePheLeu ProLeuAla LeuIle LeuPheAla
220 225 230
att gtg agcattggg ctcaccatc cggaaccgt ggtctg ggcgggcag 1434
Ile Val SerIleGly LeuThrIle ArgAsnArg GlyLeu GlyGlyGln
235 240 245
gca ggc ccgcagagg gccatgcgt gtgctggcc atggtg gtggccgtc 1482
Ala Gly ProGlnArg AlaMetArg ValLeuAla MetVal ValAlaVal
250 255 260

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-9-
tac acc atc tgc ttc ttg ccc agc ate atc ttt gge atg get tcc atg 1530
Tyr Thr Ile Cys Phe Leu Pro Ser Ile Ile Phe Gly Met Ala Ser Met
265 270 275 280
gtg get ttc tgg ctg tec gec tgc cga tce etg gac ctc tgc aca cag 1578
Val Ala Phe Trp Leu Ser Ala Cys Arg Ser Leu Asp Leu Cys Thr Gln
285 290 295
ctc ttc cat ggc tcc ctg gcc ttc acc tac ctc aac agt gtc ctg gac 1626
Leu Phe His Gly Ser Leu Ala Phe Thr Tyr Leu Asn Ser Val Leu Asp
300 305 310
ccc gtg ctc tac tgc ttc tct agc ccc aac ttc ctc cac cag agc cgg 1674
Pro Val Leu Tyr Cys Phe Ser Ser Pro Asn Phe Leu His Gln Ser Arg
315 320 325
gcc ttg ctg ggc ctc acg cgg ggc cgg cag ggc cca gtg agc gac gag 1722
Ala Leu Leu Gly Leu Thr Arg Gly Arg Gln Gly Pro Val Ser Asp Glu
1$ 330 335 340
agc tcc tac caa ccc tcc agg cag tgg cgc tac cgg gag gcc tct agg 1770
Ser Ser Tyr Gln Pro Ser Arg Gln Trp Arg Tyr Arg Glu Ala Ser Arg
345 350 355 360
aag gcg gag gcc ata ggg aag ctg aaa gtg cag ggc gag gtc tct ctg 1818
Lys Ala Glu Ala Ile Gly Lys Leu Lys Val Gln Gly Glu Val Ser Leu
365 370 375
gaa aag gaa ggc tcc tcc cag ggc tga gggccagctg cagggctgca 1865
Glu Lys Glu Gly Ser Ser Gln Gly
380 385
2$ gcgctgtggg ggtaagggct gccgcgctct ggcctggagg gacaaggcca gcacacggtg1925
cctcaaccaa ctggacaagg gatggcggca gaccaggggc caggccaaag cactggcagg1985
actcatgtgg gtggcaggga gagaaaccca cctaggcctc tcagtgtgtc caggatggca2045
ttcccagaat gcaggggaga gcaggatgcc gggtggagga gacaggcaag gtgccgttgg2105
cacaccagct cagacagggg cctgcgcagc tgcaggggac agacgccaat cactgtcaca2165
gcagagtcac cttagaaatt ggacagctgc atgttctgtg ctctccagtt tgtcccttcc2225
aatattaata aacttccctt ttaaatatat ttatttgcag accaatatct gtctttaatt2285
ctaacctggg actgtcagta ggcgtcaaag tgagcgcccc agtgaaggaa ccttggagag2345
agtgggagca ttcccagcct tccaggggga ctcgtcttcc agactttgga gcccgcatgt2405
ctgaagcaga ctctttcttg gtag 2429
3$ Another preferred embodiment comprises a purified and isolated
polypeptide designated CON203, comprising the complete amino acid sequence set
forth in SEQ m NO: 8. This amino acid sequence was deduced from a
polynucleotide sequence encoding CON203 (SEQ ID NO: 7), as set forth below:
ttgaatttag gtgacactat agaagagcta tgacgtcgca tgcacgcgta cgtaagctcg 60
gaattcggct cgagctgaac taatgactgc cgccataaga agacagagag aactgagtat 120

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cctcccaaag gtgacactgg aagca atg aac acc aca gtg atg caa ggc ttc 172
Met Asn Thr Thr Val Met Gln Gly Phe
1 5
aac aga tct gag cgg tgc ccc aga gac act cgg ata gta cag ctg gta 220
S Asn ArgSer GluArgCys ProArg AspThrArg IleValGln LeuVal
15 20 25
ttc ccagcc ctctacaca gtggtt ttcttgacc ggcatcctg ctgaat 268
Phe ProAla LeuTyrThr ValVal PheLeuThr GlyIleLeu LeuAsn
30 35 40
1~ act ttgget ctgtgggtg tttgtt cacatcccc agctcctcc accttc 316
Thr LeuAla LeuTrpVal PheVal HisIlePro SerSerSer ThrPhe
45 50 55
atc atctac ctcaaaaac actttg gtggccgac ttgataatg acactc 364
Ile IleTyr LeuLysAsn ThrLeu ValAlaAsp LeuIleMet ThrLeu
IS 60 65 70
atg cttcct ttcaaaatc ctctct gactcacac ctggcaccc tggcag 412
Met LeuPro PheLysIle LeuSer AspSerHis LeuAlaPro TrpGln
75 80 85
ctc agaget tttgtgtgt cgtttt tcttcggtg atattttat gagacc 460
2~ Leu ArgAla PheValCys ArgPhe SerSerVal IlePheTyr GluThr
90 95 100 105
atg tatgtg ggcatcgtg ctgtta gggctcata gcctttgac agattc 508
Met TyrVal GlyIleVal LeuLeu GlyLeuIle AlaPheAsp ArgPhe
110 115 120
25 ctc aagatc atcagacct ttgaga aatattttt ctaaaaaaa cctgtt 556
Leu LysIle IleArgPro LeuArg AsnIlePhe LeuLysLys ProVal
125 130 135
ttt gcaaaa acggtctca atcttc atctgggtc tttttggtc ttcatc 604
Phe AlaLys ThrValSer IlePhe IleTrpVal PheLeuVal PheIle
140 145 150
tcc ctg cca aat atg atc ttg agc aac aag gaa gca aca cca tcg tct 652
Ser Leu Pro Asn Met Ile Leu Ser Asn Lys Glu Ala Thr Pro Ser Ser
155 160 165
gtg aaa aag tgt get tcc tta aag ggg cct ctg ggg ctg aaa tgg cat 700
35 Val Lys Lys Cys Ala Ser Leu Lys Gly Pro Leu Gly Leu Lys Trp His
170 175 180 185
caa atg gta aat aac ata tgc cag ttt att ttc tgg act ggt ttt atc 748
Gln Met Val Asn Asn Ile Cys Gln Phe Ile Phe Trp Thr Gly Phe Ile
190 195 200
40 cta atg ctt gtg ttt tat gtg gtt att gca aaa aaa gta tat gat tct 796
Leu Met Leu Val Phe Tyr Val Val Ile Ala Lys Lys Val Tyr Asp Ser
205 210 215

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tat aga aag tcc aaa agt aag gac aga aaa aac aac aaa aag ctg gaa 844
Tyr Arg LysSerLys SerLysAsp ArgLysAsn AsnLysLys LeuGlu
220 225 230
ggc aaa gtatttgtt gtcgtgget gtcttcttt gtgtgtttt getcca 892
G.ly Lys ValPheVal ValValAla ValPhePhe ValCysPhe AlaPro
235 240 245
ttt cat tttgccaga gttccatat actcacagt caaaccaac aataag 940
Phe His PheAlaArg ValProTyr ThrHisSer GlnThrAsn AsnLys
250 255 260 265
act gac tgtagactg caaaatcaa ctgtttatt getaaagaa acaact 988
Thr Asp CysArgLeu GlnAsnGln LeuPheIle AlaLysGlu ThrThr
270 275 280
ctc ttt ttggcagca actaacatt tgtatggat cccttaata tacata 1036
Leu Phe LeuAlaAla ThrAsnIle CysMetAsp ProLeuIle TyrIle
IS 285 290 295
ttc tta tgt aaa aaa ttc aca gaa aag cta cca tgt atg caa ggg aga 1084
Phe Leu Cys Lys Lys Phe Thr Glu Lys Leu Pro Cys Met Gln Gly Arg
300 305 310
aag acc aca gca tca agc caa gaa aat cat agc agt cag aca gac aac 1132
Lys Thr Thr Ala Ser Ser Gln Glu Asn His Ser Ser Gln Thr Asp Asn
315 320 325
ata acc tta ggc tga caactgtaca tagggttaac ttctatttat tgatgagact 1187
Ile Thr Leu Gly
330
tccgtagata atgtggaaat caaatttaac caagaaaaaa agattggaac aaatgctctc1247
ttacatttta tttatcctgg tgtccaggaa aagattatat taaatttaaa tccacataga1307
tctattcata agctgaatga accattacct aagagaatgc aacaggatac caatggccac1367
tagaggcata ttccttcttc tttttttttt gttaaatttc aagagcattc actttacatt1427
tggaaagact aaggggaacg gttatcctac aaacctccct tcaacacctt ttacatt 1484
Another preferred embodiment comprises a purified and isolated
polypeptide designated CON198, comprising the complete amino acid sequence set
forth in SEQ ID NO: 10. This amino acid sequence was deduced from a
polynucleotide sequence encoding CON198 (SEQ ID NO: 9), as set forth below:
atg atg gtg gat ccc aat ggc aat gaa tcc agt get aca tac ttc atc 48
Met Met Val Asp Pro Asn Gly Asn Glu Ser Ser Ala Thr Tyr Phe Ile
1 5 10 15
cta ata ggc ctc cct ggt tta gaa gag get cag ttc tgg ttg gcc ttc 96
Leu Ile Gly Leu Pro Gly Leu Glu Glu Ala Gln Phe Trp Leu Ala Phe
20 25 30
cca ttg tgc tcc ctc tac ctt att get gtg cta ggt aac ttg aca atc 144
Pro Leu Cys Ser Leu Tyr Leu Ile Ala Val Leu Gly Asn Leu Thr Ile
35 40 45

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- 12-
atc tac att gtg cgg act gag cac agc ctg cat gag ccc atg tat ata 192
Ile Tyr Ile Val Arg Thr Glu His Ser Leu His Glu Pro Met Tyr Ile
50 55 60
ttt ctt tgc atg ctt tca ggc att gac atc ctc atc tcc acc tca tcc 240
Phe Leu Cys Met Leu Ser Gly Ile Asp Ile Leu Ile Ser Thr Ser Ser
65 70 75 80
atg ccc aaa atg ctg gcc atc ttc tgg ttc aat tcc act acc atc cag 288
Met Pro Lys Met Leu Ala Ile Phe Trp Phe Asn Ser Thr Thr Ile Gln
85 90 95
ttt gat get tgt ctg cta cag atg ttt gcc atc cac tcc tta tct ggc 336
Phe Asp Ala Cys Leu Leu Gln Met Phe Ala Ile His Ser Leu Ser Gly
1$ 100 105 110
atg gaa tcc aca gtg ctg ctg gcc atg get ttt gac cgc tat gtg gcc 384
Met Glu Ser Thr Val Leu Leu Ala Met Ala Phe Asp Arg Tyr Val Ala
115 120 125
atc tgt cac cca ctg cgc cat gcc aca gta ctt acg ttg cct cgt gtc 432
Ile Cys His Pro Leu Arg His Ala Thr Val Leu Thr Leu Pro Arg Val
130 135 140
acc aaa att ggt gtg get get gtg gtg cgg ggg get gca ctg atg gca 480
Thr Lys Ile Gly Val Ala Ala Val Val Arg Gly Ala Ala Leu Met Ala
145 150 155 160
ccc ctt cct gtc ttc atc aag cag ctg ccc ttc tgc cgc tcc aat atc 528
Pro Leu Pro Val Phe Ile Lys Gln Leu Pro Phe Cys Arg Ser Asn Ile
165 170 175
ctt tcc cat tcc tac tgc cta cac caa gat gtc atg aag ctg gcc tgt 576
Leu Ser His Ser Tyr Cys Leu His Gln Asp Val Met Lys Leu Ala Cys
180 185 190
gat gat atc cgg gtc aat gtc gtc tat ggc ctt atc gtc atc atc tcc 624
Asp Asp Ile Arg Val Asn Val Val Tyr Gly Leu Ile Val Ile Ile Ser
195 200 205
gcc att ggc ctg gac tca ctt ctc atc tcc ttc tca tat ctg ctt att 672
Ala Ile Gly Leu Asp Ser Leu Leu Ile Ser Phe Ser Tyr Leu Leu Ile
210 215 220
4$ ctt aag act gtg ttg ggc ttg aca cgt gaa gcc cag gcc aag gca ttt 720
Leu Lys Thr Val Leu Gly Leu Thr Arg Glu Ala Gln Ala Lys Ala Phe
225 230 235 240
ggc act tgc gtc tct cat gtg tgt get gtg ttc ata ttc tat gta cct 768
$0 Gly Thr Cys Val Ser His Val Cys Ala Val Phe Ile Phe Tyr Val Pro
245 250 255
ttc att gga ttg tcc atg gtg cat cgc ttt agc aag cgg cgt gac tct 816
Phe Ile Gly Leu Ser Met Val His Arg Phe Ser Lys Arg Arg Asp Ser
55 260 265 270
ccg ctg ccc gtc atc ttg gcc aat atc tat ctg ctg gtt cct cct gtg 864
Pro Leu Pro Val Ile Leu Ala Asn Ile Tyr Leu Leu Val Pro Pro Val
275 280 285
GO
ctc aac cca att gtc tat gga gtg aag aca aag gag att cga cag cgc 912
Leu Asn Pro Ile Val Tyr Gly Val Lys Thr Lys Glu Ile Arg Gln Arg
290 295 300

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atc ett cga ctt ttc cat gtg gcc aca cac get tca gag ccc tag 957
Ile Leu Arg Leu Phe His Val Ala Thr His Ala Ser Glu Pro
305 310 315
It will be appreciated that SEQ LD NO: 10 contains methionine
S residues at positions 1 and 2. Translation of the relevant mRNA sequences
may occur
beginning from either or both methionines, which can be determined for a
particular
cell source by purifying expressed CON198 protein and performing amino-
terminal
sequencing thereon. CON 198 polypeptides beginning at either Met, or Metz of
SEQ
~ NO: 10 are intended a polypeptides of the invention.
Another preferred embodiment comprises a purified and isolated
polypeptide designated CON 197, comprising the complete amino acid sequence
set
forth in SEQ ID NO: 12. This amino acid sequence was deduced from a
polynucleotide sequence encoding CON197 (SEQ ID NO: 11), as set forth below:
1
IS ATGGAAAGCGAGAACAGAAGAGTGATAAGAGAATTCATCCTCCTTGGTCTGACCCAGTCTCAAGATATT
M E S E N R R V I R E F I L L G L T Q S Q D I
CAGCTCCTGGTCTTTGTGCTAGTTTTAATATTCTACTTCATCATCCTCCCTGGAAATTTTCTCATTATT
ZO Q L L V F V L V L I F Y F I I L P G N F L I I
ZS
139
TTCACCATAAAGTCAGACCCTGGGCTCACAGCCCCCCTCTATTTCTTTCTGGGCAACTTGGCCTTCCTG
F T I K S D P G L T A P L Y F F L G N L A F L
208
GATGCATCCTACTCCTTCATTGTGGCTCCCCGGATGTTGGTGGACTTCCTCTCTGCGAAGAAGATAATC
D A S Y S F I V A P R M L V D F L S A K K I I
30 277
TCCTACAGAGGCTGCATCACTCAGCTCTTTTTCTTGCACTTCCTTGGAGGAGGGGAGGGATTACTCCTT
S Y R G C I T Q L F F L H F L G G G E G L L L
346
3S GTTGTGATGGCCTTTGACCGCTACATCGCCATCTGCCGGCCTCTGCACTATCCTACTGTCATGAACCCT
V V M A F D R Y I A I C R P L H Y P T V M N P
415
AGAACCTGCTATGCAATGATGTTGGCTCTGTGGCTTGGGGGTTTTGTCCACTCCATTATCCAGGTGGTC
4O R T C Y A M M L A L W L G G F V H S I I Q V V
4S
484
CTCATCCTCCGCTTGCCTTTTTGTGGCCCAAACCAGCTGGACAACTTCTTCTGTGATGTCCCACAGGTC
L I L R L P F C G P N Q L D N F F C D V P Q V
553
ATCAAGCTGGCCTGCACCGACACATTTGTGGTGGAGCTTCTGATGGTCTTCAACAGTGGCCTGATGACA
I K L A C T D T F V V E L L M V F N S G L M T
S0 622
CTCCTGTGCTTTCTGGGGCTTCTGGCCTCCTATGCAGTCATTCTTTGTCGCATACGAGGGTCTTCTTCT
L L C F L G L L A S Y A V I L C R I R G S S S

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GAGGCAAAAAACAAGGCCATGTCCACGTGCATCACCCATATCATTGTTATATTCTTCATGTTTGGACCT
E A K N K A M S T C I T H I I V I F F M F G P
S 760
GGCATCTTCATCTACACGCGCCCCTTCAGGGCTTTCCCAGCTGACAAGGTGGTTTCTCTCTTCCACACA
G I F I Y T R P F R A F P A D K V V S L F H T
829
IO GTGATTTTTCCTTTGTTGAATCCTGTCATTTATACCCTTCGCAACCAGGAAGTGAAAGCTTCCATGAAA
V I F P L L N P V I Y T L R N Q E V K A S M K
898
AAGGTGTTTAATAAGCACATAGCCTGAAAAAGGGCGCAAP,AAAAAAAAGAATAAAAATAGACTGTAGAA
IS K V F N K H I A
967
TTTTT
Another preferred embodiment comprises a purified and isolated
20 polypeptide designated CON202, comprising the complete amino acid sequence
set
forth in SEQ >D NO: 14. This amino acid sequence was deduced from a
2S
polynucleotide sequence encoding CON202 (SEQ ID NO: I3), as set forth below:
1
TGCTTCCCCATAAGGTAACAGCTTTGTTAGCNCTGTCTGACATCATTGCTTGTTNACTTAAGAACTGAT
AGGTNTTTTTTTTTTTTTTTTTTCAGATATTCTGATGGCAAAACAAGTGGAAGAAAAGAGGAAGCATGA
139
3O CTGCAGATCAGATCAGTTCTCTTTGTGGATTATATTTTCAGTAAAATGTATGGATCTATCTTTTCCTTG
3S
208
TTCTTATATCTAGATCATGAGACTTGACTGAGGCTGTATCCTTATCCTCCATCCATCTATGGCGAACTA
M A N Y
277
TAGCCATGCAGCTGACAACATTTTGCAAAATCTCTCGCCTCTAACAGCCTTTCTGAAACTGACTTCCTT
S H A A D N I L Q N L S P L T A F L K L T S L
40 346
GGGTTTCATAATAGGAGTCAGCGTGGTGGGCAACCTCCTGATCTCCATTTTGCTAGTGAAAGATAAGAC
G F I I G V S V V G N L L I S I L L V K D K T
415
4S CTTGCATAGAGCACCTTACTACTTCCTGTTGGATCTTTGCTGTTCAGATATCCTCAGATCTGCAATTTG
L H R A P Y Y F L L D L C C S D I L R S A I C
484
TTTCCCATTTGTGTTCAACTCTGTCAAAAATGGTTCTACCTGGACTTATGGGACTCTGACTTGCAAAGT
S O F P F V F N S V K N G S T W T Y G T L T C K V
553
GATTGCCTTTCTGGGGGTTTTGTCCTGTTTCCACACTGCTTTCATGCTCTTCTGCATCAGTGTCACCAG
I A F L G V L S C F H T A F M L F C I S V T R
SS
622
ATATTTAGCTATCGCCCATCACCGCTTCTATACAAAGAGGCTGACCTTTTGGACGTGTCTGGCTGTGAT
Y L A I A H H R F Y T K R L T F W T C L A V I

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CTGTATGGTGTGGACTCTGTCTGTGGCCATGGCATTTCCCCCGGTTTTAGACGTGGGCACTTACTCATT
C M V W T L S V A M A F P P V L D V G T Y S F
S 760
CATTAGGGAGGAAGATCAATGCACCTTCCAACACCGCTCCTTCAGGGCTAATGATTCCTTAGAATTTAT
I R E E D Q C T F Q H R S F R A N D S L G F M
829
1O GCTGCTTCTTGCTCTCATCCTCCTAGCCACACAGCTTGTCTACCTCAAGCTGATATTTTTCGTCCACGA
L L L A L I L L A T Q L V Y L K L I F F V H D
898
TCGAAGAAAAATGAAGCCAGTCCAGTTTGTAGCAGCAGTCAGCCAGAACTGGACTTTTCATGGTCCTGG
1S R R K M K P V Q F V A A V S Q N W T F H G P G
967
AGCCAGTGGCCAGGCAGCTGCCAATTGGCTAGCAGGATTTGGAAGGGGTCCCACACCACCCACCTTGCT
A S G Q A A A N W L A G F G R G P T P P T L L
1036
GGGCATCAGGCAAAATGCAAACACCACAGGCAGAAGAAGGCTATTGGTCTTAGACGAGTTCAAAATGGA
G I R Q N A N T T G R R R L L V L D E F K M E
2S 1105
GAAAAGAATCAGCAGAATGTTCTATATAATGACTTTTCTGTTTCTAACCTTGTGGGGCCCCTACCTGGT
K R I S R M F Y I M T F L F L T L W G P Y L V
1174
GGCCTGTTATTGGAGAGTTTTTGCAAGAGGGCCTGTAGTACCAGGGGGATTTCTAACAGCTGCTGTCTG
A C Y W R V F A R G P V V P G G F L T A A V W
3S 1243
GATGAGTTTTGCCCAAGCAGGAATCAATCCTTTTGTCTGCATTTTCTCAAACAGGGAGCTGAGGCGCTG
M S F A Q A G I N P F V C I F S N R E L R R C
1312
4O TTTCAGCACAACCCTTCTTTACTGCAGAAAATCCAGGTTACCAAGGGAACCTTACTGTGTTATATGAGG
F S T T L L Y C R K S R L P R E P Y C V I
Still another preferred embodiment comprises a purified and isolated
polypeptide designated CON222, comprising the complete amino acid sequence set
forth in SEQ ID NO: 16. This amino acid sequence was deduced from a
4S polynucleotide sequence encoding CON222 (SEQ ID NO: 1S), as set forth
below:
1 ATGTTTAGACCTCTTGTGAATCTCTCTCACATATATTTTAAGAAATTCCAGTACTGTGGGTATGCA
M F R P L V N L S H I Y F K K F Q Y C G Y A
67 CCACATGTTCGCAGCTGTAAACCAAACACTGATGGAATTTCATCTCTAGAGAATCTCTTGGCAAGC
P H V R S C K P N T D G I S S L E N L L A S
SO 133 ATTATTCAGAGAGTATTTGTCTGGGTTGTATCTGCAGTTACCTGCTTTGGAAACATTTTTGTCATT
I I Q R V F V W V V S A V T C F G N I F V I
199 TGCATGCGACCTTATATCAGGTCTGAGAACAAGCTGTATGCCATGTCAATCATTTCTCTCTGCTGT
C M R P Y I R S E N K L Y A M S I I S L C C
265 GCCGACTGCTTAATGGGAATATATTTATTCGTGATCGGAGGCTTTGACCTAAAGTTTCGTGGAGAA
S S A D C L M G I Y L F V I G G F D L K F R G E

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331 TACAATAAGCATGCGCAGCTGTGGATGGAGAGTACTCATTGTCAGCTTGTAGGATCTTTGGCCATT
Y N K H A Q L W M E S T H C Q L V G S L A I
397 CTGTCCACAGAAGTATCAGTTTTACTGTTAACATTTCTGACATTGGAAAAATACATCTGCAT'TGTC
L S T E V S V L L L T F L T L E K Y I C I V
S 463 TATCCTTTTAGATGTGTGAGACCTGGAAAATGCAGAACAATTACAGTTCTGATTCTCATTTGGATT
Y P F R C V R P G K C R T I T V L I L I W I
529 ACTGGTTTTATAGTGGCTTTCATTCCATTGAGCAATAAGGAATTTTTCAAAAACTACTATGGCACC
T G F I V A F I P L S N K E F F K N Y Y G T
595 AATGGAGTATGCTTCCCTCTTCATTCAGAAGATACAGAAAGTATTGGAGCCCAGATTTATTCAG1'G
1 N G V C F P L H S E D T E S I G A Q I Y S V
O
661 GCAATTTTTCTTGGTATTAATTTGGCCGCATTTATCATCATAGTTTTTTCCT'ATGGF1AGCATGTTT
A I F L G I N L A A F I I I V F S Y G S M F
727 TATAGTGTTCATCAAAGTGCCATAACAGCAACTGAAATACGGAATCAAGTTAAAAA.AGAGATGATC
Y S V H Q S A I T A T E I R N Q V K K E M I
IS 793 CTTGCCAAACGTTTTTTCTTTATAGTATTTACTGATGCATTATGCTGGATACCCATTTTTGTAGTG
L A K R F F F I V F T D A L C W I P I F V V
859 AAATTTCTTTCACTGCTTCAGGTAGAAATACCAGGTACCATAACCTCTTGGGTAGTGATTTTTATT
K F L S L L Q V E I P G T I T S W V V I F I
925 CTGCCCATTAACAGTGCTTTGAACCCAATTCTCTATACTCTGACCACAAGACCATTTAAAGAAATG
ZO L P I N S A L N P I L Y T L T T R P F K E M
991 ATTCATCGGTTTTGGTATAACTACAGACAAAGAAAATCTATGGACAGCAAAGGTCAGAAAACATAT
I H R F W Y N Y R Q R K S M D S K G Q K T Y
1057 GCTCCATCATTCATCTGGGTGGAAATGTGGCCACTGCAGGAGATGCCACCTGAGTTAATGAAGCCG
A P S F I W V E M W P L Q E M P P E L M K P 1123
ZS GACC TTTTCACATACCCCTGTGAAATGTCACTGATTTCTCAATCAACGAGACTCAATTCCTATTCA
D L F T Y P C E M S L I S Q S T R L N S Y S
1189 TGA 1191
Another preferred embodiment comprises a purified and
isolated
30 polypeptide
designated
CON21S,
comprising
the
complete
amino
acid
sequence
set
forth in SEQ ID NO: 18. This amino acid sequence was deduced
from a
polynucleotide
sequence
encoding
CON21
S
(SEQ
ID
NO:
17),
as
set
forth
below:
atg ggg ttc aac ttg acg ctt gca aaa tta cca aat aac gag
ctg cac 48
Met Gly Phe Asn Leu Thr Leu Ala Lys Leu Pro Asn Asn Glu
Leu His
3S 1 5 10 15
ggc caa gag agt cac aat tca ggc aac agg agc gac ggg cca gga aag 96
Gly Gln Glu Ser His Asn Ser Gly Asn Arg Ser Asp Gly Pro Gly Lys
20 25 30
aac acc acc ctt cac aat gaa ttt gac aca att gtc ttg cca gtg ctt 144
Asn Thr Thr Leu His Asn Glu Phe Asp Thr Ile Val Leu Pro Val Leu
35 40 45
tat ctc att ata ttt gtg gca agc atc ttg ctg aat ggt tta gca gtg 192
4S Tyr Leu Ile Ile Phe Val Ala Ser Ile Leu Leu Asn Gly Leu Ala Val
55 60

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tgg atc ttc ttc cac att agg aat aaa acc agc ttc ata ttc tat ctc 240
Trp Ile Phe Phe His Ile Arg Asn Lys Thr Ser Phe Ile Phe Tyr Leu
65 70 75 80
aaa aac ata gtg gtt gca gac ctc ata atg acg ctg aca ttt cca ttt 288
Lys Asn Ile Val Val Ala Asp Leu Ile Met Thr Leu Thr Phe Pro Phe
85 90 95
cga ata gtc cat gat gca gga ttt gga cct tgg tac ttc aag ttt att 336
Arg Ile Val His Asp Ala Gly Phe Gly Pro Trp Tyr Phe Lys Phe Ile
100 105 110
ctc tgc aga tac act tca gtt ttg ttt tat gca aac atg tat act tcc 384
Leu Cys Arg Tyr Thr Ser Val Leu Phe Tyr Ala Asn Met Tyr Thr Ser
115 120 125
atc gtg ttc ctt ggg ctg ata agc att gat cgc tat ctg aag gtg gtc 432
Ile Val Phe Leu Gly Leu Ile Ser Ile Asp Arg Tyr Leu Lys Val Val
130 135 140
aag cca ttt ggg gac tct cgg atg tac agc ata acc ttc acg aag gtt 480
Lys Pro Phe Gly Asp Ser Arg Met Tyr Ser Ile Thr Phe Thr Lys Val
145 150 155 160
2S tta tct gtt tgt gtt tgg gtg atc atg get gtt ttg tct ttg cca aac 528
Leu Ser Val Cys Val Trp Val Ile Met Ala Val Leu Ser Leu Pro Asn
165 170 175
atc atc ctg aca aat ggt cag cca aca gag gac aat atc cat gac tgc 576
Ile Ile Leu Thr Asn Gly Gln Pro Thr Glu Asp Asn Ile His Asp Cys
180 185 190
tca aaa ctt aaa agt cct ttg ggg gtc aaa tgg cat acg gca gtc acc 624
Ser Lys Leu Lys Ser Pro Leu Gly Val Lys Trp His Thr Ala Val Thr
3$ 195 200 205
tat gtg aac agc tgc ttg ttt gtg gcc gtg ctg gtg att ctg atc gga 672
Tyr Val Asn Ser Cys Leu Phe Val Ala Val Leu Val Ile Leu Ile Gly
210 215 220
tgt tac ata gcc ata tcc agg tac atc cac aaa tcc agc agg caa ttc 720
Cys Tyr Ile Ala Ile Ser Arg Tyr Ile His Lys Ser Ser Arg Gln Phe
225 230 235 240
ata agt cag tca agc cga aag cga aaa cat aac cag agc atc agg gtt 768
Ile Ser Gln Ser Ser Arg Lys Arg Lys His Asn Gln Ser Ile Arg Val
245 250 255
gtt gtg get gtg ttt ttt acc tgc ttt cta cca tat cac ttg tgc aga 816
Val Val Ala Val Phe Phe Thr Cys Phe Leu Pro Tyr His Leu Cys Arg
260 265 270
att cct ttt act ttt agt cac tta gac agg ctt tta gat gaa tct gca 864
Ile Pro Phe Thr Phe Ser His Leu Asp Arg Leu Leu Asp Glu Ser Ala
275 280 285
caa aaa atc cta tat tac tgc aaa gaa att aca ctt ttc ttg tct gcg 912
Gln Lys Ile Leu Tyr Tyr Cys Lys Glu Ile Thr Leu Phe Leu Ser Ala
290 295 300

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tgt aat gtt tgc ctg gat cca ata att tac ttt ttc atg tgt agg tca 960
Cys Asn Val Cys Leu Asp Pro Ile Ile Tyr Phe Phe Met Cys Arg Ser
305 310 315 320
ttt tca aga agg ctg ttc aaa aaa tca aat atc aga acc agg agt gaa 1008
Phe Ser Arg Arg Leu Phe Lys Lys Ser Asn Ile Arg Thr Arg Ser Glu
325 330 335
agc atc aga tca ctg caa agt gtg aga aga tcg gaa gtt ctc ata tat 1056
Ser Ile Arg Ser Leu Gln Ser Val Arg Arg Ser Glu Val Leu Ile Tyr
340 345 350
tat gat tat act gat gtg tag 1077
Tyr Asp Tyr Thr Asp Val
1S 355
Another preferred embodiment comprises a purified and isolated
polypeptide designated CON217, comprising the complete amino acid sequence set
forth in SEQ ID NO: 20. This amino acid sequence was deduced from a
polynucleotide sequence encoding CON217 (SEQ ID NO: 19), as set forth below:
2O -41 C ATGGCATCCC CAGCCTAGCT CCCAATCCCA CTTTGGCACG
1 ATGTTAGCCAACAGCTCCTCAACCAACAGTTCTGTTCTCCCGTGTCCTGACTACCGACCTACCCAC
M L A N S S S T N S S V L P C P D Y R P T H
67 CGCCTGCACTTGGTGGTCTACAGCTTGGTGCTGGCTGCCGGGCTCCCCCTCAACGCGCTAGCCCTC
R L H L V V Y S L V L A A G L P L N A L A L
2S 133 TGGGTCTTCCTGCGCGCGCTGCGCGTGCACTCGGTGGTGAGCGTGTACATGTGTAACCTGGCGGCC
W V F L R A L R V H S V V S V Y M C N L A A
199 AGCGACCTGCTCTTCACCCTCTCGCTGCCCGTTCGTCTCTCCTACTACGCACTGCACCACTGGCCC
S D L L F T L S L P V R L S Y Y A L H H W P
265 TTCCCCGACCTCCTGTGCCAGACGACGGGCGCCATCTTCCAGATGAACATGTACGGCAGCTGCATC
3 F P D L L C Q T T G A I F Q M N M Y G S C I
O
331 TTCCTGATGCTCATCAACGTGGACCGCTACGCCGCCATCGTGCACCCGCTGCGACTGCGCCACCTG
F L M L I N V D R Y A A I V H P L R L R H L
397 CGGCGGCCCCGCGTGGCGCGGCTGCTCTGCCTGGGCGTGTGGGCGCTCATCCTGGTGTTTGCCGTG
R R P R V A R L L C L G V W A L I L V F A V
3S 463 CCCGCCGCCCGCGTGCACAGGCCCTCGCGTTGCCGCTACCGGGACCTCGAGGTGCGCCTATGCTTC
P A A R V H R P S R C R Y R D L E V R L C F
529 GAGAGCTTCAGCGACGAGCTGTGGAAAGGCAGGCTGCTGCCCCTCGTGCTGCTGGCCGAGGCGCTG
E S F S D E L W K G R L L P L V L L A E A L
595 GGCTTCCTGCTGCCCCTGGCGGCGGTGGTCTACTCGTCGGGCCGAGTCTTCTGGACGCTGGCGCGC
4O G F L L P L A A V V Y S S G R V F W T L A R
661 CCCGACGCCACGCAGAGCCAGCGGCGGCGGAAGACCGTGCGCCTCCTGCTGGCTAACCTCGTCATC
P D A T Q S Q R R R K T V R L L L A N L V I
727 TTCCTGCTGTGCTTCGTGCCCTACAACAGCACGCTGGCGGTCTACGGGCTGCTGCGGAGCAAGCTG
F L L C F V P Y N S T L A V Y G L L R S K L
~1S 793 GTGGCGGCCAGCGTGCCTGCCCGCGATCGCGTGCGCGGGGTGCTGATGGTGATGGTGCTGCTGGCC
V A A S V P A R D R V R G V L M V M V L L A

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859 GGCGCCAACTGCGTGCTGGACCCGCTGGTGTACTACTTTAGCGCCGAGGGCTTCCGCAACACCCTG

G A N C V L D P L V Y F S A E G R N T
Y F L
925 CGCGGCCTGGGCACTCCGCACCGGGCCAGGACCTCGGCCACCArICGGGACGCGGGCGGCGCTCGCG

R G L G T P H R A R S A T N G T A A L
T R A
S 991 CAATCCGAAAGGTCCGCCGTCACCACCGACGCCACCAGGCCGG
ATGCCGCCAGTCAGGGGCTGCTC
Q S E R S A V T T D T R P D A A Q G L
A S L
1057 CGACCCTCCGACTCCCACTCTCTGTCTTCCTTCACACAGTGTC
CCCAGGATTCCGCCCTCTGAACA
R P S D S H S L S S T Q C P Q D A L
F S
1123 CACATGCCAT TGCGCTGTCCGTGCCCGACTCCCAACGCCTCTCGTTCTGGGAGGCTTACA
IO 1183 GGGTGTACAC ACAAGAAGGTGGGCTGGGCACTTGGACCTTTGGGTGGCAATTCCAGCTTA
1243 GCAACGCAGA AGAGTACAAAGTGTGGAAGCCAGGGCCCAGGGAAGGCAGTGCTGCTGGAA
1303 ATGGCTTCTT TAAACTGTGAGCACGCAGAGCACCCCTTCTCCAGCGGTGGGAAGTGATGC
1363 AGAGAGCCCA CCCGTGCAGAGGGCAGAAGAGGACGAAATGC'CTTTGGGTGGGCAGGGCAT
1423 TAAACTGCTA AAAGCTGGTTAGATGGAACAGAAAATGGGCATTCTGGATCTAAACCGCCA
IS 1483 CAGGGGCCTG AGAGCTGAAGAGCACCAGGTTTGGTGGACAAAGCTACTGAGATGCCTGTT
1543 CATCTGCTGA CTTCTGTCTAGGCTCATGGATGCCACCCCCTTTCATTTCGGCCTAGGCTT
1603 CCCCTGCTCA CCACTGAGGCCTAATACAAGAGTTCCTATGGACAGAACTACATTCTTTCT
1663 CGCATAGTGA CTTGTGACAATTTAGACTTGGCATCCAGCATGGGATAGTTGGGGCAAGGC
1723 AAAACTAACT TAGAGTTTCCCCCTCAACAACATCCAAGTCCAAACCCTTTTTAGGTTATC
2O 1783 CTTTCTTCCA TCACATCCCCTTTTCCAGGCCTCCTCCATTTTAGGTCCTTAATATTCTTT
1843 CTTTTTCTCT CTCTCTCGTTTCTCTCTTCTCTCTCCTCTCCTCTCCTCTCTC'rTCTCCTC
1903 TTCTCTCTCT CTCCCTCTCTCTCCTTTGTCCAGAGTAAGGATAAAATTCTTTCTACTAAA
1963 GCACTGGTTC TCAAACTTTTTGGTCTCAGACCCCACTCTTAG.~1AATTGAGGATCTCAAAG
2023 AGCTTTGCTT ATATTTTGTTCTTTTGATACTTACCATACTAGAAATTAAAGCGAATACAT
2S 2083 TTTTAAAATA AATACACATGCACACATTACATTAGCCATGGGAGCAATAATGTCACCACA
2143 CACACTTCAT GAAGCCTCTGGAAAACTCTACAGTATACTTGTGAGAGAATGAGAGTGAAA
2203 GGGACAAATA ACATCTGTGTAGCAGTATTATGAAAATAGCTTGACCTTGTGGACTTCCTC
2263 AGAGGGTTGG TCCCTGGATCACACTTTGAGAACCATACTTGTCCTGAAGTATTGGAGTTC
2323 ATGTCTAACT TCTTCCCAGGGCATTATGTACAGTGCTTTTTATTACTGTGGGGAGAGGGC
3O 2383 AGTGCTAAAT AAATTAATCACTACTGATAAAAAAAAAAAAP~AAAAAAAAAAAAAAlIA
Although SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 provide
for particular human sequences, the invention is intended to include within
its scope
other human allelic variants; non-human mammalian forms of GPCR polypeptides,
and other vertebrate forms of GPCR polypeptides.
3S It will be appreciated that extracellular epitopes are particularly useful
for generating and screening for antibodies and other binding compounds that
bind to
receptors such as GPCR polypeptides. Thus, in another preferred embodiment,
the
invention provides a purified and isolated polypeptide comprising at least one
extracellular domain of a GPCR polypeptide of the invention. By "extracellular
40 domain", is it meant the amino terminal extracellular domain or an
extracellular loop
that spans two membrane domains.

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A purified and isolated polypeptide comprising the N-terminal
extracellular domain of GPCR polypeptides of the invention is highly
preferred. Also
preferred is a purified and isolated polypeptide comprising a GPCR seven
transmembrane receptor fragment selected from the group consisting of the
N-terminal extracellular domain of GPCR polypeptides of the invention,
transmembrane domains of GPCR polypeptides of the invention, extracellular
loops
connecting transmembrane domains of GPCR polypeptides of the invention,
intracellular loops connecting transmembrane domains of GPCR polypeptides of
the
invention, the C-terminal cytoplasmic domain of GPCR polypeptides, and fusions
thereof. Such fragments may be continuous portions of the native receptor.
However,
it will also be appreciated that knowledge of the GPCR gene and protein
sequences as
provided herein permits recombining of various domains that are not contiguous
in
the native protein.
1n another embodiment, the invention provides purified and isolated
polynucleotides (e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations
thereof, single or double stranded) that comprise a nucleotide sequence
encoding an
amino acid sequence of the polypeptides of the invention. Another embodiment
provides a purified and isolated polynucleotide encoding the amino acid
sequence of
the polypeptide of the invention fused to a heterologous tag amino acid
sequence.
Such polynucleotides are useful for recombinantly expressing the receptor and
also for
detecting expression of the receptor in cells (e.g., using Northern
hybridization and in
situ hybridization assays, and Western studies). Polynucleotides encoding
polypeptides of the invention also are useful to design antisense and other
molecules
for the suppression of GPCR polypeptides expression in a cultured cell or
animal (for
therapeutic purposes or to provide a model for diseases characterized by
aberrant
GPCR polypeptide expression). Such polynucleotides are also useful to design
antisense and other molecules for the suppression of GPCR polypeptide
expression in
a cultured cell or tissue or in an animal, for therapeutic purposes or to
provide a model
for diseases characterized by aberrant GPCR polypeptide expression.
Specifically
excluded from the definition of polynucleotides of the invention are entire
isolated
chromosomes of native host cells. A preferred polynucleotide set forth in any
one of

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the SEQ (D NOS: 1, 3, 5, 7, 9, 1 I, 13, 15, 17, and 19 con-esponds to a
naturally
occurring GPCR seduence. It will be appreciatec( that numerous other sequences
exist that also encode GPCR polypeptides having the amino acid sequence set
out in
SEQ 1D NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 due to the well-known
degeneracy
of the universal genetic code. All such sequences represent polynucleotides of
the
invention.
The invention also provides a purified and isolated polynuclcotide
comprising a nucleotide sequence that encodes a mammalian seven transmembrane
receptor, wherein the polynucleotide hybridizes to a nucleotide sequence set
forth in
any one of SEQ 1D NOS: l, 3, 5, 7, 9, 11, 13, 15, 17, or 19 or the non-coding
strand
complementary thereto, under the following hybridization conditions:
(a) hybridization for 16 hours at 42°C in a hybridization solution
comprising
50% formamide, 1% SDS, 1 M NaCI, l0% Dextran sulphate; and
(b) washing 2 times for 30 minutes at 60°C in a wash solution
comprising
0.1% SSC, 1% SDS. Polynucleotides that encode a human allelic variant are
highly
preferred.
A highly preferred polynucleotide of the invention comprises the
sequence set forth in SEQ ID NO: l, which comprises a human CON193 encoding
DNA sequence:
ntggttgttggaccattaaaatgcattatggaatttttaaaagttgggggagagggagac60
agtaaaaataacctatattttctcttgtttttttttttttaactctaggaaagcccagac120
aaattttgagctatttcataacctaccagacttatcatgctaacactgaataaaacagac180
ctaataccagcttcatttattctgaatggagtcccaggactggaagacacacaactctgg240
atttccttcccattctgctctatgtatgttgtggctatggtagggaattgtggactcctc300
tacctcattcactatgaggatgccctgcacaaacccatgtactacttcttggccatgctt360
tcctttactgaccttgttatgtgctctagtacaatccctaaagccctctgcatcttctgg420
tttcatctcaaggacattggatttgatgaatgccttgtccagatgttcttcatccacacc480
ttcacagggatggagtctggggtgcttatgcttatggccctggatcgctatgtggccatc540
tgctaccccttacgctattcaactatcctcaccaatcctgtaattgcaaaggttgggact600
gccaccttcctgagaggggtattactcattattccctttactttcctcaccaagcgcctg660
ccctcctgcagaggcaatatacttccccatacctactgtgaccacatgtctgtagccaaa720
ttgtcctgtggtaatgtcaaggtcaatgccatctatggtctgatggttgccctcctgatt780
gggggctttgacatactgtgtatcaccatctcctataccatgattctccgggcagtggtc840
agcctctcctcagcagatgctcggcagaaggcctttaatacctgcactgcccacatttgt900
gccattgttttctcctatactccagctttcttctccttcttttcccaccgctttggggaa960
cacataatccccccttcttgccacatcattgtagccaatatttatctgctcctaccaccc1020
actatgaaccctattgtctatggggtgaaaaccaaacagatacgagactgtgtcataagg1080

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atcctttcag gttctaagga taccaaatcc tacagcatgt gaatgaacac ttgccaggag 1140
tgagaagaga aggaaagaat tacttctatt tgcctcttat gcaggagttc ataaaatctt 1200
tctggaagta ctgtattgat cacaaaatgg agtttgntga ctggtgcatt ctcaataagt 1260
accttgggaa tctnacatca ctggaaggcc caccacattt ctataaat 1308
Also prefen-ed is a polynucleotide comprising nucleotides 157-1 1 19 of
SEQ >D NO: l, which represent the portion of SEQ LD NO: 1 that encodes CON 193
amino acids.
Another highly prefen-ed polynucleotide of the invention comprises the
sequence set forth in SEQ ID NO: 3, which comprises a human CON166 encoding
DNA sequence:
atggatgaaacaggaaatctgacagtatcttctgccacatgccatgacactattgatgac60
ttccgcaatcaagtgtattccaccttgtactctatgatctctgttgtaggcttctttggc120
aatggctttgtgctctatgtcctcataaaaacctatcacaagaagtcagccttccaagta180
tacatgattaatttagcagtagcagatctactttgtgtgtgcacactgcctctccgtgtg240
gtctattatgttcacaaaggcatttggctctttggtgacttcttgtgccgcctcagcacc300
tatgctttgtatgtcaacctctattgtagcatcttctttatgacagccatgagctttttc360
cggtgcattgcaattgtttttccagtccagaacattaatttggttacacagaaaaaagcc420
aggtttgtgtgtgtaggtatttggatttttgtgattttgaccagttctccatttctaatg480
gccaaaccacaaaaagatgagaaaaataataccaagtgctttgagcccccacaagacaat540
caaactaaaaatcatgttttggtcttgcattatgtgtcattgtttgttggctttatcatc600
ccttttgttattataattgtctgttacacaatgatcattttgaccttactaaaaaaatca660
atgaaaaaaaatctgtcaagtcataaaaaggctataggaatgatcatggtcgtgaccgct720
gcctttttagtcagtttcatgccatatcatattcaacgtaccattcaccttcatttttta780
cacaatgaaactaaaccctgtgattctgtccttagaatgcagaagtccgtggtcataacc840
2$ ttgtctctggctgcatccaattgttgctttgaccctctcctatatttcttttctgggggt900
aactttaggaaaaggctgtctacatttagaaagcattctttgtccagcgtgacttatgta960
cccagaaagaaggcctctttgccagaaaaaggagaagaaatatgtaaagtatag 1014
The final three nucleotides of this sequence represent a stop codon.
Still another highly preferred polynucleotide of the invention
comprises the sequence set forth in SEQ >D NO: 5, which comprises a human
CON103 encoding DNA sequence:
ggggcctact tcaccgtgta cccggacttg ggaccatcac agacttcaga accatcagga 60
acctgggagc aactgaaagc tgaactacag tgggctttca gacacacagc aggctgcgga 120
gcacaaatag gactggttcc ctccaggcca ccagcagggc ggtggaggtc ttcactgact 180
ccctgcctac ctctcaggac aatgtccttt tggctccaca gtccctgaag ccagagctgg 240
tgggggcagg gaggcagcca ccagcctcta tatgtagtgg aggagggggt gtccagggag 300
ggctgcatga tcctgagagc ccccacctca cccggctgga ctatcctccc acttcagggt 360
ttctctgggc ttccatcttg cccctgctga gccctgcttc ctcctctacc agcagcacaa 420
cccccaggct gggctcagag acctcatgtg gtgggatcac tcagtacccc gaggcggagg 480
gaaggaggga gggctgcagg gttccccttg gcctgcaaac aggaacacag ggtgtttctc 540
agtggctgcg agaatgctga tgaaaacccc aggatgttgt gtcaccgtgg tggccagctg 600
atagtgccaa tcatcccact ttgccctgag cactcctgca ggggtagaag actccagaac 660

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cttctctcag geceatggec atggaa cat ctg tct 714
caagcagcec ctt aac age
CCd tCt tCt CtC tCC tCt gttCtC CCC ttC CCC 762
CCC tCC CCt tCC tCt
tea ccc tct get ccc gcc tttace gtg ggg tct 810
tcc tct act ggg tcc
gga ggg tgc cac ccc tct tcctcg gtg gcc ctg 858
ccc ace ctg tct ttc
$ gca cca ctg gee ctg ttt gtcctg ctg ggg agt 906
ate gag ggc gtg aac
ttg gcc ttc ate ttc ate cacacg ccc ace aac 954
ctc tgc egg tgg tcc
acg gtg etg gtc age gtg gccget ttc ctg age 1002
ttc ctg gac ctc ate
aac ctg ctc ege gtg tae taectc cat ace cgc 1050
cce gac ctc gag tgg
ttt ggg get gcc tgc gtc aacctc atg tr_c aac 1098
get aaa ttc ctg ace
cgc acg age gtt gtc ctc acagcc gca aac tae 1146
gcc ttc ate ctc cgc
ctg aag gtg cag ccc cac gtgctg cgt tcc ggg 1194
gtg cac age get gtg
gca get egg gtg gcc gga ctctgg ggc ctg ctc 1242
gcc ggg gtg ate ctc
aac ggg ctg ctC ctg ace ttctcc ccc tgc age 1290
cac age ggc tcc ctc
tae agg gge acg aag tcg geetcg cgc cac gca 1338
gtg cce ctc tgg cag
1$ ctg tae ctg gag ttc ctg ccactg ctc ctc get 1386
ctg ttc gcg ate ttt
att gtg att ggg cte ate eggaac ggt gge cag 1434
age ace egt ctg ggg
gca ggc cag agg gcc cgt gtgctg atg gtg gtc 1482
ccg atg gcc gtg gcc
tae ace tgc tte ttg age ateate ggc get atg 1530
ate ece ttt atg tcc
gtg get tgg ctg tee tge cgatee gac tgc cag 1578
ttc gee etg cte aca
ete tte gge tee etg tte acetae aac gtc gac 1626
cat gee ctc agt etg
ccc gtg tae tgc ttc age cccaac ctc cag egg 1674
ctc tet ttc cac age
gee ttg gge ctc acg ggc eggcag cca age gag 1722
ctg egg ggc gtg gac
age tcc caa ccc tcc eag tggcgc egg gcc agg 1770
tae agg tae gag tct
aag gcg gcc ata ggg ctg aaagtg ggc gtc ctg 1818
gag aag cag gag tct
2$ gaa aag ggc tcc tcc ggc tgagggccagctg ctgca 1865
gaa cag caggg
gcgctgtggg ggtaagggct ggcctggagg gacaaggcca cggtg1925
gccgcgctct gcaca
cctcaaccaa ctggacaagg gaccaggggc caggccaaag cactggcagg 1985
gatggcggca
actcatgtgg gtggcaggga cctaggcctctcagtgtgtc caggatggca2045
gagaaaccca
ttcccagaat gcaggggaga gggtggagga gacaggcaag gtgccgttgg 2105
gcaggatgcC
cacaccagct cagacagggg tgcaggggac agacgccaat cactgtcaca 2165
cctgcgcagc
gcagagtcac cttagaaatt atgttctgtg ctctccagtt tgtcccttcc 2225
ggacagctgc
aatattaata aacttccctt ttatttgcag accaatatet gtctttaatt2285
ttaaatatat
ctaacctggg actgtcagta tgagcgccccagtgaaggaa ccttggagag2345
ggcgtcaaag
agtgggagca ttcccagcct ctcgtcttcc agactttgga gcccgcatgt 2405
tccaggggga
3$ ctgaagcaga ctctttcttg 2429
gtag
Also preferred is a polynucleotide comprising nucleotides 691-1842 of SEQ >D
NO:
$, which represent the portion of SEQ LD NO: $ that encodes CON103 amino
acids.
Nucleotides 1843-184$ represent a stop codon.
Another highly preferred polynucleotide of the invention comprises the
sequence set forth in SEQ ID NO: 7, which comprises a CON203-encoding DNA
sequence:
ttgaatttag gtgacactat agaagagcta tgacgtcgca tgcacgcgta cgtaagctcg 60
gaattcggct cgagctgaac taatgactgc cgccataaga agacagagag aactgagtat 120
cctcccaaag gtgacactgg aagcaatgaa caccacagtg atgcaaggct tcaacagatc 180

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tgagcggtgccccagagacactcggatagtacagctggtattcccagccctctacacagt240
ggttttcttgaccggcatcctgctgaatactttggctctgtgggtgtttgttcacatccc300
cagctcctccaccttcatcatctacctcaaaaacactttggtggccgacttgataatgac360
actcatgcttcctttcaaaatcctctctgactcacacctggcaccctggcagctcagagc420
ttttgtgtgtcgtttttcttcggtgatattttatgagaccatgtatgtgggcatcgtgct480
gttagggctcatagcctttgacagattcctcaagatcatcagacctttgagaaatatttt540
tctaaaaaaacctgtttttgcaaaaacggtctcaatcttcatctgggtctttttggtctt600
catctccctgccaaatatgatcttgagcaacaaggaagcaacaccatcgtctgtgaaaaa660
gtgtgcttccttaaaggggcctctggggctgaaatggcatcaaatggtaaataacatatg720
ccagtttattttctggactggttttatcctaatgcttgtgttttatgtggttattgcaaa780
aaaagtatatgattcttatagaaagtccaaaagtaaggacagaaaaaacaacaaaaagct840
ggaaggcaaagtatttgttgtcgtggctgtcttctttgtgtgttttgctccatttcattt900
tgccagagttccatatactcacagtcaaaccaacaataagactgactgtagactgcaaaa960
tcaactgtttattgctaaagaaacaactctctttttggcagcaactaacatttgtatgga1020
tcccttaatatacatattcttatgtaaaaaattcacagaaaagctaccatgtatgcaagg1080
gagaaagaccacagcatcaagccaagaaaatcatagcagtcagacagacaacataacctt1140
aggctgacaactgtacatagggttaacttctatttattgatgagacttccgtagataatg1200
tggaaatcaaatttaaccaagaaaaaaagattggaacaaatgctctcttacattttattt1260
atcctggtgtccaggaaaagattatattaaatttaaatccacatagatctattcataagc1320
tgaatgaaccattacctaagagaatgcaacaggataccaatggccactagaggcatattc1380
cttcttcttttttttttgttaaatttcaagagcattcactttacatttggaaagactaag1440
gggaacggttatcctacaaacctcccttcaacaccttttacatt 1484
Also preferred is a polynucleotide comprising nucleotides 146-1144 of SEQ ~
NO:
7, which represent the portion of SEQ >D NO: 7 that encodes CON203 amino
acids.
Nucleotides 1145-1147 represent a stop codon.
Another highly preferred polynucleotide of the invention comprises the
sequence set forth in SEQ )D NO: 9, which comprises a human CON198 encoding
DNA sequence:
ATGATGGTGG ATCCCAATGG CAATGAATCC AGTGCTACAT ACTTCATCCT AATAGGCCTC 60
3O CCTGGTTTAG AAGAGGCTCA GTTCTGGTTG GCCTTCCCAT TGTGCTCCCT CTACCTTATT 120
GCTGTGCTAG GTAACTTGAC AATCATCTAC ATTGTGCGGA CTGAGCACAG CCTGCATGAG 180
CCCATGTATA TATTTCTTTG CATGCTTTCA GGCATTGACA TCCTCATCTC CACCTCATCC 240
ATGCCCAAAA TGCTGGCCAT CTTCTGGTTC AATTCCACTA CCATCCAGTT TGATGCTTGT 300
CTGCTACAGA TGTTTGCCAT CCACTCCTTA TCTGGCATGG AATCCACAGT GCTGCTGGCC 360
3S ATGGCTTTTG ACCGCTATGT GGCCATCTGT CACCCACTGC GCCATGCCAC AGTACTTACG 420
TTGCCTCGTG TCACCAAAAT TGGTGTGGCT GCTGTGGTGC GGGGGGCTGC ACTGATGGCA 480
CCCCTTCCTG TCTTCATCAA GCAGCTGCCC TTCTGCCGCT CCAATATCCT TTCCCATTCC 540
TACTGCCTAC ACCAAGATGT CATGAAGCTG GCCTGTGATG ATATCCGGGT CAATGTCGTC 600
TATGGCCTTA TCGTCATCAT CTCCGCCATT GGCCTGGACT CACTTCTCAT CTCCTTCTCA 660
4O TATCTGCTTA TTCTTAAGAC TGTGTTGGGC TTGACACGTG AAGCCCAGGC CAAGGCATTT 720
GGCACTTGCG TCTCTCATGT GTGTGCTGTG TTCATATTCT ATGTACCTTT CATTGGATTG 780
TCCATGGTGC ATCGCTTTAG CAAGCGGCGT GACTCTCCGC TGCCCGTCAT CTTGGCCAAT 840
ATCTATCTGC TGGTTCCTCC TGTGCTCAAC CCAATTGTCT ATGGAGTGAA GACAAAGGAG 900
ATTCGACAGC GCATCCTTCG ACTTTTCCAT GTGGCCACAC ACGCTTCAGA GCCCTAG 957

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The last three nucleotides of this sequence represent a stop codon.
Still another A
highly preferred
polynucleotide
of the invention
comprises ID NO: 11, which
the sequence comprises a human
set forth
in SEQ
CON 197 oding DNA sequence:
enc
S ATGGAAAGCG AGAACAGAAG AGTGATAAGAGAATTCATCC TCCTTGGTCTGACCCAGTCT60
CAAGATATTC AGCTCCTGGT CTTTGTGCTAGTTTTAATAT TCTACTTCATCATCCTCCCT120
GGAAATTTTC TCATTATTTT CACCATAAAGTCAGACCCTG GGCTCACAGCCCCCCTCTAT180
TTCTTTCTGG GCAACTTGGC CTTCCTGGATGCATCCTACT CCTTCATTGTGGCTCCCCGG240
ATGTTGGTGG ACTTCCTCTC TGCGAAGAAGATAATCTCCT ACAGAGGCTGCATCACTCAG300
1O CTCTTTTTCT TGCACTTCCT TGGAGGAGGGGAGGGATTAC TCCTTGTTGTGATGGCCTT~_r360
GACCGCTACA TCGCCATCTG CCGGCCTCTGCACTATCCTA CTGTCATGAACCCTAGAACC420
TGCTATGCAA TGATGTTGGC TCTGTGGCTTGGGGGTTTTG TCCACTCCATTATCCAGGTG480
GTCCTCATCC TCCGCTTGCC TTTTTGTGGCCCAAACCAGC TGGACAACTTCTTCTGTGAT540
GTCCCACAGG TCATCAAGCT GGCCTGCACCGACACATTTG TGGTGGAGCTTCTGATGGTC600
IS TTCAACAGTG GCCTGATGAC ACTCCTGTGCTTTCTGGGGC TTCTGGCCTCCTATGCAGTC660
ATTCTTTGTC GCATACGAGG GTCTTCTTCTGAGGCAAAAA ACAAGGCCATGTCCACGTGC720
ATCACCCATA TCATTGTTAT ATTCTTCATGTTTGGACCTG GCATCTTCATCTACACGCGC780
CCCTTCAGGG CTTTCCCAGC TGACAAGGTGGTTTCTCTCT TCCACACAGTGATTTTTCCT840
TTGTTGAATC CTGTCATTTA TACCCTTCGCAACCAGGAAG TGAAAGCTTCCATGAAAAAG900
2O GTGTTTAATA AGCACATAGC CTGA
924
The last
three nucleotides
of this
sequence
represent
a stop
codon.
Another highly preferred
polynucleotide
of the invention
comprises the
sequence forth in SEQ 1D
set NO: 13, which comprises
a human CON202
encoding
DNA sequence:
2S 1 TGCTTCCCCATAAGGTAACAGCTTTGTTAGCNCTGTCTGACATCATTGCT
51 TGTTWACTTAAGAACTGATAGGTYTTTTTTTTTTTTTTTTTTCAGATATT
101 CTGATGGCAAAACAAGTGGAAGAAAAGAGGAAGCATGACTGCAGATCAGA
151 TCAGTTCTCTTTGTGGATTATATTTTCAGTAAAATGTATGGATCTATCTT
201 TTCCTTGTTCTTATATCTAGATCATGAGACTTGACTGAGGCTGTATCCTT
3O 251 ATCCTCCATCCATCTATGGCGAACTATAGCCATGCAGCTGACAACATTTT
301 GCAAAATCTCTCGCCTCTAACAGCCTTTCTGAAACTGACTTCCTTGGGTT
351 TCATAATAGGAGTCAGCGTGGTGGGCAACCTCCTGATCTCCATTTTGCTA
401 GTGAAAGATAAGACCTTGCATAGAGCACCTTACTACTTCCTGTTGGATCT
451 TTGCTGTTCAGATATCCTCAGATCTGCAATTTGTTTCCCATTTGTGTTCA
3S 501 ACTCTGTCAAAAATGGTTCTACCTGGACTTATGGGACTCTGACTTGCAAA
551GTGATTGCCTTTCTGGGGGTTTTGTCCTGTTTCCACACTGCTTTCATGCT
601CTTCTGCATCAGTGTCACCAGATATTTAGCTATCGCCCATCACCGCTTCT
651ATACAAAGAGGCTGACCTTTTGGACGTGTCTGGCTGTGATCTGTATGGTG
701TGGACTCTGTCTGTGGCCATGGCATTTCCCCCGGTTTTAGACGTGGGCAC

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751 TTACTCATTCATTAGGGAGGAAGATCAATGCACCTTCCAACACCGCTCCT
801 TCAGGGCTAATGATTCCTTAGGATTTATGCTGCTTCTTGCTCTCATCCTC
851 CTAGCCACACAGCTTGTCTACCTCAAGCTGATATTTTTCGTCCACGATCG
901 AAGAAAAATGAAGCCAGTCCAGTTTGTAGCAGCAGTCAGCCAGAACTGGA
S 951 CTTTTCATGGTCCTGGAGCCAGTGGCCAGGCAGCTGCCAATTGGCTAGCA
1001 GGATTTGGAAGGGGTCCCACACCACCCACCTTGCTGGGCATCAGGCAAAA
1051 TGCAAACACCACAGGCAGAAGAAGGCTATTGGTCTTAGACGAGTTCAAAA
1101 TGGAGAAAAGAATCAGCAGAATGTTCTATATAATGACTTTTCTGTTTCTA
1151 ACCTTGTGGGGCCCCTACCTGGTGGCCTGTTATTGGAGAGTTTTTGCAAG
IO 1201 AGGGCCTGTAGTACCAGGGGGATTTCTAACAGCTGCTGTCTGGATGAGTT
1251 TTGCCCAAGCAGGAATCAATCCTTTTGTCTGCATTTTCTCAAACAGGGAG
1301 CTGAGGCGCTGTTTCAGCACAACCCTTCTTTACTGCAGAAAATCCAGGTT
1351 ACCAAGGGAACCTTACTGTGTTATATGAGG
Also preferred comprising -1375 of SEQ
is a polynucleotide nucleotides ID NO:
266
1 13, SEQ ID 13 that
S which NO: encodes
represent CON202
the amino
portion acids.
of
Nucleotides 78 represent
1376-13 a stop
codon.
Another highly preferred polynucleotide of the invention comprises the
sequence set forth in SEQ ID NO: 1 S, which comprises a human CON222 encoding
DNA
sequence:
2O 1 ATGTTTAGACCTCTTGTGAATCTCTCTCACATATATTTTAAGAAATTCCA
51 GTACTGTGGGTATGCACCACATGTTCGCAGCTGTAAACCAAACACTGATG
101 GAATTTCATCTCTAGAGAATCTCTTGGCAAGCATTATTCAGAGAGTATTT
151 GTCTGGGTTGTATCTGCAGTTACCTGCTTTGGAAACATTTTTGTCATTTG
201 GATGCGACCTTATATCAGGTCTGAGAACAAGCTGTATGCCATGTCAATCA
2S 251 TTTCTCTCTGCTGTGCCGACTGCTTAATGGGAATATATTTATTCGTGATC
301 GGAGGCTTTGACCTAAAGTTTCGTGGAGAATACAATAAGCATGCGCAGCT
351 GTGGATGGAGAGTACTCATTGTCAGCTTGTAGGATCTTTGGCCATTCTGT
401 CCACAGAAGTATCAGTTTTACTGTTAACATTTCTGACATTGGAAAAATAC
451 ATCTGCATTGTCTATCCTTTTAGATGTGTGAGACCTGGAAAATGCAGAAC
3O 501 AATTACAGTTCTGATTCTCATTTGGATTACTGGTTTTATAGTGGCTTTCA
551 TTCCATTGAGCAATAAGGAATTTTTCAAAAACTACTATGGCACCAATGGA
601 GTATGCTTCCCTCTTCATTCAGAAGATACAGAAAGTATTGGAGCCCAGAT
651 TTATTCAGTGGCAATTTTTCTTGGTATTAATTTGGCCGCATTTATCATCA
701 TAGTTTTTTCCTATGGAAGCATGTTTTATAGTGTTCATCAAAGTGCCATA
3S 751 ACAGCAACTGAAATACGGAATCAAGTTAAAAAAGAGATGATCCTTGCCAA
801 ACGTTTTTTCTTTATAGTATTTACTGATGCATTATGCTGGATACCCATTT
851 TTGTAGTGAAATTTCTTTCACTGCTTCAGGTAGAAATACCAGGTACCATA
901 ACCTCTTGGGTAGTGATTTTTATTCTGCCCATTAACAGTGCTTTGAACCC
951 AATTCTCTATACTCTGACCACAAGACCATTTAAAGAAATGATTCATCGGT
4O 1001 TTTGGTATAACTACAGACAAAGAAAATCTATGGACAGCAAAGGTCAGAAA
1051 ACATATGCTCCATCATTCATCTGGGTGGAAATGTGGCCACTGCAGGAGAT
1101 GCCACCTGAGTTAATGAAGCCGGACCTTTTCACATACCCCTGTGAAATGT

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1151 CACTGATTTC TCAATCAACG AGACTCAATT CCTATTCA
The last three nucleotides of this sequence represent a slop colon.
Another highly preferred polynucleotide of the invention comprises the
sequence set forth in SEQ CD NO: l7, which comprises a human CON21S encoding
S DNA sequence. Also preferred is a polynucleotiiie comprising the portion of
SEQ ~
NO: 17 set forth below, which represent the portion of SEQ ID NO: 17 that
encodes
CON21S amino acids (the last three nucleotides represent a stop colon).
ATGGGGTTCAACTTGACGCTTGCAAAATTACCAAATAACGAGCTGCACGGCCAAGAGAGT60
CACAATTCAGGCAACAGGAGCGACGGGCCAGGAAAGAACACCACCCTTCACAATGAATTT120
1O GACACAATTGTCTTGCCAGTGCTTTATCTCATTATATTTGTGGCAAGCATCTTGCTGAAT180
GGTTTAGCAGTGTGGATCTTCTTCCACATTAGGAATAAAACCAGCTTCATATTCTATCTC240
AAAAACATAGTGGTTGCAGACCTCATAATGACGCTGACATTTCCATTTCGAATAGTCCAT300
GATGCAGGATTTGGACCTTGGTACTTCAAGTTTATTCTCTGCAGATACACTTCAGTTTTG360
TTTTATGCAAACATGTATACTTCCATCGTGTTCCTTGGGCTGATAAGCATTGATCGCTAT420
IS CTGAAGGTGGTCAAGCCATTTGGGGACTCTCGGATGTACAGCATAACCTTCACGAAGGTT480
TTATCTGTTTGTGTTTGGGTGATCATGGCTGTTTTGTCTTTGCCAAACATCATCCTGACA540
AATGGTCAGCCAACAGAGGACAATATCCATGACTGCTCAAAACTTAAAAGTCCTTTGGGG600
GTCAAATGGCATACGGCAGTCACCTATGTGAACAGCTGCTTGTTTGTGGCCGTGCTGGTG660
ATTCTGATCGGATGTTACATAGCCATATCCAGGTACATCCACAAATCCAGCAGGCAATTC720
ZO ATAAGTCAGTCAAGCCGAAAGCGAAAACATAACCAGAGCATCAGGGTTGTTGTGGCTGTG780
TTTTTTACCTGCTTTCTACCATATCACTTGTGCAGAATTCCTTTTACTTTTAGTCACTTA840
GACAGGCTTTTAGATGAATCTGCACAAAAAATCCTATATTACTGCAAAGAAATTACACTT900
TTCTTGTCTGCGTGTAATGTTTGCCTGGATCCAATAATTTACTTTTTCATGTGTAGGTCA960
TTTTCAAGAAGGCTGTTCAAAAAATCAAATATCAGAACCAGGAGTGAAAGCATCAGATCA1020
ZS CTGCAAAGTGTGAGAAGATCGGAAGTTCTCATATATTATGATTATACTGATGTGTAG 1077
Another preferred polynucleotide of the invention comprises the
portion of the sequence set forth in SEQ B7 NO: 19 which comprises a human
CON217 encoding DNA sequence:
1 ATGTTAGCCAACAGCTCCTC TCTGTTCTCCCGTGTCCTGACTACCGACCT
AACCAACAGT
3O 61 ACCCACCGCCTGCACTTGGTGGTCTACAGCTTGGTGCTGGCTGCCGGGCTCCCCCTCAAC
121 GCGCTAGCCCTCTGGGTCTTCCTGCGCGCGCTGCGCGTGCACTCGGTGGTGAGCGTGTAC
181 ATGTGTAACCTGGCGGCCAGCGACCTGCTCTTCACCCTCTCGCTGCCCGTTCGTCTCTCC
241 TACTACGCACTGCACCACTGGCCCTTCCCCGACCTCCTGTGCCAGACGACGGGCGCCATC
301 TTCCAGATGAACATGTACGGCAGCTGCATCTTCCTGATGCTCATCAACGTGGACCGCTAC
3S 361 GCCGCCATCGTGCACCCGCTGCGACTGCGCCACCTGCGGCGGCCCCGCGTGGCGCGGCTG
421 CTCTGCCTGGGCGTGTGGGCGCTCATCCTGGTGTTTGCCGTGCCCGCCGCCCGCGTGCAC
481 AGGCCCTCGCGTTGCCGCTACCGGGACCTCGAGGTGCGCCTATGCTTCGAGAGCTTCAGC
541 GACGAGCTGTGGAAAGGCAGGCTGCTGCCCCTCGTGCTGCTGGCCGAGGCGCTGGGCTTC
601 CTGCTGCCCCTGGCGGCGGTGGTCTACTCGTCGGGCCGAGTCTTCTGGACGCTGGCGCGC
4O 661 CCCGACGCCACGCAGAGCCAGCGGCGGCGGAAGACCGTGCGCCTCCTGCTGGCTAACCTC
721 GTCATCTTCCTGCTGTGCTTCGTGCCCTACAACAGCACGCTGGCGGTCTACGGGCTGCTG
781 CGGAGCAAGCTGGTGGCGGCCAGCGTGCCTGCCCGCGATCGCGTGCGCGGGGTGCTGATG

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841 G'i'GATGGTGCTGCTGGCCGGCGCCAACTGCGTGCTGGACC CGCTGGTGTACTACTTTAGC
901 GCCGAGGGCTTCCGCAACACCCTGCGCGGCCTGGGCACTC CGCACCGGGCCAGGACCTCG
951 GCCACCAACGGGACGCGGGCGGCGCTCGCGCAATCCGAAA GGTCCGCCGTCACCACCGAC
1021 GCCACCAGGCCGGATGCCGCCAGTCAGGGGCTGCTCCGAC CCTCCGACTCCCACTCTCTG
S 1081 TCTTCCTTCACACAGTGTCCCCAGGATTCCGCCCTCTGA
The last s sequence
three represent
nucleotides a stop
of thi colon.
The invention also includes polynucleotides differing from the
sequences set forth in SEQ ID NOS: 1, 3, S, 7, 9, 1 l, 13, 1 S, 17 and 19 and
from their
complementary strand by at least one nucleotide.
In a related embodiment, the invention provides vectors comprising a
polynucleotide of the invention. Such vectors are useful, e.g., for amplifying
the
polynucleotides in host cells to create useful quantities thereof. In
preferred
embodiments, the vector is an expression vector wherein the polynucleotide of
the
invention is operatively linked to a polynucleotide comprising an expression
control
1 S sequence. Such vectors are useful for recombinant production of
polypeptides of the
invention.
In another related embodiment, the invention provides host cells that
are transformed or transfected (stably or transiently) with a polynucleotide
of the
invention or vectors of the invention. As stated above, such host cells are
useful for
amplifying the polynucleotides and also for expressing the GPCR seven
transmembrane receptor polypeptides or fragments thereof encoded by the
polynucleotides. Such host cells are useful in assays as described herein.
In still another related embodiment, the invention provides a method
for producing a seven transmembrane receptor polypeptide (or fragment thereof)
of
2S the invention comprising the steps of growing a host cell of the invention
in a nutrient
medium and isolating the polypeptide or variant thereof from the cell or the
medium.
Since the GPCR polypeptides are seven transmembrane receptors, it will be
appreciated that, for some applications, such as certain activity assays, the
preferable
isolation may involve isolation of cell membranes containing the polypeptide
embedded therein, whereas for other applications a more complete isolation may
be
preferable.
In still another embodiment, the invention provides antibodies that are
specific for the GPCR seven transmembrane receptors of the invention. Antibody

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specificity is described in greater detail below. However, it should be
emphasized
that antibodies that can be generated from polypeptides that have previously
been
described in the literature and that are capable of fortuitously cross-
reacting with the
GPCR polypcptides of the invention (e.g., due to the .fortuitous existence of
a similar
epitope in both polypeptides) are considered "cross-reactive" antibodies. Such
cross-
reactive antibodies are not antibodies that are "specific" for the GPC-R
polypeptides.
The determination of whether an antibody is specific for a GPCR polypeptide or
is
cross-reactive with another known receptor is made using Western blotting
assays or
several other assays well known in the literature. For identifying cells that
express
GPCR polypeptides and also for modulating GPCR -ligand binding activity,
antibodies that specifically bind to an extracellular epitope of one of the
GPCR seven
transmembrane receptors of the present invention are preferred.
In one preferred variation, the invention provides monoclonal
antibodies. Hybridomas that produce such antibodies also are intended as
aspects of
the invention. In yet another variation, the invention provides a humanized
antibody.
Humanized antibodies are useful for ire vivo therapeutic indications.
In another variation, the invention provides a cell-free composition
comprising polyclonal antibodies, wherein at least one of the antibodies is an
antibody
of the invention specific for a GPCR polypeptide of the present invention.
Antisera
isolated from an animal is an exemplary composition, as is a composition
comprising
an antibody fraction of an antisera that has been resuspended in water or in
another
diluent, excipient, or carrier.
In still another related embodiment, the invention provides
anti-idiotypic antibodies specific for an antibody that is specif c for a GPCR
polypeptide of the present invention.
It is well known that antibodies contain relatively small antigen
binding domains that can be isolated chemically or by recombinant techniques.
Such
domains are useful GPCR binding molecules themselves, and also may be
reintroduced into human antibodies, or fused to toxins or other polypeptides.
Thus, in
still another embodiment, the invention provides a polypeptide comprising a
fragment
of a GPCR-specific antibody, wherein the fragment and the polypeptide bind to
a

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GPCR seven transmembrane receptor of the present invention. By way of non-
limiting example, the invention provides polypeptides that are single chain
antibodies
and CDR-grafted antibodies.
Also within the scope of the invention are compositions comprising
polypeptides, polynucleotides, or antibodies of the invention that have been
formulated with, e.g., a pharmaceutically acceptable carrier.
The invention also provides methods of using antibodies of the
invention. For example, the invention provides a method for modulating ligand
binding of a GPCR seven transmembrane receptor of the present invention
comprising
the step of contacting the seven transmembrane receptor with an antibody
specific for
the seven transmembrane receptor, under conditions wherein the antibody binds
the
receptor.
GPCR polypeptides are expressed in the brain, providing an indication
that aberrant GPCR polypeptide signaling activity may correlate with one or
more
1 S neurological disorders. The invention also provides a method for treating
a
neurological disorder comprising the step of administering to a mammal in need
of
such treatment an amount of an antibody-like polypeptide of the invention that
is
sufficient to modulate ligand binding of a GPCR seven transmembrane receptor
of the
present invention in neurons of the mammal. In addition to administration of
antibody-like polypeptides, administration of natural ligands for GPCR
polypeptides
as well as modulators of GPCR polypeptide activity, such as small molecules
that
mimic, agonize or antagonize ligand-mediated GPCR polypeptide signaling, are
contemplated. The expression pattern provides an indication that such
molecules will
have utility for treating neurological and/or psychiatric diseases, including
but not
limited to schizophrenia, depression, anxiety, bipolar disease, affective
disorders,
attention deficit hyperactivity disorder/attention deficit disorder
(ADHD/ADO),
epilepsy, neuritis, neurasthenia, neuropathy, neuroses, Alzheimer's disease,
Parkinson's disease, migraine, senile dementia, and the like. Treatment of
individuals
having any of these disorders is contemplated as an aspect of the invention.
Thus, in yet another embodiment, the invention provides genetic
screening procedures that entail analyzing a person's genome -- in particular
their

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alleles for GPCR's of the invention -- to determine whether the individual
possesses a
genetic characteristic found in other individuals that are considered to be
afflicted
with, or at risk for, developing a mental disorder or disease of the brain
that is
suspected of having a hereditary component. For example, in one embodiment,
the
invention provides a method for determining a potential for developing a
disorder
affecting the brain in a human subject comprising the steps of analyzing the
coding
sequence of one or more GPCR genes from the human subject; and determining
development potential for the disorder in said human subject from the
analyzing step.
More particularly, the invention provides a method of screening a
human subject to diagnose a disorder affecting the brain or genetic
predisposition
therefor, comprising the steps o~ (a) assaying nucleic acid of a human subject
to
determine a presence or an absence of a mutation altering the amino acid
sequence,
expression, or biological activity of at least one seven transmembrane
receptor that is
expressed in the brain, wherein the seven transmembrane receptor comprises an
amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6,
8,
10, 12, 14, 16, 18, and 20, or an allelic variant thereof, and wherein the
nucleic acid
corresponds to the gene encoding the seven transmembrane receptor; and (b)
diagnosing the disorder or predisposition from the presence or absence of said
mutation, wherein the presence of a mutation altering the amino acid sequence,
expression, or biological activity of allele in the nucleic acid correlates
with an
increased risk of developing the disorder. In preferred variations, the seven
transmembrane receptor is CON202 comprising an amino acid sequence set forth
in
SEQ ID NO: 14, or an allelic variant thereof, and the disease is
schizophrenia.
By "human subject" is meant any human being, human embryo, or
human fetus. It will be apparent that methods of the present invention will be
of
particular interest to individuals that have themselves been diagnosed with a
disorder
affecting the brain or have relatives that have been diagnosed with a disorder
affecting
the brain.
By "screening For an increased risk" is meant determination of whether
a genetic variation exists in the human subject that correlates with a greater
likelihood
of developing a disorder affecting the brain than exists for the human
population as a

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whole, or for a relevant racial or ethnic human sub-population to which the
individual
belongs. Both positive and negative determinations (i.c., dete~7~oinations
that a genetic
predisposition marker is present or is absent) are intended to fall within the
scope of
screening methods of the invention. In preferred embodiments, the presence of
a
mutation altering the sequence or expression of at least one CON202 seven
transmembrane receptor allele in the nucleic acid is correlated with an
increased risk
of developing schizophrenia, whereas the absence of such a mutation is
reported as a
negative determination.
The "assaying" step of the invention may involve any techniques
available for analyzing nucleic acid to determine its characteristics,
including but not
limited to well-known techniques such as single-strand conformation
polymorphism
analysis (SSCP) [Orita et al., Proc Natl. Acad. Sci. USA, 86: 2766-2770
(1989)];
heteroduplex analysis [White et al., Genomics, l 2: 301-306 (1992)];
denaturing
gradient gel electrophoresis analysis [Fischer et al., Proc. Natl. Acad. Sci.
USA, 80:
1579-1583 (1983); and Riesner et al., Electrophoresis, 10: 377-389 (1989)];
DNA
sequencing; RNase cleavage [Myers et al., Science, 230: 1242-1246 (1985)];
chemical
cleavage of mismatch techniques [Rowley et al., Genomics, 30: 574-582 (1995);
and
Roberts et al., Nucl. Acids Res., 25: 3377-3378 (1997)]; restriction fragment
length
polymorphism analysis; single nucleotide primer extension analysis [Shumaker
et al.,
Hum. Mutat., 7: 346-354 (1996); and Pastinen et al., Genome Res., 7: 606-614
(1997)]; 5' nuclease assays [Pease et cal., Proc. Natl. Acad. Sci. USA,
91:5022-5026
( 1994)]; DNA Microchip analysis [Ramsay, G., Nature Biotechnology, 16: 40-48
(1999); and Chee et al., U.S. Patent No. 5,837,832]; and ligase chain reaction
[Whiteley et al., U.S. Patent No. 5,521,065]. [See generally, Schafer and
Hawkins,
Nature Biotechnology, 16: 33-39 (1998).] All of the foregoing documents are
hereby
incorporated by reference in their entirety.
Thus, in one preferred embodiment involving screening CON202
sequences, for example, the assaying step comprises at least one procedure
selected
from the group consisting of: (a) determining a nucleotide sequence of at
least one
colon of at least one CON202 allele of the human subject; (b) performing a
hybridization assay to determine whether nucleic acid from the human subject
has a

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nucleotide sequence identical to or different from one or more reference
scduences;
(c) performing a polynucleotide migration assay to detern~ine whether nucleic
acid
from the human subject has a nucleotide sequence identical to or different
from one or
more reference sequences; and (d) performing a restriction endonuclease
digestion to
detenmine whether nucleic acid from the human subject has a nucleotide
sequence
identical to or different from one or more reference sequences.
In a highly preferred embodiment, the assaying involves sequencing of
nucleic acid to determine nucleotide sequence thereof, using any available
sequencing
technique. [See, e.g., Sanger et al., Proc. Natl. Acad. Sci. (USA), 74: 5463-
5467
( 1977) (dideoxy chain termination method); Mirzabekov, TIBTECH, 12: 27-32 (
1994)
(sequencing by hybridization); Drmanac et al., Nature Biotechnology, 16: 54-58
(1998); U.S. Patent No. 5,202,231; and Science, 260: 1649-1652 (1993)
(sequencing
by hybridization); Kieleczawa et al., Science, 258: 1787-1791 ( 1992)
(sequencing by
primer walking); (Douglas et al., Biotechniques, 14: 824-828 (1993) (Direct
sequencing of PCR products); and Akane et al., Biotechniques 16: 238-241
(1994);
Maxam and Gilbert, Meth. Enzymol., 65: 499-560 (1977) (chemical termination
sequencing), all incorporated herein by reference.] The analysis may entail
sequencing
of the entire seven transmembrane receptor gene genomic DNA sequence, or
portions
thereof; or sequencing of the entire seven transmembrane receptor coding
sequence or
portions thereof. In some circumstances, the analysis may involve a
determination of
whether an individual possesses a particular allelic variant, in which case
sequencing
of only a small portion of nucleic acid -- enough to determine the sequence of
a
particular codon characterizing the allelic variant -- is sufficient. This
approach is
appropriate, for example, when assaying to determine whether one family member
inherited the same allelic variant that has been previously characterized for
another
family member, or, more generally, whether a person's genome contains an
allelic
variant that has been previously characterized and con-elated with a mental
disorder
having a heritable component.
In another highly preferred embodiment, the assaying step comprises
performing a hybridization assay to determine whether nucleic acid from the
human
subject has a nucleotide sequence identical to or different from one or more
reference

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sequences. In a preferred embodiment, the hybridization involves a
determination of
whether nucleic acid derived from the human subject will hybridize with one or
more
oligonucleotides, wherein the oligonucleotides have nucleotide sequences that
correspond identically to a portion of the GPCR gene sequence taught herein,
such as
the CON202 coding sequence set forth in SEQ ID NO: 14, or that correspond
identically except for one mismatch. The hybridization conditions are selected
to
differentiate between perfect sequence complementarity and imperfect matches
differing by one or more bases. Such hybridization experiments thereby can
provide
single nucleotide polymorphism sequence information about the nucleic acid
from the
human subject, by virtue of knowing the sequences of the oligonucleotides used
in the
experiments.
Several of the techniques outlined above involve an analysis wherein
one performs a polynucleotide migration assay, e.g., on a polyacrylamide
electrophoresis gel (or in a capillary electrophoresis system), under
denaturing or non-
denaturing conditions. Nucleic acid derived from the human subject is
subjected to
gel electrophoresis, usually adjacent to (or co-loaded with) one or more
reference
nucleic acids, such as reference GPCR-encoding sequences having a coding
sequence
identical to all or a portion of SEQ >D NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or
19 (or
identical except for one known polymorphism). The nucleic acid from the human
subject and the reference sequences) are subjected to similar chemical or
enzymatic
treatments and then electrophoresed under conditions whereby the
polynucleotides
will show a differential migration pattern, unless they contain identical
sequences.
[See generally Ausubel et al. (eds.), Current Protocols in Molecular Biology,
New
York: John Wiley & Sons, Inc. (1987-1999); and Sambrook et al., (eds.),
Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor, New York: Cold Spring Harbor
Laboratory Press (1989), both incorporated herein by reference in their
entirety.]
In the context of assaying, the term "nucleic acid of a human subject"
is intended to include nucleic acid obtained directly from the human subject
(e.g.,
DNA or RNA obtained from a biological sample such as a blood, tissue, or other
cell
or fluid sample); and also nucleic acid derived from nucleic acid obtained
directly
from the human subject. By way of non-limiting examples, well known procedures

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exist for Creatltlg cDNA that IS COlllplenlentaCy to RNA derived from a
biological
sample from a human subject, and for amplifying (e.g., via polymerise chain
reaction
(PCR)) DNA or RNA derived from a biological sample obtained from a human
subject. Any such derived polynucleotide which retains relevant nucleotide
sequence
information of the human subject's own DNA/RNA is intended to fall within the
definition of "nucleic acid of a human subject" for the purposes of the
present
invention.
In the context of assaying, the term "mutation" includes addition,
deletion, and/or substitution of one or more nucleotides in the GPCR gene
sequence
(e.g., as compared to the seven transmembrane receptor-encoding sequences set
forth
in SEQ m NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19) and other polymorphisms
that occur
in introns (where introns exist) and that are identifiable via sequencing,
restriction
fragment length polymorphism, or other techniques. The various activity
examples
provided herein permit determination of whether a mutation modulates activity
of the
relevant receptor in the presence or absence of various test substances.
In a related embodiment, the invention provides methods of screening
a person's genotype with respect to GPCR's of the invention, and correlating
such
genotypes with diagnoses for disease or with predisposition for disease (for
genetic
counseling). For example, the invention provides a method of screening for a
CON202 hereditary schizophrenia genotype in a human patient, comprising the
steps
of: (a) providing a biological sample comprising nucleic acid from the
patient, the
nucleic acid including sequences corresponding to said patient's CON202
alleles; (b)
analyzing the nucleic acid for the presence of a mutation or mutations; (c)
determining
a CON202 genotype from the analyzing step; and (d) correlating the presence of
a
mutation in a CON202 allele with a hereditary schizophrenia genotype. In a
preferred
embodiment, the biological sample is a cell sample containing human cells that
contain genomic DNA of the human subject. The analyzing can be performed
analogously to the assaying described in preceding paragraphs. For example,
the
analyzing comprises sequencing a portion of the nucleic acid (e.g., DNA or
RNA), the
portion comprising at least one codon of the CON202 alleles.

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Although more time consuming and expensive than methods involving
nucleic acid analysis, the invention also may be practiced by assaying protein
of a
human subject to determine the presence or absence of an amino acid sequence
variation in GPCR protein from the human subject. Such protein analyses may be
performed, e.g., by fragmenting GPCR protein via chemical or enzymatic methods
and sequencing the resultant peptides; or by Western analyses using an
antibody
having specificity for a particular allelic variant of the GPCR.
The invention also provides materials that are useful for performing
methods of the invention. For example, the present invention provides
oligonucleotides useful as probes in the many analyzing techniques described
above.
In general, such oligonucleotide probes comprise 6, 7, 8, 9, 10 , 11, 12, 13,
14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39,
40, 41,.42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides that have a sequence
that is
identical, or exactly complementary, to a portion of a human GPCR gene
sequence
taught herein (or allelic variant thereof), or that is identical or exactly
complementary
except for one nucleotide substitution. In a preferred embodiment, the
oligonucleotides have a sequence that corresponds in the foregoing manner to a
human GPCR coding sequence taught herein, and in particular, the coding
sequences
set forth in SEQ ID NOs: l, 3, 5, 7, 9, 11, 13, 15, 17, or 19. In one
variation, an
oligonucleotide probe of the invention is purified and isolated. In another
variation,
the oligonucleotide probe is labeled, e.g., with a radioisotope, chromophore,
or
fluorophore. In yet another variation, the probe is covalently attached to a
solid
support. [See generally Ausubel et al. And Sambrook et al., supra.]
In a related embodiment, the invention provides kits comprising
reagents that are useful for practicing methods of the invention. For example,
the
invention provides a kit for screening a human subject to diagnose
schizophrenia or a
genetic predisposition therefor, comprising, in association: (a) an
oligonucleotide
useful as a probe for identifying polymorphisms in a human CON202 seven
transmembrane receptor gene, the oligonucleotide comprising 6-50 nucleotides
that
have a sequence that is identical or exactly complementary to a portion of a
human
CON202 gene sequence or CON202 coding sequence, except for one sequence

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di fference selected from the group consisting of a nucleotide addition, a
nucleotide
deletion, or nucleotide substitution; and (b) a media packaged with the
oligonucleotide containing information identifying polymorphisms identifyable
with
the probe that correlate with schizophrenia or a genetic predisposition
therefor.
Exemplary information-containing media include printed paper package inserts
or
packaging labels; and magnetic and optical storage media that are readable by
computers or machines used by practitioners who perform genetic screening and
counseling services. The practitioner uses the information provided in the
media to
correlate the results of the analysis with the oligonucleotide with a
diagnosis. In a
preferred variation, the oligonucleotide is labeled.
In still another embodiment, the invention provides methods of
identifying those allelic variants of GPCR's of the invention that correlate
with mental
disorders. For example, the invention provides a method of identifying a seven
transmembrane allelic variant that correlates with a mental disorder,
comprising steps
of: (a) providing a biological sample comprising nucleic acid from a human
patient
diagnosed with a mental disorder, or from the patient's genetic progenitors or
progeny; (b) analyzing the nucleic acid for the presence of a mutation or
mutations in
at least one seven transmembrane receptor that is expressed in the brain,
wherein the
at least one seven transmembrane receptor comprises an amino acid sequence
selected
from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and
20, or an
allelic variant thereof, and wherein the nucleic acid includes sequence
corresponding
to the gene or genes encoding the at least one seven transmembrane receptor;
(c)
determining a genotype for the patient for the at least one seven
transmembrane
receptor from said analyzing step; and (d) identifying an allelic variant that
correlates
with the mental disorder from the determining step. To expedite this process,
it may
be desirable to perform linkage studies in the patients (and possibly their
families) to
correlate chromosomal markers with disease states. The chromosomal
localization
data provided herein facilitates identifying an involved GPCR with a
chromosomal
m arker.
The foregoing method can be performed to correlate GPCR's of the
invention to a number of disorders having hereditary components that are
causative or

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that predispose persons to the disorder. For example, in one preferred
variation, the
disorder is schizophrenia, and the at least one seven transmembrane receptor
comprises CON202 having an amino acid sequence set forth in SEQ ID NO: l4, or
an
allelic variant thereof.
Also contemplated as part of the invention are polynucleotides that
comprise the allelic variant sequences identified by such methods, and
polypeptides
encoded by the allelic variant sequences, and oligonucleotide and oligopeptide
fragments therof that embody the mutations that have been identified. Such
materials
are useful in in vitro cell-free and cell-based assays for idenifying lead
compounds
and therapeutics for treatment of the disorders. For example, the variants are
used in
activity assays, binding assays, and assays to screen for activity modulators
described
herein. In one preferred embodiment, the invention provides a purified and
isolated
polynucleotide comprising a nucleotide sequence encoding a CON202 receptor
allelic
variant identified according to the methods described above; and an
oligonucleotide
that comprises the sequences that differentiate the allelic variant from the
CON202
sequences set forth in SEQ ~ NOs: 13 and 14. The invention also provides a
vector
comprising the polynucleotide (preferably an expression vector); and a host
cell
transformed or transfected with the polynucleotide or vector. The invention
also
provides an isolated cell line that is expressing the allelic variant GPCR
polypeptide;
purified cell membranes from such cells; purified polypeptide; and synthetic
peptides
that embody the allelic variation amino acid sequence. In one particular
embodiment,
the invention provides a purified polynucleotide comprising a nucleotide
sequence
encoding a CON202 seven transmembrane receptor protein of a human that is
affected
with schizophrenia; wherein said polynucleotide hybridizes to the complement
of
SEQ ID NO: 13 under the following hybridization conditions: (a) hybridization
for 16
hours at 42°C in a hybridization solution comprising SO% formamide, 1%
SDS, 1 M
NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at
60°C in a wash
solution comprising O.lx SSC and 1% SDS; and wherein the polynucleotide
encodes
a CON202 amino acid sequence that differs from SEQ ID NO: 14 at at least one
residue.

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An examplary assay for using the allelic variants is a method for
identifying a modulator of C'.ON202 biological activity, comprising the steps
of: (a)
contacting a cell expressing the allelic variant in the presence and in the
absence of a
putative modulator compound; (b) measuring CON202 biological activity in the
cell;
and (c) identifying a putative modulator compound in view of decreased or
increased
CON202 biological activity in the presence versus absence of the putative
modulator.
In still another example, the invention provides for a method of
diagnosing schizophrenia or a susceptibility to schizophrenia comprising the
steps of:
determining the presence or amount of expression of CON202 polypeptide as set
out
as SEQ ~ NO: 14 or the polypeptide encoded by the nucleic acid molecule having
SEQ ID NO: 13 in a sample; and comparing the level of CON202 polypeptide in a
biological, tissue or cellular sample from normal subjects or the subject at
an earlier
time, wherein the susceptibility to schizophenia is based on the presence or
amount of
CON202 polypeptide expression.
The invention also provides for a method of treating schizophrenia
comprising the step of administering to a human diagnosed with schizophrenia
an
amount of a modulator of CON202 receptor activity sufficient to modulate
CON202
receptor activity or CON202 ligand binding in said human.
The invention also provides assays to identify compounds that bind
GPCR seven transmembrane receptors. One such assay comprises the steps of: (a)
contacting a composition comprising one of the GPCR seven transmembrane
receptor
polypeptides of the invention with a compound suspected of binding a GPCR
polypeptide of the invention; and (b) measuring binding between the compound
and
the GPCR polypeptide. In one variation, the composition comprises a cell
expressing
a GPCR polypeptide of the invention on its surface. In another variation, an
isolated
GPCR polypeptide of the invention or cell membranes comprising a GPCR
polypeptide of the invention are employed. The binding may be measured
directly,
e.g., using a labeled compound, or may be measured indirectly by several
techniques,
including measuring intracellular signaling of a GPCR polypeptide of the
invention
induced by the compound (or measuring changes in the level of GPCR polypeptide
signaling).

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The invention also provides a method for identifying a modulator of
binding between a GPCR seven transmembrane receptor of the invention and a
GPCR
polypeptide binding partner, comprising the steps of: (a) contacting a GPCR
polypeptide binding partner and a composition comprising one of the GPCR seven
transmembrane receptors of the invention in the presence and in the absence of
a
putative modulator compound; (b) detecting binding between the binding partner
and
the GPCR polypeptide of the invention; and (c) identifying a putative
modulator
compound in view of decreased or increased binding between the binding partner
and
the GPCR polypeptide in the presence of the putative modulator, as compared to
binding in the absence of the putative modulator.
GPCR polypeptide binding partners that stimulate GPCR seven
transmembrane receptors of the present invention are useful as agonists in
disease
states characterized by insufficient GPCR polypeptide signaling (e.g., as a
result of
insufficient expression of active GPCR polypeptide ligand). GPCR polypeptide
binding partners that block ligand-mediated GPCR polypeptide signaling are
useful as
GPCR polypeptide antagonists to treat disease states characterized by
excessive
GPCR polypeptide signaling.
Additional features and variations of the invention will be apparent to
those skilled in the art from the entirety of this application, including the
detailed
description, and all such features are intended as aspects of the invention.
Likewise,
features of the invention described herein can be re-combined into additional
embodiments that also are intended as aspects of the invention, irrespective
of
whether the combination of features is specifically mentioned above as an
aspect or
embodiment of the invention. Also, only such limitations which are described
herein
as critical to the invention should be viewed as such; variations of the
invention
lacking limitations which have not been described herein as critical are
intended as
aspects of the invention.
In addition to the foregoing, the invention includes, as an additional
aspect, all embodiments of the invention narrower in scope in any way than the
variations specifically mentioned above. Although the applicants) invented the
full
scope of the claims appended hereto, the claims appended hereto are not
intended to

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encompass within their scope the prior art work of others. 'hhercfore, in the
event that
statutory prior art within the scope of a claim is brought to the attention of
the
applicants by a Patent Office or other entity or individual, the applicants)
reserve the
right to exercise amendment rights under applicable patent laws to redefine
the
subject matter of such a claim to specifically exclude such statutory prior
art or
obvious variations of statutory prior art from the scope of such a claim.
Variations of
the invention defined by such amended claims also are intended as aspects of
the
invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides purified and isolated polynucleotides
(e.g., DNA sequences and RNA transcripts, both sense and complementary
antisense
strands, both single and double stranded, including splice variants thereof)
encoding
human G protein-coupled receptors referred to herein as GPCR polypeptides. DNA
polynucleotides of the invention include genomic DNA, cDNA, and DNA that has
been chemically synthesized in whole or in part. "Synthesized" as used herein
and
understood in the art, refers to polynucleotides produced by purely chemical,
as
opposed to enzymatic, methods. "Wholly" synthesized DNA sequences are
therefore
produced entirely by chemical means, and "partially" synthesized DNAs embrace
those wherein only portions of the resulting DNA were produced by chemical
means.
Genomic DNA of the invention comprises the protein coding region
for a polypeptide of the invention and is also intended to include allelic
variants
thereof. It is widely understood that, for many genes, genomic DNA is
transcribed
into RNA transcripts that undergo one or more splicing events wherein intron
(i.e.,
non-coding regions) of the transcripts are removed, or "spliced out." RNA
transcripts
that can be spliced by alternative mechanisms, and therefore be subject to
removal of
different RNA sequences but still encode a GPCR polypeptide of the present
invention, are referred to in the art as splice variants which are embraced by
the
invention. Splice variants comprehended by the invention therefore are encoded
by
the same original genomic DNA sequences but arise from distinct mRNA
transcripts.
Allelic variants are modified forms of a wild type gene sequence, the
modification

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resulting from recombination dLll'lllg chromosomal segregation or exposure to
conditions which give rise to genetic mutation. Allelic variants, like wild
type genes,
are naturally occurring sequences (as opposed to non-naturally occun-ing
variants
which arise from in vits~o manipulation).
The invention also comprehends cDNA that is obtained through
reverse transcription of an RNA polynucleotide encoding a GPCR of the present
invention (conventionally followed by second strand synthesis of a
complementary
strand to provide a double-stranded DNA).
A preferred DNA sequence encoding a human GPCR polypeptide is set
out in SEQ ID NO: 1, wherein nucleotides 157 to 1122 represent the CON193
coding
sequence, with termination codon (surrounded by upstream and downstream
untranslated sequences). Another preferred DNA sequence encoding a human GPCR
polypeptide is set out in SEQ ~ NO: 3, wherein nucleotides 1 to 1014 represent
the
CON166 coding sequence and stop codon. Still another preferred DNA sequence
encoding a human GPCR polypeptide is set out in SEQ ID NO: 5, wherein
nucleotides 691 to 1845 represent the CON103 coding sequence with stop codon
(surrounded by upstream and downstream untranslated sequences). Another
preferred DNA sequence encoding a human GPCR polypeptide is set out in SEQ ID
NO: 7, wherein nucleotides 146 to 1147 represent the CON203 coding sequence
with
stop codon (surrounded by upstream and downstream untranslated sequences). A
preferred DNA sequence encoding a human GPCR polypeptide is set out in SEQ ID
NO: 9, wherein nucleotides 1 to 957 represent the CON198 coding sequence with
stop
codon. Another preferred DNA sequence encoding a human GPCR polypeptide is set
out in SEQ ID NO: 11, wherein nucleotides 1 to 924 represent the CON197 coding
sequence with stop codon (followed by downstream untranslated sequences). A
preferred DNA sequence encoding a human GPCR polypeptide is set out in SEQ ID
NO: 13, wherein nucleotides 266 to 1378 represent the CON202 coding sequence
and
termination codon (surrounded by upstream and downstream untranslated
sequences).
A preferred DNA sequence encoding a human GPCR polypeptide is set out in SEQ
ID
NO: 15, wherein nucleotides 1 to 1191 represent the CON222 coding sequence and
termination codon. A preferred DNA sequence encoding a human GPCR polypeptide

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is set out in SEQ ID NO: 17, wherein nucleotides 13 to 1089 represent the
CON215
coding seduence and termination colon (surrounded by upstream and downstream
untranslated sequences). A prefers -ed DNA sequence encoding a human GPCR
polypeptide is set out in SEQ ID NO: 19, wherein nucleotides 42 to l 157
represent
the CON217 coding sequence (surrounded by upstream and downstream untranslated
sequences). The foregoing sequences without their termination colons also
comprise
preferred sequences.
The worker of skill in the art will readily appreciate that the preferred
DNA of the invention comprises a double stranded molecule, for example the
~ molecule having any one of the sequences set forth in SEQ )17 NOS: l, 3, 5,
7, 9, 11,
13, 15, 17, or 19 (or coding portions thereof) along with the complementary
molecule
(the "non-coding strand" or "complement") having a sequence deducible from the
sequence of SEQ LD NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 according to
Watson-
Crick base pairing rules for DNA. Also preferred are other polynucleotides
encoding
the GPCR polypeptides of the invention set forth in SEQ ID NOS: 2, 4, 6, 8,
10, 12,
14, 16, 18 and 20 which differ in sequence from the polynucleotide of SEQ ID
NOS:
1, 3, 5, 7, 9, 11, 13, 15, 17, or 19, respectively, by virtue of the well-
known
degeneracy of the universal genetic code.
The invention further embraces species, preferably mammalian,
homologs of the human GPCR DNAs. Species homologs, sometimes referred to as
"orthologs," in general, share at least 35%, at least 40%, at least 45%, at
least 50%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least
90%, at least 95%, at least 98%, or at least 99% homology with human DNA of
the
invention. Percent sequence "homology" with respect to polynucleotides of the
invention is defined herein as the percentage of nucleotide bases in the
candidate
sequence that are identical to nucleotides in the GPCR sequence set forth in
any one
of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 after aligning the
sequences and
introducing gaps, if necessary, to achieve the maximum percent sequence
identity.
The polynucleotide sequence information provided by the invention
makes possible large scale expression of the encoded polypeptide by techniques
well
known and routinely practiced in the art. Polynucleotides of the invention
also permit

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identif canon and isolation of polynucleotides encoding related GPC.R
polypeptides,
such as human allelic variants and species homologs, by well known techniques
including Southern and/or Northern hybridization, and polymerise chain
reaction
(PCR). Examples of related polynucleotides include human and non-human genomic
sequences, including allelic variants, as well as polynucleotides encoding
polypeptides
homologous to GPCR polypeptides and stmcturally related the polypeptides
sharing
one or more biological, immunological, and/or physical properties of the GPCR
polypeptides. Non-human species genes encoding proteins homologous to GPCR
polypeptides can also be identified by Southern and/or PCR analysis and are
useful in
animal models for GPCR-related disorders. Knowledge of the sequence of a human
GPCR DNA also makes possible, through use of Southern hybridization or
polymerise chain reaction (PCR), the identification of genomic DNA sequences
encoding GPCR expression control regulatory sequences such as promoters,
operators, enhancers, repressors, and the like. Polynucleotides of the
invention are
also useful in hybridization assays to detect the capacity of cells to express
GPCR
polypeptides. Polynucleotides of the invention may also be the basis for
diagnostic
methods useful for identifying a genetic alterations) in a GPCR locus that
underlies a
disease state or states, which information is useful both for diagnosis and
for selection
of therapeutic strategies.
The disclosure herein of full length polynucleotides encoding GPCR
polypeptides of the present invention makes readily available to the worker of
ordinary skill in the art every possible fragment of the full length
polynucleotides.
The invention therefore provides fragments of GPCR-encoding polynucleotides
comprising at least 14-15, and preferably at least 18, 20, 25, 50, or 75
consecutive
nucleotides of a polynucleotide encoding GPCR polypeptides. Preferably,
fragment
polynucleotides of the invention comprise sequences unique to the GPCR-
encoding
polynucleotide sequence, and therefore hybridize under highly stringent or
moderately
stringent conditions only (i.e., "specifically") to polynucleotides encoding
GPCR
polypeptides (or fragments thereof). Polynucleotide fragments of genomic
sequences
of the invention comprise not only sequences unique to the coding region, but
also
include fragments of the full length sequence derived from introns, regulatory
regions,

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and/or other non-translated sequences. Sequences unique to polynucleotides of
the
invention are recognizable through sequence cmnparison to other Known
polynucleotides, and can be identified through use of alignment programs
routinely
utilized in the art, e.g., those made available in public sequence databases.
Such
sequences also are recognizable from Southern and Northern hybridization
analyses to
determine the number of fragments of genomic DNA and RNA to which a
polynucleotide will hybridize. Polynucleotides of the invention can be labeled
in a
manner that permits their detection, including radioactive, fluorescent, and
enzymatic
labeling.
Fragment polynucleotides are particularly useful as probes for
detection of full length or other fragment GPCR polynucleotides. One or more
fragment polynucleotides can be included in kits that are used to detect the
presence
of a polynucleotide encoding a GPCR polypeptide, or used to detect variations
in a
polynucleotide sequences encoding GPCR polypeptides.
The invention also embraces DNAs encoding GPCR polypeptides
which DNAs hybridize under moderately stringent or high stringency conditions
to
the non-coding strand, or complement, of the polynucleotide in any one of SEQ
ID
NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19.
Exemplary highly stringent hybridization conditions are as follows:
hybridization at 42°C in a hybridization solution comprising 50%
formamide, 1%
SDS, 1 M NaCI, 10% Dextran sulfate, and washing twice for 30 minutes at
60°C in a
wash solution comprising O.lx SSC and 1% SDS. It is understood in the art that
conditions of equivalent stringency can be achieved through variation of
temperature
and buffer, or salt concentration as described Ausubel, et al. (Eds.),
Protocols in
Molecular Biology, John Wiley & Sons (1994), pp. 6Ø3 to 6.4.10.
Modifications in
hybridization conditions can be empirically determined or precisely calculated
based
on the length and the percentage of guanosine/cytosine (GC) base pairing of
the
probe. The hybridization conditions can be calculated as described in Sambrook
et
al., (Eds.), Molecular Cloning: A Lcaboratory Munuc~l, Cold Spring Harbor
Laboratory
Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.

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Autonomously replicating recombinant expression constructs such as
plasmid and viral DNA vectors incorporating polynucleotidcs of the invention
are also
provided. Expression constructs wherein GPCR-encoding polynucleotides are
operatively linked to an endogenous or exogenous expression control DNA
sequence
and a transcription terminator are also provided. Expression control DNA
sequences
include promoters, enhancers, and operators, and are generally selected based
on the
expression systems in which the expression constrict is to be utilized.
Preferred
promoter and enhancer sequences are generally selected for the ability to
increase
gene expression, while operator sequences are generally selected for the
ability to
regulate gene expression. Expression constructs of the invention may also
include
sequences encoding one or more selectable markers that permit identification
of host
cells bearing the construct. Expression constructs may also include sequences
that
facilitate, and preferably promote, homologous recombination in a host cell.
Preferred
constricts of the invention also include sequences necessary for replication
in a host
cell.
Expression constructs are preferably utilized for production of an
encoded protein, but also may be utilized simply to amplify GPCR-encoding
polynucleotide sequences.
According to another aspect of the invention, host cells are provided,
including prokaryotic and eukaryotic cells, comprising a polynucleotide of the
invention (or vector of the invention) in a manner which permits expression of
the
encoded GPCR polypeptide. Polynucleotides of the invention may be introduced
into
the host cell as part of a circular plasmid, or as linear DNA comprising an
isolated
protein coding region or a viral vector. Methods for introducing DNA into the
host
cell well known and routinely practiced in the art include transformation,
transfection,
electroporation, nuclear injection, or fusion with carriers such as liposomes,
micelles,
ghost cells, and protoplasts. Expression systems of the invention include
bacterial,
yeast, fungal, plant, insect, invertebrate, and mammalian cells systems.
Host cells of the invention are a valuable source of immunogen for
development of antibodies specifically immunoreactive with GPCR polypeptides.
Host cells of the invention are also useful in methods for large scale
production of

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GPCR polypeptides wherein the cells are grown in a suitable culture medium
anti the
desired polypeptide products are isolated .from the cells or from the medium
in which
the cells are grown by purification methods known in the art, e.g.,
conventional
chromatographic methods including immunoaffinity chromatography, receptor
affinity chromatography, hydrophobic interaction chromatography, lectin
affinity
chromatography, size exclusion filtration, cation or anion exchange
chromatography,
high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like.
Still
other methods of purification include those wherein the desired protein is
expressed
and purif ed as a fusion protein having a specific tag, label, or chelating
moiety that is
recognized by a specific binding partner or agent. The purified protein can be
cleaved
to yield the desired protein, or be left as an intact fusion protein. Cleavage
of the
fusion component may produce a form of the desired protein having additional
amino
acid residues as a result of the cleavage process.
Knowledge of GPCR DNA sequences allows for modification of cells
to permit, or increase, expression of endogenous GPCR. Cells can be modified
(e.g.,
by homologous recombination) to provide increased expression by replacing, in
whole
or in part, the naturally occurring GPCR promoter with all or part of a
heterologous
promoter so that the cells express GPCR polypeptides at higher levels. The
heterologous promoter is inserted in such a manner that it is operatively
linked to
endogenous GPCR polypeptide encoding sequences. [See, for example, PCT
International Publication No. WO 94/12650, PCT International Publication No.
WO
92/20808, and PCT International Publication No. WO 91/09955.] It is also
contemplated that, in addition to heterologous promoter DNA, amplifiable
marker
DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl
phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or
intron
DNA may be inserted along with the heterologous promoter DNA. If linked to the
GPCR coding sequence, amplification of the marker DNA by standard selection
methods results in co-amplification of the GPCR coding sequences in the cells.
The DNA sequence information provided by the present invention also
makes possible the development through, e.g. homologous recombination or
"knock-out" strategies [Capecchi, Science 244: 1288-1292 (1989)], of animals
that

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_ ~g _
fail to express functional GPCR polypeptides or that express a variant of GPCR
polypeptides. Such animals (especially small laboratory animals such as rats,
rabbits,
and mice) are useful as models for studying the in vivo activities of GPCR
polypeptides and modulators of GPCR polypeptides.
Also made available by the invention are anti-sense polynucleotides
which recognize and hybridize to polynucleotides encoding GPCR polypeptides.
Full
length and fragment anti-sense polynucleotides are provided. Fragment anti-
sense
molecules of the invention include those which specifically recognize and
hybridize to
GPCR RNA (as detemined by sequence comparison of DNA encoding GPCR
polypeptides to DNA encoding other known molecules). Identification of
sequences
unique to GPCR-encoding polynucleotides, can be deduced through use of any
publicly available sequence database, and/or through use of commercially
available
sequence comparison programs. The uniqueness of selected sequences in an
entire
genome can be further verified by hybridization analyses. After identification
of the
desired sequences, isolation through restriction digestion or amplification
using any of
the various polymerise chain reaction techniques well known in the art can be
performed. Antisense polynucleotides are particularly relevant to regulating
expression of GPCR polypeptides by those cells expressing GPCR mRNA.
Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides)
capable of specifically binding to GPCR expression control sequences or GPCR
RNA
are introduced into cells (e.g., by a viral vector or colloidal dispersion
system such as
a liposome). The antisense nucleic acid binds to the GPCR target nucleotide
sequence
in the cell and prevents transcription or translation of the target sequence.
Phosphorothioate and methylphosphonate antisense oligonucleotides are
specifically
contemplated for therapeutic use by the invention. The antisense
oligonucleotides
may be further modified by poly-L-lysine, transferrin polylysine, or
cholesterol
moieties at their 5' end. Suppression of GPCR polypeptide expression at either
the
transcriptional or translational level is useful to general cellular and/or
animal models
for diseases characterized by aberrant expression. Suppression of GPCR
polypeptide
expression at either the transcriptional or translational level is useful to
generate

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_~c)_
cellular animal models for diseases characterized by aberrant GPCR polypeptide
expression.
The GPCR polynucleotidc and polypeptide sequences taught in the
present invention facilitate the design of novel transcription factors for
modulating
S GPCR polypeptide expression in native cells and animals, and cells
transformed or
transfected with GPCR polynucleotides. For example, the Cyst-His2 zinc finger
proteins, which bind DNA via their zinc finger domains, have been shown to be
amenable to structural changes that lead to the recognition of different
target
sequences. These artificial zinc finger proteins recognize specific target
sites with
high affinity and low dissociation constants, and are able to act as gene
switches to
modulate gene expression. Knowledge of the particular GPCR target sequence of
the
present invention facilitates the engineering of zinc finger proteins specific
for the
target sequence using known methods such as a combination of structure-based
modeling and screening of phage display libraries [Segal et al., Proc Natl
Acad Sci
1 S USA 96: 2758-2763 (1999); Liu et al., Proc Natl Acad Sci USA 94: 5525-30
(1997);
Greisman and Pabo Science 275: 657-61 (1997); Choo et al., JMoI Biol 273: 525-
32
( 1997)]. Each zinc finger domain usually recognizes three or more base pairs.
Since
a recognition sequence of 18 base pairs is generally sufficient in length to
render it
unique in any known genome, a zinc finger protein consisting of 6 tandem
repeats of
zinc fingers would be expected to ensure specificity for a particular sequence
[Segal et
al., Proc Natl Acad Sci USA 96: 2758-2763 (1999)]. The artificial zinc finger
repeats,
designed based on GPCR polynucleotide sequences, are fused to activation or
repression domains to promote or suppress GPCR polypeptides expression [Liu et
al.,
Proc Natl Acad Sci USA 94: 5525-30 (1997)]. Alternatively, the zinc finger
domains
can be fused to the TATA box-binding factor (TBP) with varying lengths of
linker
region between the zinc finger peptide and the TBP to create either
transcriptional
activators or repressors [Kim et al., Proc Natl Acad Sci USA 94: 3616-3620
(1997)].
Such proteins, and polynucleotides that encode them, have utility for
modulating
GPCR polypeptide expression in vivo in both native cells, animals and humans;
and/or cells transfected with GPCR polynulcoeitde-encoding sequences. The
novel
transcription factor can be delivered to the target cells by transfecting
constructs that

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express the transcription factor (gene therapy), or by introducing the
protein.
Cngineered zinc finger proteins can also be designed to bind EZNA sequences
for use
in therapeutics as alternatives to antisense or catalytic RNA methods [McColl
et crl.,
Proc Ncatl Acacl Sci USA 96:9521-6 (1999); Wu et al., Proc Natl Acucl Sci USA
92:344-348 (1995)]. The present invention contemplates methods of designing
such
transcription factors based on the gene sequence of the invention, as well as
customized zinc finger proteins, that are useful to modulate GPCR polypeptide
expression in cells (native or transformed) whose genetic complement includes
these
sequences.
The invention also provides purified and isolated mammalian GPCR
polypeptides encoded by a polynucleotide of the invention. Presently preferred
is a
human GPCR polypeptide comprising the amino acid sequence set out in any one
of
SEQ LD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20.
The invention also embraces polypeptides that have at least 99%, at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least
70%, at least
65%, at least 60%, at least 55% or at least 50% identity and/or homology to a
preferred polypeptide of the invention. Percent amino acid sequence "identity"
with
respect to the preferred polypeptide of the invention is defined herein as the
percentage of amino acid residues in the candidate sequence that are identical
with the
residues in a GPCR polypeptide sequence after aligning both sequences and
introducing gaps, if necessary, to achieve the maximum percent sequence
identity, and
not considering any conservative substitutions as part of the sequence
identity.
Percent sequence "homology" with respect to the preferred polypeptide of the
invention is defined herein as the percentage of amino acid residues in the
candidate
sequence that are identical with the residues in a GPCR sequence after
aligning the
sequences and introducing gaps, if necessary, to achieve the maximum percent
sequence identity, and also considering any conservative substitutions as part
of the
sequence identity.
In one aspect, percent homology is calculated as the percentage of
amino acid residues in the smaller of two sequences which align with identical
amino
acid residue in the sequence being compared, when four gaps in a length of 100
amino

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acids may be introduced to maximize alignment [Dayhoff, in ~ltla.s of Protein
Sec/crence crnrd ftrercture, Vol. 5, p. 124, National Biochemical Research
Foundation,
Washington, D.C. (1972), incorporated herein by reference].
Polypeptides of the invention may be isolated from natural cell
sources or may be chemically synthesized, but are preferably produced by
recombinant procedures involving host cells of the invention. Use of mammalian
host
cells is expected to provide for such post-translational modi fications (e.g.,
glycosylation, truncation, lipidation, and phosphorylation) as may be needed
to confer
optimal biological activity on recombinant expression products of the
invention.
Glycosylated and non-glycosylated forms of GPCR polypeptides are embraced.
The invention also embraces variant (or analog) GPCR polypeptides.
In one example, insertion variants are provided wherein one or more amino acid
residues supplement a GPCR amino acid sequence. Insertions may be located at
either or both termini of the protein, or may be positioned within internal
regions of
the GPCR amino acid sequence. Insertional variants with additional residues at
either
or both termini can include for example, fusion proteins and proteins
including amino
acid tags or labels.
Insertion variants include GPCR polypeptides wherein one or more
amino acid residues are added to a GPCR amino acid sequence, or to a
biologically
active fragment thereof.
Variant products of the invention also include mature GPCR
polypeptide products, i.e., GPCR polypeptide products wherein leader or signal
sequences are removed, with additional amino terminal residues. The additional
amino terminal residues may be derived from another protein, or may include
one or
more residues that are not identifiable as being derived from a specific
proteins.
GPCR polypeptide products with an additional methionine residue at position -1
(Met-'-GPCR) are contemplated, as are variants with additional methionine and
lysine
residues at positions -2 and -1 (Met-z-Lys ~-GPCR). Variants of GPCR
polypeptide
with additional Met, Met-Lys, Lys residues (or one or more basic residues in
general)
are particularly useful for enhanced recombinant protein production in
bacterial host
cell.

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The invention also embraces GPCR polypeptide variants hamng
additional amino acid residues which result from use of specific expression
systems.
For example, use of commercially available vectors that express a desired
polypeptide
as part of glutathione-S-transferase (GS T ) fusion product provides the
desired
polypeptide having an additional glycine residue at position -1 after cleavage
of the
GST component from the desired polypeptide. Variants which result from
expression
in other vector systems are also contemplated.
Insertional variants also include fusion proteins wherein the amino
and/or carboxy termini of a GPCR polypeptide is fused to another polypeptide.
In another aspect, the invention provides deletion variants wherein one
or more amino acid residues in a GPCR polypeptide are removed. Deletions can
be
effected at one or both termini of the GPCR polypeptide, or with removal of
one or
more residues within the GPCR amino acid sequence. Deletion variants,
therefore,
include all fragments of a GPCR polypeptide.
The invention also embraces polypeptide fragments of the sequence set
out in SEQ B7 NO: 2 wherein the fragments maintain biological (e.g., ligand
binding
and/or intracellular signaling) or immunological properties of a GPCR
polypeptide.
Fragments comprising at least 5, 10, 15, 20, 25, 30, 35, or 40 consecutive
amino acids
of SEQ ID NO: 2 are comprehended by the invention. Preferred polypeptide
fragments display antigenic properties unique to or specific for human GPCR
and its
allelic and species homologs. Fragments of the invention having the desired
biological and immunological properties can be prepared by any of the methods
well
known and routinely practiced in the art.
In still another aspect, the invention provides substitution variants of
GPCR polypeptides. Substitution variants include those polypeptides wherein
one or
more amino acid residues of a GPCR polypeptide are removed and replaced with
alternative residues. In one aspect, the substitutions are conservative in
nature,
however, the invention embraces substitutions that are also non-conservative.
Conservative substitutions for this purpose may be defined as set out in
Tables A, B,
or C below.

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Variant polypeptides include those wherein conservative substitutions
have been introduced by modification of polynuclcotides encoding polypeptides
of the
invention. Amino acids can be classified according to physical properties and
contribution to secondary and tertiary protein structure. A conservative
substitution is
recognized in the art as a substitution of one amino acid for another amino
acid that
has similar properties. Exemplary conservative substitutions are set out in
Table A
(from WO 97/09433, page 10, published March 13, 1997 (PCT/GB96/02197, filed
9/6/96), immediately below.
Table A
Conservative Substitutions 1
SIDE CHAIN
CHARACTERISTIC AMINO ACID
Aliphatic
Non-polar GA P I L V
Polar - uncharged C S T M N Q
Polar - charged D E K R
Aromatic H F W Y
Other N Q D E
Alternatively, conservative amino acids can be grouped as described in
Lehninger,
[Bioclzenzistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-
77J as
set out in Table B, immediately below.

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Table B
Conservative Substitutions It
SIDE CHAIN
CHARACTERISTIC AMINO ACID
Non-polar (hydrophobic)
A. Aliphatic: A L I V
P
B. Aromatic: F W
C. Sulfur-containing: Nl
D. Borderline: G
Uncharged-polar
A. Hydroxyl: S T Y
B. Amides: N Q
C. Sulfhydryl: C
D. Borderline: G
Positively Charged (Basic): K R H
Negatively Charged (Acidic): DE
As still an another alternative, exemplary conservative substitutions are set
out in
Table C, immediately below.

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Table C
Conservative Substitutions I II
Ori i~ nil Exemplary Substitution
Residue
Ala (A) Val, Leu, Ile
Arg (R) Lys, Gln, Asn
Asn (N) Gln, His, Lys, Arg
Asp (D) Glu
Cys (C) Ser
Gln (Q) Asn
Glu (E) Asp
His (H) Asn, Gln, Lys, Arg
Ile (I) Leu, Val, Met, Ala,
Phe,
Leu (L) Ile, Val, Met, Ala,
Phe
Lys (K) Arg, Gln, Asn
Met (M) Leu, Phe, Ile
Phe (F) Leu, Val, Ile, Ala
Pro (P) Gly
Ser (S) Thr
Thr (T) Ser
Trp (W) Tyr
Tyr (Y) Trp, Phe, Thr, Ser
Val (V) Ile, Leu, Met, Phe,
Ala
GPCR polypeptide variants that display ligand binding properties of
native GPCR polypeptides and are expressed at higher levels, and variants that
provide for constitutive active receptor are particularly useful in assays of
the

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-56-
invention. Such variants also are useful in cellular and animal models for
diseases
characterized by aberrant GPCR polypeptide expression/activity.
Lt should be understood that the definition of polypeptides of the
invention is intended to include polypeptides bearing modifications other than
insertion, deletion, or substitution of amino acid residues. By way of
example, the
modifications may be covalent in nature, and include for example, chemical
bonding
with polymers, lipids, other organic, and inorganic moieties. Such derivatives
may be
prepared to increase circulating half life of a polypeptide, or may be
designed to
improve targeting capacity for the polypeptide to desired cells, tissues, or
organs.
Similarly, the invention further embraces GPCR polypeptides that have
been covalently modified to include one or more water soluble polymer
attachments
such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
W a related embodiment, the present invention provides compositions
comprising purified polypeptides of the invention. Preferred compositions
comprise,
in addition to the polypeptide of the invention, a pharn~aceutically
acceptable (i.e.,
sterile and non-toxic) liquid, semisolid, or solid diluents that serve as
pharmaceutical
vehicles, excipients, or media. Any diluent known in the art may be used.
Exemplary
diluents include, but are not limited to, water, saline solutions,
polyoxyethylene
sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate,
talc,
alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol,
calcium
phosphate, mineral oil, and cocoa butter.
Also comprehended by the present invention are antibodies (e.g.,
monoclonal and polyclonal antibodies, single chain antibodies, chimeric
antibodies,
bifunctional/bispecif c antibodies, humanized antibodies, human antibodies,
and
complementary determining region (CDR)-grafted antibodies, including compounds
which include CDR sequences which specifically recognize a polypeptide of the
invention) speci fic for GPCR polypeptides of the invention or fragments
thereof.
Preferred antibodies of the invention are human antibodies which can be
produced
and identified according to methods described in W093/11236, published .lune
20,
1993, which is incorporated herein by reference in its entirety. Antibody
fragments,
including Fab, Fab', F(ab')Z, and F~, are also provided by the invention. The
term

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"specific for," when used to describe antibodies of the invention, indicates
that the
variable regions of the antibodies of the invention recognize and bind G:PCR
polypeptides exclusively (i.e., able to distinguish GPCR polypeptides from
other
known GPCR polypeptides by virtue of measurable differences in binding
affinity,
despite the possible existence of localized sequence identity, homology, or
similarity
between GPCR polypeptides and such polypeptides). It will be understood that
specific antibodies may also interact with other proteins (for example, S.
caureus
protein A or other antibodies in EL1SA techniques) through interactions with
sequences outside the variable region of the antibodies, and in particular, in
the
constant region of the molecule. Screening assays to determine binding
specificity of
an antibody of the invention are well known and routinely practiced in the
art. For a
comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A
Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor , NY
(1988), Chapter 6. Antibodies that recognize and bind fragments of the GPCR
polypeptides of the invention are also contemplated, provided that the
antibodies are,
first and foremost, specific for GPCR polypeptides. Antibodies of the
invention can
be produced using any method well known and routinely practiced in the art.
Non-human antibodies may be humanized by any methods known in
the art. In one method, the non-human CDRs are inserted into a human antibody
or
consensus antibody framework sequence. Further changes can then be introduced
into
the antibody framework to modulate affinity or immunogenicity.
Antibodies of the invention are useful for, for example, therapeutic
purposes (by modulating activity of GPCR polypeptides), diagnostic purposes to
detect or quantitate GPCR polypeptides, as well as purification of GPCR
polypeptides. Kits comprising an antibody of the invention for any of the
purposes
described herein are also comprehended. In general, a kit of the invention
also
includes a control antigen for which the antibody is immunospecific.
Specific binding molecules, including natural ligands and synthetic
compounds, can be identified or developed using isolated or recombinant GPCR
polypeptide products, GPCR polypeptide variants, or preferably, cells
expressing such
products. Binding partners are useful for purifying GPCR polypeptide products
and

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detection or quantification of GPCR polypeptide products in fluid and tissue
samples
using known immunological procedures. Binding molecules are also manifestly
useful in modulating (i.e., blocking, inhibiting or stimulating) biological
activities of
GPCR polypeptides, especially those activities involved 111 signal
transduction.
The DNA and amino acid seduence information provided by the
present invention also makes possible identification of binding partner
compounds
with which a GPCR polypeptide or polynucleotide will interact. Methods to
identify
binding partner compounds include solution assays, in vitro assays wherein
GPCR
polypeptides are immobilized, and cell based assays. Identification of binding
partner
compounds of GPCR polypeptides provides candidates for therapeutic or
prophylactic
intervention in pathologies associated with GPCR polypeptide normal and
aberrant
biological activity.
The invention includes several assay systems for identifying GPCR
polypeptide binding partners. In solution assays, methods of the invention
comprise
1 s the steps of (a) contacting a GPCR polypeptide with one or more candidate
binding
partner compounds and (b) identifying the compounds that bind to the GPCR
polypeptide. Identification of the compounds that bind the GPCR polypeptide
can be
achieved by isolating the GPCR polypeptide/binding partner complex, and
separating
the GPCR polypeptide from the binding partner compound. An additional step of
characterizing the physical, biological, and/or biochemical properties of the
binding
partner compound is also comprehended in another embodiment of the invention.
In
one aspect, the GPCR polypeptide/binding partner complex is isolated using a
antibody immunospecific for either the GPCR polypeptide or the candidate
binding
partner compound.
2s In still other embodiments, either the GPCR polypeptide or the
candidate binding partner compound comprises a label or tag that facilitates
its
isolation, and methods of the invention to identify binding partner compounds
include
a step of isolating the GPCR polypeptide/binding partner complex through
interaction
with the label or tag. An exemplary tag of this type is a poly-histidine
sequence,
generally around six histidine residues, that permits isolation of a compound
so
labeled using nickel chelation. Other labels and tags, such as the FLAG~ tag

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(Eastman Kodak, Rochester, NY), well known and routinely used in the art, are
embraced by the invention.
fn one variation of an in vitro assay, the invention provides a method
comprising the steps of (a) contacting an immobilized GPCR polypeptide with a
candidate binding partner compound and (b) detecting binding of the candidate
compound to GPCR polypeptide. In an alternative embodiment, the candidate
binding partner compound is immobilized and binding of GPCR polypeptide is
detected. Immobilization is accomplished using any of the methods well known
in
the art, including covalent bonding to a support, a bead, or a chromatographic
resin, as
well as non-covalent, high affinity interaction such as antibody binding, or
use of
streptavidin/biotin binding wherein the immobilized compound includes a biotin
moiety. Detection of binding can be accomplished (i) using a radioactive label
on the
compound that is not immobilized, (ii) using a fluorescent label on the non-
immobilized compound, (iii) using an antibody immunospecific for the non-
immobilized compound, (iv) using a label on the non-immobilized compound that
excites a fluorescent support to which the immobilized compound is attached,
as well
as other techniques well known and routinely practiced in the art.
The invention also provides cell-based assays to identify binding
partner compounds of a GPCR polypeptide. In one embodiment, the invention
provides a method comprising the steps of contacting a GPCR polypeptide
expressed
on the surface of a cell with a candidate binding partner compound and
detecting
binding of the candidate binding partner compound to the GPCR polypeptide. In
a
preferred embodiment, the detection comprises detecting a calcium flux or
other
physiological cellular events caused by the binding of the molecule.
Agents that modulate (i.e., increase, decrease, or block) GPCR
polypeptide activity or expression may be identified by incubating a putative
modulator with a cell expressing a GPCR polypeptide or polynucleotide and
determining the effect of the putative modulator on GPCR polypeptide activity
or
expression. The selectivity of a compound that modulates the activity of GPCR
polypeptides can be evaluated by comparing its effects on GPCR polypeptides to
its
effect on other G coupled-protein receptor compounds. Selective modulators may

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include, for example, antibodies and other proteins, peptides, or organic
molecules
which specifically bind to a G coupled-protein receptor polypeptide or a G
coupled-
protein receptor-encoding nucleic acid. Modulators of GPCR polypeptide
activity
will be therapeutically useful in treatment of diseases and physiological
conditions in
which normal or aberrant GPCR polypeptide activity is involved.
Methods of the invention to identify modulators include variations on
any of the methods described above to identify binding partner compounds, the
variations including tecl-miques wherein a binding partner compound has been
identified and the binding assay is carried out in the presence and absence of
a
candidate modulator. A modulator is identified in those instances where
binding
between the GPCR polypeptide and the binding partner compound changes in the
presence of the candidate modulator compared to binding in the absence of the
candidate modulator compound. A modulator that increases binding between the
GPCR polypeptide and the binding partner compound is described as an enhancer
or
activator, and a modulator that decreases binding between the GPCR polypeptide
and
the binding partner compound is described as an inhibitor.
The invention also comprehends high throughput screening (HTS)
assays to identify compounds that interact with or inhibit biological activity
(i.e.,
inhibit enzymatic activity, binding activity, etc.) of a GPCR polypeptide. HTS
assays
permit screening of large numbers of compounds in an efficient manner. Cell-
based
HTS systems are contemplated to investigate GPCR receptor-ligand interaction.
HTS
assays are designed to identify "hits" or "lead compounds" having the desired
property, from which modifications can be designed to improve the desired
property.
Chemical modification of the "hit" or "lead compound" is often based on an
identifiable structure/activity relationship between the "hit" and the GPCR
polypeptide.
Mutations in the GPCR gene that result in loss of nornial function of
the GPCR gene product underlie GPCR polypeptide-related human disease states.
The invention comprehends gene therapy to restore activity to treat those
disease
states. Delivery of a functional GPCR gene to appropriate cells is effected ex
vivo, in
situ, or in vivo by use of vectors, and more particularly viral vectors (e.g.,
adenovirus,

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adeno-associated virus, or a retrovirus), or e_r vivo by use of physical DNA
transfer
methods (e.~;., liposomes or chemical treatments). See, for example, Anderson,
Natr.~re, supplement to vol. 392, no. GG79, pp.25-20 (1998). For additional
reviews of
gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma,
fcientific Americczr~: G8-84 (1990); and Miller, Nature, 357: 455-4G0 (1992).
Alternatively, it is contemplated that in other human disease states,
preventing the
expression of or inhibiting the activity of GPCR polypeptides of the invention
will be
useful in treating the disease states. It is contemplated that antisense
therapy or gene
therapy could be applied to negatively regulate the expression of GPCR
polypeptides
of the invention.
Additional features of the invention will be apparent from the
following Examples.
EXAMPLE 1
Cloning of G Protein-Coupled Receptors
The Incyte and Genbank expressed sequence tag (EST) databases were
searched with the NCBI program Blastall using either the transmembrane VI
region of
known dopamine receptors (leading to the identification of CON193, CON1 GG,
CON103 and CON 203) or all known GPCR's except olfactory and opsin receptors
(leading to the identification of CON198, CON197, CON202, CON222, CON215) as
query sequences, to find patterns suggestive of novel G protein-coupled
receptors.
Positive hits from the find-pattern program were further analyzed with the GCG
program BLAST to determine which ones were the most likely candidates to
encode a
GPCR, using the standard (default) alignment produced by BLAST as a guide.
A. Cloning of CON193 G Protein-Coupled Receptor
A.1. Database Search Results
Searching identified Clone 3091220H1 in the Incyte database as an
interesting candidate sequence. The 3091220H1 Clone was obtained and sequenced
directly using an ABI377 fluorescence-based sequencer (Perkin-Elmer/Applied
Biosystems Division, PE/ABD, Foster City, CA) and the ABI PR1SM''~' Ready

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Dye-Deoxy Tern~inator kit with Taq FSTM polymerise. Each ABI cycle sequencing
reaction contained about 0.5 pg of plasmid DNA. Cycle-sequencing was perfonoed
using an initial denaturation at 98°C for 1 minute, followed by SO
cycles using the
following parameters: 98°C for 30 seconds, annealing at 50°C for
30 seconds, and
S extension at 60°C for 4 minutes. Temperature cycles and times were
controlled by a
Perkin-Elmer 9600 thermocycler. Extension products were purified using
CentriflexT'~ gel filtration cartridges (Advanced Genetic Technologies Corp.,
Gaithersburg, MD). Each reaction product was loaded by pipette onto the
column,
which was then centrifuged in a swinging bucket centrifuge (Sorvall model
RT6000B
tabletop centrifuge) at 1500 x g for 4 minutes at room temperature. Column-
purified
samples were dried under vacuum for about 40 minutes and then dissolved in 5
p1 of a
DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml
Blue Dextrin). The samples were then heated to 90°C for three minutes
and loaded
into the gel sample wells for sequence analysis using the ABI377 sequencer.
Sequence analysis was done by importing ABI377 files into the Sequencer
program
(Gene Codes, Ann Arbor, MI). Generally, sequence reads of 700 by were
obtained.
Potential sequencing errors were minimized by obtaining sequence information
from
both DNA strands and by re-sequencing difficult areas using primers annealing
at
different locations until all sequencing ambiguities were removed.
From the sequence it was deduced that Clone 3091220H1 contained only an
amino-terminal fragment of a putative GPCR corresponding to the third through
the
seventh transmembrane regions (3TM-7TM) of a GPCR. Referring to SEQ ID NO: 1,
the nucleotide sequence of Clone 3091220H1 corresponds to nucleotides 404 to
1308
of what was eventually determined to be the complete sequence of a novel seven-
transmembrane receptor designated CON193. A database search with this partial
sequence showed a 56% match to members of the olfactory receptor gene family,
e.g.,
the gene encoding mouse odorant receptor S 19.
A.2 Screening of a Genomic Phage Library to Obtain a Full-Length GPCR
Clone:
The PCR technique was used to prepare a genomic fragment for use as
a probe specific for the genomic CON193 Clone. Based on the complete sequence
of
Clone 3091220H1, two oligonucleotide primers were designed: Primer LW1282: 5'-

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TAATACCTGCACTGCCCAC-3' (SEQ ID NO: 21; see nucleotides 876-894 of SEQ
ID NO: l ) and Primer LW 1283: 5'-TCTTTCCTTCTC'fTCTCACTCC-3' (SEQ LD
NO: 22 see nucleotides 1 137-1158 of SEQ ID NO: l ). These primers were
designed
to amplify a 283 base-pair fragment of genomic DNA containing a portion of the
CON l93 coding region found in Clone 3091220H 1 (assuming the absence of
introns
in this region).
Initially, a suitable human genomic library constructed in EMBL3
SP6/T7 (Clontech Laboratories) was amplified to provide the materials required
for
screening. Two microliters of the human genomic library (approximately 10$
plaque-
forming units per milliliter; Clontech Laboratories, catalog number HL1067J)
were
added to 6 ml of an overnight culture of K802 cells (Clontech Laboratories),
and 250
p,1 aliquots were distributed into each of 24 tubes. The tubes were incubated
at 37°C
for 15 minutes, and then 7 ml of 0.8% agarose (i.e., top agarose) at
SO°C were added
to each tube. After mixing, the contents of the tubes were poured onto 150 mm
LB
plates and incubated overnight at 37°C to allow clone amplification,
evident as plaque
formation (typically, confluent lysis was observed rather than discrete
plaques). To
each plate, 5 ml of SM phage buffer (0.1 M NaCI, 8.1 ~,M MgS04~7H20, 50 mM
Tris-HC1 (pH 7.5), and 0.0001 % gelatin) was added and the top agarose was
removed
by scraping with a microscope slide. Top agarose slurnes containing phage were
then
placed in individual 50 ml centrifuge tubes. A drop of chloroform was added
and
each tube was placed in a 37°C shaker for 15 minutes, followed by
centrifuging at
2,750 x g for 15 minutes. The supernatants were isolated and separately stored
at 4°C
as 24 stock solutions of amplified library clones.
As noted above, polymerase chain reaction (PCR) was selected as a
technique for screening the phage library. Each PCR reaction was done in a 20
p.1
reaction volume containing 8.84 ~,l HZO, 2 p,1 lOX PCR buffer II (Perkin-
Elmer), 2 ~,l
25 mM MgClz, 0.8 p1 dNTP mixture (dATP, dCTP, dGTP, dCTP, each at 10 mM),
0.12 p,1 primer LW1282 (approximately 1 p,g/pl), 0.12 p.1 primer LW1283
(approximately 1 pg/pl), 0.12 p,1 AmpliTaq Gold polymerase (5 Units/~,1, with
"Units"
as defined by the supplier, Perkin-Elmer) and 2 p,1 of phage from one of the
24 stock
tubes. The PCR reaction involved 1 cycle at 95°C for 10 minutes and
80°C for 20

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minutes, followed by 22 cycles at 95°C for 30 seconds, 72-S l °C
for 2 minutes (72°C
for this stage of the second cycle, with a decrease of one degree for this
stage in each
succeeding cycle), 72°C for one minute, followed by 30 cycles at
95°C for 15
seconds, 50°C for 30 seconds, and 72°C for one minute.
Following PCR cycling, the contents from each reaction tube were
loaded onto a 2% agarose gel and electrophoresed adjacent to known size
standards to
screen for PCR products of the expected size, indicative of a clone containing
the 283
by portion of Clone 3091220H 1 amplified by the two selected primers. A
positive
signal (i.e., a fragment of the expected size) was found in one of the 24 PCR
reactions, thereby identifying a single stock genomic library tube containing
positive
clones.
From the original genomic library tube that had given a PCR product
of the correct size, a 5 p,1 phage aliquot was used to establish a set of five
serial
dilutions (1/100, v/v) that were plated and incubated in the same manner as
described
for the amplification of the phage library. Following incubation, BA85
nitrocellulose
filters (Schleicher & Schuell) were placed on top of each of the plates for 1
hour to
adsorb phage from the plaques that had formed in the top agarose during
incubation.
Each filter was then gently removed, placed phage side up in an individual
petri dish,
and covered with 4 ml of SM buffer for 15 minutes to elute the phage. One
milliliter
of SM containing eluted phage was removed from each plate and used to set up a
PCR
reaction as described above. The plate containing the most dilute phage
solution to
yield a PCR product of the expected size was then subdivided using the
following
procedure. A BA85 filter was placed on the top agar of the plate and the
medium
with applied filter was physically divided into 24 sections. After one hour to
allow
phage adsorption to the 24 filters, each filter was removed and separately
incubated in
1 ml of SM buffer at room temperature for 15 minutes. Two microliters of each
eluted phage solution were then used as a PCR substrate. Those plate sections
yielding positive PCR results were then subdivided into 12 subsections by
removing
the top agar and incubating it in 200 p.1 of SM buffer for one hour at room
temperature. Again, 2 p,1 of the eluted phage solutions were plated and lifted
using
BA85 filters, and PCR reactions were repeated. The procedure for progressive

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dilution o.f phage was continued Lllltll a single plaque was isolated.
Subsequently, 10
p,1 of eluted phage from that single plaque were added to 100 p.1 SM and 200
p1 of
K802 cells for plating in a single petri dish as described above. A total of 7
plates
were inoculated in this manner. Following incubation at 37°C for 16
hours, the top
S agarose from each of the 7 plates was removed to recover the phage, which
were used
to prepare purified genomic phage DNA using the Qiagen Lambda Midi Kit.
The purified CON193 genomic phage DNA was sequenced using the
ABI PRISM'"'' 310 Genetic Analyzer (Perkin-Elmer/Applied Biosystems) which
uses
advanced capillary electrophoresis technology and the ABI PRISM.r"~ BigDyel"''
Terminator Cycle Sequencing Ready Reaction Kit. The cycle-sequencing reaction
contained 18 w1 of HZO, 16 p.1 of BigDye'~"' Terminator mix, 3 p.1 of genomic
phage
DNA (0.26 wg/p.l), and 3 p1 primer (25 ng/pl). The reaction was performed in a
Perkin-Elmer 9600 thermocycler at 95°C for 5 minutes, followed by 75
cycles of
95°C for 30 seconds, 55°C for 20 seconds, and 60°C for 4
minutes. The final
subclone was also sequenced using the ABI PRISM'"' 310 Genetic Analyzer. The
cycle-sequencing reaction contained 6 p,1 of HZO, 8 p1 of BigDye'"''
Terminator mix, 5
p,1 of miniprep clone DNA (0.1 p,g/p,l), and 1 p.1 primer (25 ng/p.l). The
reaction was
performed in a Perkin-Elmer 9600 thermocycler at 25 cycles of 96°C for
10 seconds,
50°C for 10 seconds, and 60°C for 4 minutes. The product of the
PCR reaction was
purified using CentriflexT"'' gel filtration cartridges, dried under vacuum,
and
dissolved in 16 p,1 of Template Suppression Reagent (PE-Applied Biosystems).
The
samples were then incubated at 95°C for 5 minutes and placed in the 310
Genetic
Analyzer. These efforts resulted in the determination of the CON193
polynucleotide
sequence set forth in SEQ ID NO:1 and the deduced amino acid sequence of the
encoded CON193 polypeptide which is set forth in SEQ ~ N0:2.
A.3 Subcloning of the Coding Region of CON193 via PCR
Additional experiments were conducted to subclone the coding region
of CON193 and place the isolated coding region into a useful vector. Two
additional
PCR primers were designed based on the coding region of CON 193. The first PCR
primer, designated Primer LW 1373, has the sequence 5'-GCATAAGCTTATGCTA-
ACACTGAATAAAACAG-3' (SEQ ID NO: 23), nucleotides 11-32 of which

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correspond to nucleotides 157-178 of SEQ lD NO: 1. The second PCR primer is
Primer LW 1374, which has the sequence 5'-GC'.ATCTCGAGTCACA-
TGCTGTAGGATTTGG-3' (SEQ ID NO: 24, nucleotides 1 1-30 of which correspond
to the complement of nucleotides 1 102-1 121 of SEQ ID NO: 1. To protect
against
exonucleolytic attack during subsequent exposure to enzymes, e.g., Taq
polymerise,
primers were routinely synthesized with a protective run of nucleotides at the
S' end
that were not necessarily complementary to the desired target.
PCR was performed in a 50 p.1 reaction containing 35 p1 HZO, 5 p,1 lOX
TT buffer (140 mM ammonium sulfate, 0.1% gelatin, 0.6 M Tris-tricine, pH 8.4),
5 ~,l
15 mM MgS04, 2 p1 dNTP mixture (dGTP, dATP, dTTP, and dCTP, each at 10 mM),
2 p,1 genomic phage DNA (0.26 p,g/p,l), 0.3 p.1 Primer LW1373 (1 p,g/pl), 0.3
p,1
Primer LW1374 (1 ~,g/p,l), 0.4 p,1 High Fidelity Taq polymerise (Boehringer
Mannheim). The PCR reaction was started with 1 cycle of 94°C for 2
minutes;
followed by 15 cycles at 94°C for 30 seconds, 55°C for 30
seconds, and 72°C for 1.3
1 S minutes.
The contents from the PCR reaction were loaded onto a 2% agarose
gel, fractionated and electroeluted. The DNA band of expected size was excised
from
the gel,. placed in a GenElute Agarose spin column (Supelco) and spun for 10
minutes
at maximum speed in a microcentrifuge. The eluted DNA was precipitated with
ethanol and resuspended in 6 p.1 HZO for ligation.
The PCR-amplified DNA fragment containing the CON193 coding
region was cloned into pCR2.l using a protocol standard in the art. In
particular, the
ligation reaction consisted of 6 ~,l of CON193 DNA, 1 ~,l lOX ligation buffer,
2 p,1
pCR2.1 (25 ng/p,l, Invitrogen), and 1 p,1 T4 DNA ligase (Invitrogen). The
reaction
mixture was incubated overnight at 14°C and the reaction was then
stopped by heating
at 65°C for 10 minutes. Two microliters of the ligation reaction were
transformed
into One Shot cells (lnvitrogen) and plated onto ampicillin plates. A single
colony
containing an insert was used to inoculate a 5 ml culture of LB medium. The
culture
was grown for 18 hours and the plasmid DNA was purified using the Concert
Rapid
Plasmid Miniprep System (GibcoBRL) and sequenced. Following confirmation of
the
sequence, pCR-CON193 was identiFed, and a 50 ml culture of LB medium was

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inoculated and recombinant plasmid DNA was purified LISIIIg d Qiagen Plasmid
Midi
Kit to yield purified pCR-CON193.
B. Cloning of CON166 G Protein-Coupled Receptor
S B.I Database Search Results
The database searching identified clone 2553280H 1 in the lncyte
database as an interesting candidate sequence. The 2553280H 1 clone was
obtained
and sequenced directly using an ABI377 fluorescence-based sequencer and the
ABI
PR1SM~' ~' Ready Dye-Deoxy Terminator kit with Taq FSTM polymerase as
described
above for CON 193 in Example 1A.1. From the sequence it was deduced that clone
2553280H1 contained 349 nucleotides of a GPCR coding region comprising a
carboxy-terminal fragment of a putative GPCR corresponding to the sixth and
seventh
transmembrane regions (6TM and 7TM). In addition, clone 2553280H1 contained
1.2 kb of the 3' untranslated sequence of that GPCR. Referring to SEQ 11.7 NO:
3, the
nucleotide sequence of Clone 2553280H1 corresponds to nucleotides 663 to 1,014
of
what was eventually determined to be the complete sequence of a novel seven-
transmembrane receptor that was designated CON166. A database search with this
partial sequence showed a 44% match to an activated T cell-specific G protein-
coupled receptor.
B2. Screening of a Genomic Phage Library to Obtain a
Full-Length GPCR Clone
The PCR technique was used to prepare a genomic fragment for use as
a probe specific for the genomic CON166 clone. Based on the complete sequence
of
clone 2553280H1, two oligonucleotide primers were designed: Primer LW1278: 5'-
ACCGCTGCCTTTTTAGTC-3' (SEQ ID NO: 28; see nucleotides 715 to 732 of SEQ
ID NO: 3 and Primer LW 1279: 5'-CCTTCTTTCTGGGTACATAAGTC-3' (SEQ >D
NO: 29; see the reverse complement of nucleotides 951-973 of SEQ ID NO: 3).
These primers were designed to amplify a 259 base-pair fragment of genomic DNA
containing a portion of the CON166 coding region found in clone 2553280H1
(assuming the absence of introns in this region).
Initially, a suitable human genomic library constructed in EMBL
SP6/T7 was amplified to provide the materials required for screening as
described

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above for CON193 in Example IA.2. Polymerise chain reaction (PCR) was selected
as a technique for screening the phage library. Each PCR reaction was clone in
a 20 p.1
reaction volume containing 8.84 p1 HzO, 2 p1 lOX PCR buffer II (Perkin-Elmer),
2 p1
25 mM MgCI,, 0.8 p,1 dNTP mixture (dATP, dCTP, dGTP, dCTP, each at 10 mM),
0.12 p1 primer LW 1278 (approximately 1 p,g/p.l), 0.12 p,1 primer LW 1279
(approximately I pg/p.l), 0.12 ~,1 AmpliTaq Gold polymerise (5 Units/~.1, with
"Units"
as defined by the supplier, Perkin-Elmer) and 2 p.1 of phage from one of the
24 stock
tubes. The PCR reaction involved 1 cycle at 95°C for 10 minutes and
80°C for 20
minutes, followed by 12 cycles at 95°C for 30 seconds, 72-61 °C
for 2 minutes (72°C
for this stage of the second cycle, with a decrease of one degree for this
stage in each
succeeding cycle), 72°C for 30 seconds, followed by 30 cycles at
95°C for IS seconds,
60°C for 30 seconds, and 72°C for 30 seconds.
Following PCR cycling, the contents from each reaction tube were
loaded onto a 2% agarose gel and electrophoresed adjacent to known size
standards to
screen for PCR products of the expected size of 259 bp, indicative of a clone
containing the portion of clone 2553280H1 amplified by the two selected
primers. A
positive signal (i.e., a fragment of the expected size) was found in one of
the 24 PCR
reactions, thereby identifying a single stock genomic library tube containing
positive
clones.
From the original genomic library tube that had given a PCR product
of the correct size, a 5 p,1 phage aliquot was used to amplify the CON166
genomic
phage DNA as described for CON 193 above in Example 1 A.2. For the
amplification
of the phage library, the plates containing the diluted phage solution were
subdivided
into 12 sections unlike that of CON193; otherwise the procedures were
identical.
The purified CON166 genomic phage DNA was sequenced using the
ABI PRISM'°'' 310 Genetic Analyzer which uses advanced capillary
electrophoresis
technology and the ABI PRISMTM BigDyeTM Terminator Cycle Sequencing Ready
Reaction Kit as described above for CON193 in Example 1 A.2. These efforts
resulted in the determination of the CON166 polynucleotide sequence set forth
in
SEQ ID NO: 3 and the deduced amino acid sequence of the encoded CON 166
polypeptide which is set forth in SEQ ID NO: 4.

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B.3 Subcloning of the Coding Region of CON166 via PCR
Additional experiments were conducted to subclone the coding region
of CON166 from the genomic clone and place the isolated coding region into a
useful
vector. Two additional PCR primers were designed based on the coding region of
CON 166. The first PCR primer, designated Primer LW 1405, has the sequence
5'-AAGCATAACATGGATGAAACAGGAAATCTG-3' (SEQ ID NO: 29,
nucleotides 10-30 of which correspond to nucleotides 1-21 of SEQ ID NO: 3). To
protect against exonucleolytic attack during subsequent exposure to enzymes,
e.g.,
Taq polymerase, primers were routinely synthesized with a protective run of
nucleotides at the 5' end that were not necessarily complementary to the
desired
target. The second PCR primer is Primer LW1406, which has the sequence 5'
AAGCATAACTATACTTTACATATTTCTTC-3' (SEQ ID NO: 30, nucleotides 9-29
of which correspond to the reverse complement of nucleotides 994-1014 of SEQ
ID
NO: 3).
PCR was performed in a 50 p1 reaction containing 34 p,1 H20, S p1 lOX
TT buffer (140 mM ammonium sulfate, 0.1% gelatin, 0.6 M Tris-tricine, pH 8.4),
5 p,1
15 mM MgS04, 2 p,1 dNTP mixture (dGTP, dATP, dTTP, and dCTP, each at 10 mM),
3 w1 genomic phage DNA (0.25 p,g/p,l), 0.3 p,1 Primer LW1405 (1 p,g/p,l), 0.3
p.1
Primer LW 1406 (1 p.g/p,l), 0.4 p.1 High Fidelity Taq polymerase (Boehringer
Mannheim). The PCR reaction was started with 1 cycle of 94°C for 2
minutes;
followed by 25 cycles at 94°C for 30 seconds, 55°C for 30
seconds, and 72°C for 1.3
minutes.
The contents from the PCR reaction were loaded onto a 2% agarose gel
and fractionated. The DNA band of expected size (1,031 bp) was excised from
the
gel, placed in a GenElute Agarose spin column (Supelco) and spun for 10
minutes at
maximum speed in a microfuge. The eluted DNA was precipitated with ethanol and
resuspended in 6 p1 Hz0 for ligation.
The PCR-amplified DNA fragment containing the CON 166 coding
region was cloned into pCR2.1 to generate pCR-CON166 using a protocol standard
in
the art. In particular, the ligation reaction was carried out as described for
CON193 in
Example 1 A.3. The resulting plasmid DNA was purif ed using the Concert Rapid

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Plasmid Miniprep System (GibcoBRL) and seduenced. Following confirmation of
the
sequence, a 50 ml culture of LB medium was inoculated with the transformed One
Shot cells, cultured, and processed using a Qiagen Plasmid Midi Kit to yield
purified
pCR-CON 166.
C. Cloning of CON103 G Protein-Coupled Receptor
C.l Database Search Results
The database searching identified clone 1581220H1 in the Incyte
database as an interesting candidate sequence. The 1581220H1 clone was
obtained
and sequenced directly using an ABI377 fluorescence-based sequencer and the
ABI
PRISM'''' Ready Dye-Deoxy Terminator kit with Taq FSTM polymerase as described
for CON 193 in Example IA. I. From the sequence it was deduced that clone
1581220H1 contained 454 nucleotides of a GPCR coding region comprising a
carboxy-terminal fragment of a putative GPCR corresponding to the sixth and
seventh
transmembrane regions (6TM and 7TM). In addition, clone 1581220H1 contained
1.2 kb of the 3' untranslated sequence of that GPCR. Referring to SEQ >D NO:
5, the
nucleotide sequence of clone 1581220H1 corresponds to nucleotides 698 to 1190
of
what was eventually determined to be the complete sequence of a novel seven-
transmembrane receptor designated CON103. A database search with this partial
sequence showed a 44% match to an activated T cell-specific G protein-coupled
receptor.
C.2 Screening of a Genomic Phage Library
to Obtain a Full-Length GPCR Clone
The PCR technique was used to prepare a genomic fragment for use as
a probe specific for the genomic CON103 clone. Based on the complete sequence
of
clone 1581220H1, two oligonucleotide primers were designed: Primer LW1280: 5'-
TCTGCACACAGCTCTTCCATGG-3' (SEQ ID NO: 32; see nucleotides 1568-1589
of SEQ >D NO: 5) and Primer LW 1281: 5'-TCCCTTGTCCAGTTGGTTGAGG-3'
(SEQ ID NO: 33; see nucleotides 1926 to 1947 of SEQ ID NO: 5. These primers
were designed to amplify a 380 base-pair fragment of genomic DNA containing a
portion of the CON103 coding region found in clone 1581220H1 (assuming the
absence of introns in this region).

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Initially, a suitable human genomic library constructed in EMBL
SP6/T7 was amplified to provide the materials required for screening as
described
above for CON193 in Example 1A.2. Polymerase chain reaction (PCR) was selected
as a technique for screening the phage library. Each PCR reaction was done in
a 20 p,1
reaction volume containing 8.84 p,1 HZO, 2 p1 l OX PCR buffer II (Perkin-
Elmer), 2 p,1
25 mM MgCl2, 0.8 p,1 dNTP mixture (dATP, dTTP, dGTP, dCTP, each at 10 mM),
0.12 p,1 primer LW 1280 (approximately 1 p,g/p.l), 0.12 ~.l primer LW 1281
(approximately 1 pg/pl), 0.12 p.1 AmpliTaq Gold polymerase (5 Units/p,l, with
"Units"
as defined by the supplier, Perkin-Elmer) and 2 p,1 of phage from one of the
24 stock
tubes. PCR amplification reactions using each one of the other 23 stock
collections of
genomic clones were performed under the same conditions. The PCR reaction
involved 1 cycle at 95°C for 10 minutes and 80°C for 20 minutes,
followed by 12
cycles at 95°C for 30 seconds, 72-61°C for 2 minutes
(72°C for this stage of the
second cycle, with a decrease of one degree for this stage in each succeeding
cycle),
72°C for one minute, followed by 30 cycles at 95°C for 1 S
seconds, 60°C for 30
seconds, and 72°C for 30 seconds.
Following PCR cycling, the contents from each reaction tube were
loaded onto a 2% agarose gel and electrophoresed adjacent io known size
standards to
screen for PCR products of the expected size of 380 bp, indicative of a clone
containing the portion of clone 1581220H1 amplified by the two selected
primers. A
positive signal (i.e., a fragment of the expected size) was found in one of
the 24 PCR
reactions, thereby identifying a single stock genomic library tube containing
positive
clones.
From the original genomic library tube that had given a PCR product
of the correct size, a 5 p,1 phage aliquot was used to amplify the CON 103
genomic
phage DNA as described above for CON 193 in Example 1 A.2. A total of 8 plates
were inoculated with eluted phage in this manner described above. Following
incubation at 37°C for 16 hours, the top agarose from each of the 8
plates was
removed to recover the phage, which were used to prepare purified genomic
phage
DNA using the Qiagen Lambda Midi Kit.

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The CON 103 clone was sequenced using the ABI PRISM~~'''' 3 ( 0
Genetic Analyzer. The cycle-sequencing reaction contained 6 ~.l ol~ H,O, 8 ~.1
of
BigDye~'''~ Terminator mix, 5 p.1 of miniprep clone .DNA (0.1 ~,g/p l), and 1
~,I primer
(25 ng/p.l). The reaction was performed in a Perkin-Elmer 9600 thennocycler at
25
cycles of 96°C for 10 seconds, SO°C for 10 seconds, and
60°C for 4 minutes. The
product of the PCR reaction was purified using CentriflexT~' gel filtration
cartridges,
dried under vacuum, and dissolved in 16 ~,1 of Template Suppression Reagent
(.PE-
Applied Biosystems). The samples were then incubated at 95°C for 5
minutes and
placed in the 310 Genetic Analyzer. These efforts resulted in the
determination of the
CON103 polynucleotide sequence set forth in SEQ ~ NO: 5 and the deduced amino
acid sequence of the encoded CON103 polypeptide which is set forth in SEQ ID
NO:
6.
C.3 Subcloning of the Coding Region of CON103 via PCR
Additional experiments were conducted to subclone the coding region
of CON 103 from the genomic clone and place the isolated coding region into a
useful
vector. Two additional PCR primers were designed based on the sequence of the
coding region of CON103: Primer LW1385 (5'-GCATAAGCT-
TCCATGGAACTTCATAACCTG-3'; SEQ m NO: 34, nucleotides 13-30 of which
correspond to nucleotides 1-18 of SEQ ~ NO: 5) and Primer LW 1386 (5'-
GCATCTCGAGTTACCCCCACAGCGCTGCAG-3'; SEQ ID NO: 35, nucleotides
11-30 of which correspond to the reverse complement of nucleotides 1171-1190
of
SEQ ID NO: 5). To protect against exonucleolytic attack during subsequent
exposure
to enzymes, e.g., Taq polymerise, primers were routinely synthesized with a
protective run of nucleotides at the 5' end that were not necessarily
complementary to
the desired target.
PCR was performed in a 50 Pl reaction containing 22.6 p1 H20, 5 p,1
lOX TT buffer (140 mM ammonium sulfate, 0.1% gelatin, 0.6 M Tris-tricine, pH
8.4),
5 p1 15 mM MgS04, 10 p.1 rapid dye (Origene), 2 p.1 dNTP mixture (dGTP, dATP,
dTTP, and dCTP, each at 10 mM), 0.5 p,1 genomic phage DNA (0.97 p,g/p,l), 0.3
p1
Primer LW1385 (1 pg/p.l), 0.3 p.1 Primer LW1386 (1 pg/p,l), and 0.4 p1 High
Fidelity
Taq polymerise (Boehringer Mannheim). The PCR reaction was started with 1
cycle

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of 94°C for 2 minutes, followed by 12 cycles at 94°C for 30
seconds, SS°C for 30
seconds, and 72°C. for 1.3 minutes.
The contents ti-om the PCR reaction were loaded onto a 2% agarose gel
and fractionated. The DNA band of expected size (1,212 bp) was excised from
the
gel, placed in a GenElutc Agarose spin column (Supelco) and spun for 10
minutes at
maximum speed in a microcentrifuge. The eluted DNA was precipitated with
ethanol
and resuspended in 6 p.1 HZO for ligation.
The PCR-amplified DNA fragment containing the CON 103 coding
region was cloned into pCR2.1 using a protocol standard in the art. In
particular, the
ligation reaction was carried out as described above for CON193 in Example 1
A.3.
The resulting plasmid DNA was purified using the Concert Rapid Plasmid
Miniprep
System (GibcoBRL) and sequenced. Following confirmation of the sequence, pCR-
CON103 was identified, and a SO ml culture of LB medium was inoculated,
cultured,
and processed using a Qiagen Plasmid Midi Kit to yield purified pCR-CON103.
1S .
D. Cloning of CON203 G Protein-Coupled Receptor
D.1 Database Search Results
The database searching identified clone 3210396H 1 in the Incyte
database as an interesting candidate sequence. The 3210396H1 clone was
obtained
and sequenced directly using an ABI377 fluorescence-based sequences and the
ABI
PR1SM'~M Ready Dye-Deoxy Terminator kit with Taq FSTM polymerise as described
above for CON 193 in Example 1A.1. From the sequence it was deduced that clone
3210396H1 contained all 1,002 nucleotides of a GPCR coding region (see SEQ ID
NO: 7). A database search with this sequence showed a 33% match to a platelet
2S activating receptor (Gene H963, GenBank Acc. No. AF002986).
D.2 Subcloning of the Coding Region of CON203 via PCR
Additional experiments were conducted to subclone the coding region
of CON203 and place the isolated coding region into a useful vector. Two
additional
PCR primers were designed based on the sequence of the coding region of
CON203:
Primer LW 1329: S'-GCATCTCGAGTCAGCCTAAGGTTATGTTG-3' (SEQ ID
NO: 36; see nucleotides 984 to 1,002 of SEQ ID NO: 7 for the reverse
complement of

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nucleotides 9-29 of SEQ ID NO: 36) and Primer LW 1377: 5'-
GCATAAGCTTATGAACACCACAGTGAffGC-3' (SEQ TD NO: 37; see
nucleotides 1-19 of SEQ 1D NO: 7 which correspond to nucleotides 11-29 of SEQ
ID
NO: 37). To protect against exonucleolytic attack during subsequent exposure
to
enzymes, e.g., Taq polymerise, primers were routinely synthesized with a
protective
run of nucleotides at the 5' end that were not necessarily complementary to
the
desired target. These primers were designed to amplify a 1,020 base-pair
fragment of
clone 3210396H1 containing the complete coding region of CON203.
PCR was performed in a 50 p,1 reaction containing 34 p1 H20, 5 P.1 l OX
TT buffer (140 mM ammonium sulfate, 0.1% gelatin, 0.6 M Tris-tricine, pH 8.4),
5 P,l
mM MgS04, 2 p,1 dNTP mixture (dGTP, dATP, dTTP, and dCTP, each at 10 mM),
3 P,1 clone 3210396H1 (miniprep DNA), 0.3 p1 Primer LW1329 (1 p,g/pl), 0.3 P1
Primer LW1377 (1 p.g/P,1), and 0.4 ~l High Fidelity Taq polymerise (Boehringer
Mannheim). The PCR reaction was started with 1 cycle of 94°C for 2
minutes,
15 followed by 12 cycles at 94°C for 30 seconds, 55°C for 30
seconds, and 72°C for 1.3
minutes.
The contents from the PCR reaction were loaded onto a 1.2% agarose
gel and fractionated. The DNA band of expected size (1,020 bp) was excised
from
the gel, placed in a GenElute Agarose spin column (Supelco) and spun for 10
minutes
at maximum speed in a microcentrifuge. The eluted DNA was precipitated with
ethanol and resuspended in 6 P,1 Hz0 for ligation.
The PCR-amplified DNA fragment containing the CON203 coding
region was cloned into pCR2.1 using a standard protocol and the Original TA
Cloning
Kit (Invitrogen). Ligation reactions were carried out as described above for
CON 193
in Example 1A.3. The resulting plasmid DNA was purified using the Concert
Rapid
Plasmid Miniprep System (GibcoBRL) and sequenced. Following confirmation of
the
sequence, pCR-C203 was identified, and a 50 ml culture of LB medium was
inoculated, cultured, and processed using a Qiagen Plasmid Midi Kit to yield
purified
pCR-C203.
The CON203 clone was sequenced using the ABI PRISM'M 310
Genetic Analyzer (P-E Applied Biosystems), which uses advanced capillary

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electrophoresis technology and the ABI PI'ISIII~~'~~~ BigDye~~'~'~ Terminator
Cycle
Sequencing Ready Reaction Kit. The cycle-sequencing reaction contained 6 p1 of
IIzO, 8 p1 of BigDye'~M Terminator mix, 5 p1 of miniprep clone DNA (0.1
pg/pl), and
1 p,1 primer (25 ng/p.l). The reaction was performed in a Perkin-Elmer 9600
thennocycler using the following conditions: 25 cycles of 96°C for 10
seconds, SO°C
for 10 seconds, and 60°C for 4 minutes. The product of the PCR reaction
was
purified using CentriflexTM gel filtration cartridges, dried under vacuum, and
dissolved in 16 p,1 of Template Suppression Reagent (PE-Applied Biosystems).
The
samples were then incubated at 95°C for 5 minutes and placed in the 310
Genetic
Analyzer.
Initially, these efforts showed that the CON203 coding region cloned
into pCR2.1 had a single by difference from the corresponding sequence of
clone
3210396H1. The single by change in the pCR2.1 clone was eliminated by
conforming that sequence to the sequence of clone 3210396H1 using the
QuikChange
Site-Directed Mutagenesis Kit (Stratagene). The method involves modification
of a
sequence during PCR amplification, for which PCR primers LW 1387 (S'-
GAGAAATATTTTTCTAAAAAAACCTGTTTTTGCAAAAACGG-3'; SEQ ID
NO: 38) and LW1388 (5'-CCGTTTTTGCAAAAACAGGTTTTTTTAGAAAA-
ATATTTCTC-3'; SEQ ID NO: 39) were used. The PCR reaction contained 40 p,1
HZO, 5 p.1 lOX proprietary Reaction Buffer (Stratagene), 1 p.1 pCR-0203 (0.125
p,g/p,l)
mini-prep DNA, 1 p.1 dNTP mixture (dGTP, dATP, dTTP, and dCTP, each at 10
mM), 1 p,1 Pfu DNA polymerase (2.5 Units/p,l), 1 p,1 LW1387 (125 ng/p,l) and 1
p,1
LW1388 (125 ng/ 1). The cycle conditions were 95°C for 30 seconds,
followed by 12
cycles at 95°C for 30 seconds, 55°C for 1 minute, and
68°C for 12 minutes. The tube
was then placed on ice for 2 minutes and 1 p,1 of Dpnl was added. The tube was
then
incubated at 37°C for one hour. One microliter of the DpnI-treated DNA
was
transformed into Epicurian coli XL1-Blue supercompetent E. coli cells.
Following
isolation of pCR-C203, the entire insert was re-sequenced, thereby
successfully
verifying repair of the single-site polymorphism. As expected, the sequence of
the
CON203 coding region determined using this pCR2.1 clone is in complete
agreement

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with the CON203 coding region sequence of SEQ ID NO: 7 which specifies the
amino acid sequence set forth in SEQ CD NO: 8.
E. Cloning of CON198 G Protein-Coupled Receptor
E.1 Database Search Results
The database searching identified Clone 3359808HI in the Incyte
database as an interesting candidate sequence. The 3359808HI clone was
obtained
and sequenced using standard techniques. From the sequence it was deduced that
Clone 3359808HI contained the entire coding region for a previously
unidentified
GPCR, which was designated "CON198." The DNA and deduced amino acid
sequences for CON198 are set forth in SEQ ID NOS: 9 and 10, respectively. A
database search with this CON198 DNA sequence showed a 61% match to the rat
putative GPCR designated RAIc [Raming et. al., Recept Channels, 6: 141-151
(1998)] and 46% identity to an olfactory receptor.
E.2 Subcloning of the Coding Region of CON198 via PCR
Additional experiments were conducted to subclone the coding region
of the CON198 clone into a useful vector. Two PCR primers were designed based
on
the coding region of CON198 for the purpose of PCR amplification of the CON198
coding sequence. The first, Primer LW 1326, from 5' to 3' (SEQ 1D NO: 42):
GCATGAATTCATGATGGTGGATCCCAATGG, includes the 5' end of the
CON198 coding sequence (underlined) as well as a EcoRI restriction site,
useful for
subsequent expression work. The second, Primer LW 1327, from 5' to 3' (SEQ ID
NO: 43): GCATCTCGAGCCTAGGGCTCTGAAGCG, includes sequence
complementary to the 3' end of the CON198 coding sequence (underlined),
preceded
by a XhoI restriction site sequence useful for subsequent cloning and
expression work.
The PCR was performed in a 50 ~.l reaction containing 34 p,1 HzO, 5 p,1
of lOX TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris-tricine,
pH
8.4), 5 p,1 of 15 mM MgS04, 2 p.1 of I 0 mM dNTPs (dATP, dCTP, dTTP, dGTP), 2
p,1
of Clone 3359808H1 mini-prep DNA (approx. 0.125 p.g/p,l), 0.3 ~.1 of Primer
LW1326 (1 ~g/pl), 0.3 p.1 of Primer LW1327 (1 p,g/p.l), and 0.5 p1 of High
Fidelity
Taq polymerase (Boehringer Mannheim). The PCR reaction was started with 1
cycle

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of 94°C for 2 minutes; followed by l2 cycles at 94°C for 30
seconds. 55°C for 30
seconds, and 72°C for l minute.
The contents from the PCR reaction were loaded onto a 1.2'% agarose
gel and electrophoresed. The DNA band of expected size was excised from the
gel,
placed in a GenElute Agarose spin column (Supelco) and spun for 10 minutes at
maximum speed in a microcentrifuge. The eluted DNA was ethanol-precipitated
and
resuspended in 6 p.1 HZO for ligation.
The purified PCR fragment containing the CON I 98 coding sequence
was ligated into a commercial vector using Invitrogen's Original TA Cloning
Kit. The
ligation reaction was carried out as described above for CON 193 in Example 1
A.3.
The resulting plasmid DNA was isolated using a Concert Rapid Plasmid Miniprep
System (GibcoBRL) and sequenced to confirm that the plasmid contained the
CON 198 insert. Sequencing of the subcloned CON 198 construct revealed that
the
PCR amplification had introduced a mutation (relative to the sequence of the
original
clone) at the nucleotide corresponding to position 204 of SEQ 1D NO: 9. A
site-directed mutagenesis experiment was performed using the QuikChange
Site-Directed Mutagenesis Kit (Stratagene) to repair the mutation.
Two primers were designed to revert the mutated A nucleotide at
position 204 back to a G nucleotide via polymerase chain reaction. Primer LW
1415
(SEQ ID NO: 44) contained the sequence:
5'-CCATGTATATATTTCTTTGCATGCTTTCAGGCATTGACATCC-3'; and
primer LW 1416 (SEQ ID NO: 45) contained the sequence:
5'-GGATGTCAATGCCTGAAAGCATGCAAAGAAATATATACATGG-3'. The
PCR reaction contained 40 p1 of HZO, 5 p,1 of l Ox Reaction buffer, 1 p1 of
mini-prep
DNA (approx. 0.125 p,g/pl) from the CON198-pCR2.1 clone (as template), 1 p,1
of
primer LW1415 (125 ng/p,l), 1 p1 of primer LW1416 (125 ng/p,l), 1 p1 of 10 mM
dNTPs, 1 p1 Pfu DNA polymerase. The PCR cycle conditions were as follows:
initial
denaturation at 95°C for 30 seconds, then 14 cycles at 95°C for
30 seconds, 55°C
annealing for 1 minute, and 68°C extension for 12 minutes. Thereafter,
the reaction
tube was placed on ice for 2 minutes.

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After PCR, I p1 of DpO was added and the tube incubated at 37°C
for
one hour to digest the methylatcd parental DNA template. One microliter of the
Dpnl-treated DNA was transformed into Epicurian coli XLl-Blue supercompetent
cells and the entire insert was re-sequenced. The resequencing confirmed that
position 204 of SEQ ID NO: 9 had been successfully reverted to a guanine
nucleotide.
Upon confirmation of the insert, the E. coli transformant was used to
inoculate a 50 ml culture of LB medium. The culture was grown for 16 hours at
37°C, and centrifuged into a cell pellet. Plasmid DNA was purified from
the pellet
using a Qiagen Plasmid Midi Kit and again sequenced to confirm successful
cloning
of the CONl 98 insert, using an ABI377 fluorescence-based sequencer and the
ABI
PRISMT"'' Ready Dye-Deoxy Terminator kit with Taq FS'~'~' polymerase as
described
abvoe for CON 193 in Example 1 A.1.
F. Cloning of CON197 G Protein-Coupled Receptor
. F.1 Database Search Results
The database searching identified Clone 866390H1 in the Incyte
database as an interesting candidate sequence. The 866390H1 clone was obtained
and
sequenced using standard techniques. From the sequence it was deduced that
Clone
866390H1 contained the entire coding region for a previously unidentified
GPCR,
which was designated "CON197." The DNA and deduced amino acid sequences for
CON197 are set forth in SEQ ID NOs: 11 and 12, respectively. A database search
with this CON197 DNA sequence showed a 42% match to an olfactory receptor.
F.2 Subcloning of the Coding Region of CON197 via PCR
Additional experiments were conducted to subclone the coding region
of the CON197 clone into a useful vector. Two PCR primers were designed based
on
the coding region of CON197 for the purpose of PCR amplification of the CON197
coding sequence. The first, Primer LW 1324, from S' to 3' (SEQ TD NO: 48):
GATCGGATCCATGGAAAGCGAGAACAG, includes the 5' end of the CON197
coding sequence (underlined) as well as a BamHI restriction site, useful for
subsequent expression work. The second, Primer LW1325, from S' to 3' (SEQ B7
NO: 49): GATCCTCGAGTCAGGCTATGTGCTTATTAAACACC, includes

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sequence complementary to the 3' end of the CON 197 coding sequence
(underlined),
preceded by a Xhol restriction site sequence useful for subsequent cloning and
expression work.
The PCR was performed in a 50 p1 reaction containing 24 p1 HZO,
10 ~,l Rapid Dye Loading buffer (Origene) 5 ~,1 l OX TT buffer (140 mM
Ammonium
Sulfate, 0.1% gelatin, O.G M Tris-tricine, pH 8.4), 5 w1 of 15 mM MgS04, 2 p1
of 10
mM dNTPs (dATP, dCTP, dTTP, dG'f P), 3 p,1 of Clone 866390H1 mini-prep DNA
(approx. 0.125 pg/wl), 0.3 p.1 of Primer LW 1324 ( 1 p.g/p,l), 0.3 p.1 of
Primer LW 1325
(1 ~.g/~1), and 0.5 p,1 of High Fidelity 'faq polymerase (Boehringer
Mannheim). The
PCR reaction was started with 1 cycle of 94°C for 2 minutes; followed
by 12 cycles at
94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1
minute.
The contents from the PCR reaction was loaded onto a 1.2% agarose
gel and electrophoresed. The DNA band of expected size was excised from the
gel,
placed in GenElute Agarose spin column (Supelco) and spun for 10 minutes at
maximum speed in a Savant microcentrifuge. The eluted DNA was ethanol-
precipitated and resuspended in 6 p,1 HZO for ligation.
The purified PCR fragment containing the CON197 coding sequence
was ligated into a commercial vector using Invitrogen's Original TA Cloning
Kit. The
resulting plasmid DNA from the culture was isolated using a Concert Rapid
Plasmid
Miniprep System (GibcoBRL) and sequenced to confirm that the plasmid contained
the CON197 insert.
Upon confirmation of the insert, the same transformant was used to
inoculate a 50 ml culture of LB medium. The culture was grown for 16 hours at
37°C, and centrifuged into a cell pellet. Plasmid DNA was purified from
the pellet
using a Qiagen Plasmid Midi Kit and again sequenced to confirm successful
cloning
of the CON197 insert, using an ABI377 fluorescence-based sequencer (Perkin
Elmer/Applied Biosystems Division, PE/ABD, Foster City, CA) and the ABI
PRISMTM Ready Dye-Deoxy Terminator kit with Taq FS'~~' polymerase as described
above for CON193 in Example 1A.1.

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G. Cloning of CON202 G Protein-Coupled Receptor
G.1 Database Search Results
The database searching identified Clone Number 1305513H1 in the
Incyte database as an interesting candidate sequence. The 13055131-11 clone
was
obtained and sequenced using an ABI377 fluorescence-based sequencer (Perkin
Elmer/Applied Biosystems Division, PE/ABD, Foster City, CA) and the ABI
PRISM~~'~' Ready Dye-Deoxy Terminator kit with Taq FS''~' polymerase as
described
above for CON193 in Example 1A.1.
Sequencing of W cyte Clone 1305513H1 revealed a sequence
corresponding to nucleotides 1054 to 1378 of SEQ ID NO: 13. Using a FORTRAN
computer program called "tmtrest.all" [Parodi et al., Comput. Appl. Biosci.,
5: 527-
535 (1994)], Clone 1305513H1 was deduced to contain two transmembrane-spanning
domains (TMVI and TMVII) and an extracellular loop for a previously
unidentified
GPCR, which was designated as "CON202". The sequence obtained was used as a
tool to identify a full length GPCR clone as described in the next section.
G.2 PCR Screening of Genomic Clones
A human genomic phage library was selected as a source from which
to attempt to clone the CON202 gene. The genomic library was amplified as
described above for CON193 in Example 1A.2.
This genomic library was screened by PCR using the primers: GV599
(5'GGCAGAAGAAGGCTATTGGTCTTAGACGAG3'; SEQ ID NO: 52), and
GV600 (5'CTGAAACAGCGCCTCAGCTCCC3'; SEQ ID NO: 53). These primers
were designed from the sequence of Clone 1305513H 1 to amplify a 253 base pair
fragment (corresponding to nucleotides 1064 to 1317 of SEQ ID NO: 13) from any
corresponding genomic clone in the library. The 20 ~.l PCR reactions each
contained
12.8 p.1 of HzO, 2 ~,1 of l Ox PCR buffer II (Perkin-Elmer), 2 ~.l of 25 mM
MgCl2, 0.8
~.1 of 10 mM dNTP's (dATP, dGTP, dCTP, dTTP), 0.12 ~,l of primer GV599
(1 p.g/ml), 0.12 ~.l of primer GV600 (1 ~,g/ml), 0.2 ,u1 AmpliTaq Gold
polymerase (5
Units/~,1, with "Units" as defined by the supplier, Perkin Elmer) and 2 ~,1 of
phage
from one of the 24 tubes. The PCR reaction consisted of 1 cycle at 95°C
for 10
minutes; then 17 cycles at 95°C for 20 seconds, 72°C for 2
minutes decreasing 1°C

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each cycle, 72°C for 30 seconds followed by 30 cycles at 95"C for 20
seconds, 55°C
for 30 seconds, and 72"C for 30 seconds.
The PCR products were visualized on a 2'% agarose gel. For those
tubes .which produced the correct sized band of 253 bp, five microliters from
each
original phage culture tube were used to amplify the CON202 genomic phage DNA
as
described above for CON 193 in Example 1A.2.
The genomic DNA from the single phage isolate, was sequenced with
the ABI PRISMTM 310 Genetic Analyzer (PE Applied Biosystems) which uses
advanced capillary electrophoresis technology and the ABI PR1SMT"' Big DyeTM
Tern~inator Cycle Sequencing Ready Reaction Kit. The cycle-sequencing reaction
contained 20 ml of HZO, 16 ml of BigDyeTM Terminator Mix, 1 ml of genomic
phage
DNA (1.l mg/ml), and 3 ml primer (25 ng/ml). The reaction was performed in a
Perkin-Elmer 9600 thermocycler at 95°C for 5 minutes, followed by 99
cycles of 95°C
for 30 seconds, SS°C for 20 seconds and 60°C for 4 minutes. The
product was
purified using a CentriflexTM gel filtration cartridge, dried under a vacuum,
then
dissolved in 16 ml of Template Suppression Reagent. The samples were heated at
95°C for 5 minutes then placed in the 310 Genetic Analyzer.
G.3 Subcloning of the Coding Region of CON202 via PCR
Additional experiments were conducted to subclone the coding region
of the CON202 clone into a more useful vector. Two PCR primers were designed
based on the coding region of CON202 for the purpose of PCR amplification of
the
CON202 coding sequence. The first, Primer LW 1482
(5'AGCTATGGCGAACTATAGCCATGCAGC3'; SEQ ID NO: 54) included the 5'
end of the CON202 coding sequence (underlined). The second, Primer LW148
(5'AGTCCTCATATAACACAGTAAGGTTCC3'; SEQ ID NO: 55) included the
sequence complementary to the 3' end of the CON202 coding sequence
(underlined).
The PCR was performed in a 50 ~.1 reaction containing 36.5 p,1 of H20,
5 p1 of lOx TT buffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris-
tricine,
pH 8.4), 5 p1 of 15 mM MgS04, 2 p,1 of 10 mM dNTP's (dATP, dCTP, dTTP, dGTP),
0.5 ~.l of CON202 genomic phage DNA (approx. 1.1 p,g/p l), 0.3 p.1 of Primer
LW 1482
(1 ~.g/pl), 0.3 p,1 of Primer LW1483 (1 ~.g/p.l), and 0.4 p1 of High Fidelity
Taq

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polymerise (l3ochringer Mannheim). The PCR reaction was started with 1 cycle
of
94°C for 2 minutes; followed by 12 cycles at 94°C for 30
seconds, 55°C for 30
seconds, and 72°C'. for 1.3 minutes.
The contents from the PCR reaction were loaded onto a 2.1 % agarose
gel and electrophoresed. The DNA band of expected size (1.1 kb) was excised
from
the gel, placed on a GenElute Agarose spin column (Supelco), and spun for 10
minutes at maximum speed in a microfuge. The eluted DNA was ethanol-
precipitated
and resuspended in 6 ~.I of HZO for ligation.
The purified PCR fragment, containing the CON202 coding sequence,
was li'gated into a commercial vector using Invitrogen's Original TA Cloning
Kit. The
ligation reaction was carried out as described above for CON193 in Example
1A.3.
The resulting plasmid DNA from the culture was isolated using a Concert Rapid
Plasmid Miniprep System (GibcoBRL) and sequenced to confirm that the plasmid
contained the CON202 insert. The resulting construct was denoted as pCR-
CON202.
The final subclone was sequenced using the ABI PR1SMTM 310
Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary
electrophoresis technology and the ABI PRISMTM Terminator Cycle Sequencing
Ready Reaction Kit. The cycle-sequencing reaction contained 6 ml of H20, 8 ml
of
BigDyeTM Terminator mix, 5 ml miniprep DNA (0.1 mg/ml), and 1 ml primer (25
ng/ml). The reaction was performed in a Perkin-Elmer 9600 thermocycler at 25
cycles of 96°C for 10 seconds, 50°C for 10 seconds, and
60°C for 4 minutes. The
product was purified using CentriflexTM gel filtration cartridges, dried under
vacuum,
then dissolved in 16 ml of Template Suppression Reagent. The samples were
heated
to 95°C for 5 minutes then placed in the 310 Genetic Analyzer.
Upon confirmation of the insert, the same transformant was used to
inoculate a 50 ml culture of LB medium. The culture was grown for 16 hours at
37°C, and centrifuged into a cell pellet. Plasmid DNA was purified from
the pellet
using a Qiagen Plasmid Midi Kit and again sequenced to confirm successful
cloning
of the CON202 insert, as described above.

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H. Cloning of CON222 G Protein-Coupled Receptor
H.1 Database Search Results
The database searching in the lncyte database identified Sequence
Number 2488822CB1 as an interesting candidate sequence. This Incyte sequence
is a
consensus sequence derived by compiling multiple, shorter contiguous
(apparently
overlapping) partial sequences from cDNA clones. A single clone known to
contain
the complete consensus sequence was not available from Incyte. The following
experiments were performed to clone a piece of human DNA which corresponds to
the region of the theoretical Incyte Sequence Number 2488822CB that was
deduced
to encode a heretofore undescribed GPCR. The human DNA and protein that was
eventually isolated is referred to herein as CON222.
H.2 Isolation of CON222 Genomic DNA using PCR
To isolate a clone of CON222, PCR primers were designed based on
the 5' and 3' ends of the open reading frame that was identified in the Incyte
Sequence
Number 2488822CB1. The first primer, designated as LW1440, has the sequence
5'AAGCGGATGTTTAGACCTCTTGTG3' (SEQ ~ NO: 60) which corresponds to
nucleotides 1 to 18 of SEQ ID NO: 15 (underlined). The second primer,
designated
LW1441, has the sequence 5'AACAGTCATGAATAGGAATTGAG3' (SEQ B7 NO:
61) which is the reverse complement of nucleotides 1173 to 1191 of SEQ ID NO:
15
(underlined).
PCR was performed in a 50 ml reaction containing 22.1 ml HZO, 10 ml
Rapid Dye Loading Buffer (Origene), 5 ml lOx TT buffer (140 mM Ammonium
Sulfate, 0.1% gelatin, 0.6 M Tris-tricine pH 8.4), 5 ml 15 mM MgS04, 2 ml 10
mM
dNTP's (dATP, dCTP, dGTP, dTTP), 5 ml human genomic DNA (0.03 mg/ml)
(Clontech, Cat# 6550-1), 0.3 ml of Primer LW1440 (1 mg/ml) (SEQ ID NO: 59),
0.3
ml of LW1441 (1 mg/ml) (SEQ ~ NO: 60), 0.4 ml High Fidelity Taq polymerase
(Boehringer Mannheim). The PCR reaction was started with 1 cycle of
94°C for 2
minutes followed by 10 cycles at 94°C for 30 seconds, 55°C for 2
minutres, 72°C for 2
minutes then 25 cycles at 94°C for 30 seconds, 55°C for 30
seconds, and 72°C for 2
minutes. The PCR reaction was loaded onto a 1.2% agarose gel. The resulting
band
was not 1.2 kB in length as expected, indicating that this method was
unsuccessful in

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identi fying an appropriate clone from the selected Clontech genomic DNA
library
containing the coding region of CON222.
A human genomic DNA phage library was selected as an alternate
source from which to attempt to clone the CON222 gene. Internal primers were
designed to attempt to isolate from a genomic library a single phage which
expresses
the complete coding region. The procedure was carried out as described above
for
CON193 in Example 1A.2.
PCR was performed to identify a phage that contained a genomic
DNA insert which corresponds to the deduced complete coding region of Incyte
Sequence Number 2488822CB1 using the primers: Primer LW1442:
5'GCCATTCTGTCCACAGAAG3' (SEQ ID NO: 58; see nucleotides 391 to 410 of
SEQ ID NO: 15) and Primer LW 1443: 5'TCAGTTGCTGTTATGGCAC3' (SEQ ID
NO: 59; see reverse complement of nucleotides 744 to 761 of SEQ >D NO: 15).
These primers were designed based on the deduced coding region of Incyte
Sequence
Number 2488822CB1, to amplify a 370 by fragment (corresponding to nucleotides
391 to 761 of SEQ m NO: 1) from any corresponding genomic clone in the
library.
The 50 p,1 PCR reactions each contained 32 p,1 of HZO, 5 ~.l of l Ox PCR gold
buffer
(PE Applied Biosystems), 5 ~.1 of 25 mM MgCh, 2 ~l of 10 mM dNTP's (dATP,
dCTP, dGTP, dTTP), 0.3 ~,l of primer LW 1442 (1 p,g/ml), 0.3 p,1 of primer LW
1443
(lp.g/ml), 0.4 ~,l AmpliTaq Gold polymerise (5 U/pl, with "Units" defined by
the
supplier; PE Applied Biosystems) and S p,1 of phage isolated human genomic DNA
(0.03 pg/p,l). The PCR reaction consisted of 1 cycle at 95°C for 10
minutes, then 17
cycles at 95°C for 20 seconds and 72°C for 2 minutes decreasing
1 degree each cycle,
and 72°C for 1 minute, followed by 30 cycles at 95°C for 20
seconds, 55°C for 30
seconds, and 72°C for 1 minute. An aliquot of the PCR reaction was
loaded onto a
1.2% agarose gel and electrophoresed. Although the internal primers were
designed
to produce a 370 by PCR fragment, the resulting band was approximately 1.4 kb
in
length.
The DNA band was excised from the gel, placed on GenElute Agarose
spin columns (Supelco) and spun for 10 minutes at maximum speed in a

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microcentrifuge. The eluted DNA was ethanol-precipitated and resuspended in 10
p1
of f-1,0 and 5 p.1 was used to sequence the PCR band.
The PCR fragment was sequenced with an ABI PRISIV1TN' 310 Genetic
Analyzer (PE Applied Biosystems) which uses advanced capillary electrophoresis
technology and the AB1 PRISMTM BigDyeTM Terminator Cycle Seduencing Ready
Reaction Kit. Each cycle-sequencing reaction contained 6 ml of H,O, 8 ml of
BigDye
Terminator mix, 5 ml PCR fragment DNA (0.2 mg/ml), and 1 ml Primer LW 1442
(25 ng/ml) and Primer LW 1443 (25 ng/ml). The reaction was performed in a
Perkin
Elmer 9600 thermocycler with 25 cycles of 96°C for 10 seconds,
50°C for 10 seconds,
and 60°C for 4 minutes. The product was purified using CentriflexT'''
gel Reagent (PE
Applied Biosystems). The samples were heated at 95°C for 5 minutes then
placed in
the 310 Genetic Analyzer.
The sequence analysis determined that there is an intron in the middle
of the 5th transmembrane-spanning domain between nucleotides 673 and 674 in
SEQ
>D NO: 15. This intron was responsible for the unexpectedly large PCR
fragment.
H.3 Isolation of Full Length cDNA
Since attempts to isolate an uninterrupted coding region from genomic DNA
were unsuccessful, a fetal brain cDNA was used to generate the complete coding
region of Incyte Sequence Number 2488833CB1. The PCR primers described above,
LW 1440 (SEQ ID NO: 60) and LW 1441 (SEQ ID NO: 61 ), which correspond to the
5' and 3' end of CON222 respectively, were used to generate the full length
coding
regi on.
The SO ~l PCR reaction contained 37.4 p,1 of HZO, 5 p1 of l Ox cDNA
PCR buffer (Clontech), 1 p,1 of 10 mM dNTP's (dATP, dCTP, dTTP, dGTP), 5 p,1
of
Marathon-Ready Fetal Brain cDNA (Clontech), 0.3 p,1 of Primer LW1440 (1
p.g/p,l),
0.3 p1 of Primer LW 1441 (1 p,g/p,l), and 1 p.1 of SOx Advantage cDNA
polymerase
(Clontech). The PCR reaction was started with 1 cycle of 94°C for 1
minute,
followed by 30 cycles at 94°C for 30 seconds, 50°C for 30
seconds, and 68°C for 3
minutes.
The contents from the PCR reaction were loaded onto a 1.2% agarose
gel and electrophoresed. The DNA band of expected size (1.2 kb) was excised
from

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the gel, placed on a GenElute Agarose spin column (Supelco), and spun for 10
minutes at maximum speed in a microfuge. The eluted DNA was ethanol-
precipitated
and resuspended in 6 p1 H,O for ligation.
H.4 Subcloning of Coding Region of CON222 via PCR
After a cDNA containing the full length CON222 open reading frame
was obtained, the coding region of CON222 was then subcloned into a more
useful
vector as follows.
The purified PCR fragment described above, containing the CON222
coding sequence, was ligated into a commercial vector using Invitrogen's
Original TA
Cloning Kit. The ligation reaction was carried out as described above for CON
193 in
Example 1 A.3. The resulting plasmid DNA from the culture was isolated using a
Concert Rapid Plasmid Miniprep System (GibcoBRL) and sequenced to confirm that
the plasmid contained the CON222 insert.
The subcloned insert in pCR2.l was sequenced using the ABI
PRISMTM 310 Genetic Analyzer (PE Applied Biosystems) which uses advanced
capillary technology and the ABI PRISM TM BigDyeTM Terminator Cycle Sequencing
Ready Reaction Kit. Each cycle-sequence reaction contained 6 ml of H20, 8 ml
of
BigDyeTM Terminator mix, 5 ml mini-prep DNA (0.1 mg/ml), and 1 ml of primer
(25
ng/ml) and was performed in a Perkin-Elmer 9600 thermocycler with 25 cycles of
96°C for I O seconds, 50°C for 10 seconds, and 60°C for 4
minutes. The product was
purified using a CentriflexTM gel filtration cartridge, vacuum dried and
dissolved in 16
ml of Template Suppression Reagent (PE Applied Biosystems). The samples were
heated at 95°C for 5 minutes then placed in the 310 Genetic Analyzer.
Upon confirmation of the insert, the same transformant was used to
inoculate a 50 ml culture of LB medium. The culture was grown for 16 hours at
37°C, and centrifuged into a cell pellet. Plasmid DNA was purified from
the pellet
using a Qiagen Plasmid Midi Kit and again sequenced to confirm successful
cloning
of the CON222 insert, as described above.

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I. Cloning of CON215 G Nrotein-Coupled Receptor
I.1 Database Search Results
The database searching identified Clone 1452259H1 in the Lncyte
database as an interesting candidate sequence. The sequence from 1452259H1
clone
was used to search the Incyte fill-length database and matched the entry
1650519CB1.
An inspection of the clones that made up 1650519CB 1 indicated that Incyte
Clone
2796157H1 probably contained the full-length coding region. Sequence analysis
of
lncyte Clone 2796157H1 indicated that it contains the entire coding region for
a
previously unidentified GPCR, which was designated "CON215", along with 12
nucleotides of 5' untranslated region, 63 nucleotides of 3' untranslated
region and a
poly A+tail. The DNA and deduced amino acid sequences for CON215 are set forth
in SEQ ID NOS: 17 and 18, respectively. A database search with this CON215
sequence showed a 47% match to the human probable G protein-coupled receptor
KIA0001.
Since the untranslated regions were relatively short, it was not
necessary to remove the coding region of CON215 from the plNCY vector (Incyte)
and the construct is referred to as pINCY-CON215. The Incyte Clone 2796157H1
was sequenced using the ABI PRISMTM 310 Genetic Analyzer (PE Applied
Biosystems) which uses advanced capillary electrophoresis technology and the
ABI
PRISMTM BigDyeTn'' Terminator Cycle Sequencing Ready Reaction Kit as described
above for CON222 in ExamplelH.4.
J. Cloning of CON217 G Protein-Coupled Receptor
J.1 Database Search Results
The Incyte database search identified EST 3700658H1 as an interesting
candidate sequence. The EST sequence No. 3700658H1 was used to search the
lncyte
full length database. This search identiFed Incyte clone No. 3356166H1 as a
clone
that potentially contained a full length GPCR corresponding to the selected
EST.
The 3356166H1 clone was obtained from W cyte and sequenced using
an ABI377 fluorescence-based sequencer ( and the ABI PRISMT"' Ready Dye-Deoxy

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Terminator kit with Taq FS'''~' polyi~erase as described above for CON193 in
Example 1A.1.
Sequencing of lncyte Clone No. 3356166H 1 revealed a 2480 basepair
sequence as shown in SEQ NO: 19. Using a FORTRAN computer program called
"tmtrest.all" [Parodi et cal., Conzpzzt. App!. Biosci., 5: 527-535 (1994)],
Clone No.
3356166H1 was deduced to contain seven transmembrane-spanning domains (TMl-
TMVII) and was designated as "CON217" (SEQ ID NO: 20). The following
experiments were performed to subclone and isolate the full length coding
sequence
of CON217 from Incyte Clone No. 3356166H 1.
J.2 Subcloning of the Coding Region of GPCR217
To subclone the full length coding sequence of CON217, PCR primers
were designed based on the 5' and 3' ends of the open reading frame that was
identified in the Incyte Clone No. 3356166H1. The first primer, designated as
LW I 448, has the sequence 5'AAGCGGTACCATGTTAGCCAACAGCTCCTC3'
(SEQ ID NO: 66) which corresponds to nucleotides 42 to 62 of SEQ ID NO: 19
(underlined). The second primer, designated LW 1449, has the sequence
5'AAGCTCTAGATCAGAGGGCGGAATCCTGG3' (SEQ ID NO: 67) which is the
reverse complement of nucleotides 1142 to 1160 of SEQ ID NO: 20 (underlined).
The primers also include recognition sequences (bold) for the restriction
enzymes
KpnI and XbaI, respectively.
PCR was performed in a 50 ml reaction containing 32.5 ml of H,O, 5
ml of l Ox Pfx Amplification buffer (GibcoBRL), S ml of l Ox PCR Enhancer
solution
(GibcoBRL), 1.5 ml of 50 mM MgS04, 2 ml of 10 mM dNTP's (dATP, dCTP, dGTP,
dTTP), 3 ml 3356166H1 mini-prep DNA (0.125 mg/ml obtained with the Concert
Rapid Plasmid Miniprep System; GibcoBRL), 0.3 ml of Primer LW1448 (1 mg/ml)
(SEQ ID NO: 3), 0.3 ml ofPrimer LW1449 (1 mg/ml) (SEQ ID NO: 4), 0.5 ml
Platinum Pfx DNA polymerise (2.5 U/ml; GibcoBRL). The PCR reaction was started
with 1 cycle of 94°C for 2 minutes followed by 25 cycles at 94°C
for 30 seconds, SS°C
for 30 seconds, 68°C for 1.3 minutes.
The contents from the PCR reaction were loaded onto a 1.2% agarose
gel and electrophoresed. The DNA band of expected size (~ 1.1 kb) was excised
from

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the gel, placed on a GenElute Agarose spin column (Supelco), and spun for 10
minutes at maximum speed in a microfuge. The eluted DNA was ethanol-
precipitated
and resuspended in 6 pt of HZO for ligation.
The purified PCR fragment, containing the CON217 coding sequence,
~~as ligated into a commercial vector designated pCR2. l using lnvitrogen's
Original
TA Cloning Kit. The ligation reaction was carried out as described above for
CON 193 in Example 1A.3. The resulting plasmid DNA from the culture was
isolated
using a Concert Rapid Plasmid Miniprep System (GibcoBRL) and sequenced to
confirm that the plasmid contained the CON217 insert and to confirm that no
errors
were introduced during PCR amplification. The resulting construct was denoted
as
pCR-CON217.
The final subclone was sequenced using the ABI PRISMTM 310
Genetic Analyzer (PE Applied Biosystems) which uses advanced capillary
electrophoresis technology and the ABI PRISMTM Terminator Cycle Sequencing
Ready Reaction Kit as described above for CON222 in Example 1H.4.
EXAMPLE 2
Analysis of G Protein-Coupled Receptor Sequence
A. CON193
The DNA and deduced amino acid sequence for CON193 are
set forth in SEQ ID NOS: 1 and 2, respectively. Beginning with the initiation
codon
(methionine), the CON193 genomic Clone contains an open reading frame of 963
nucleotides encoding 321 amino acids, followed by a stop codon. Using a
FORTRAN
computer program called "tmtrest.all" [Parodi et al., Comput. Appl. Biosci.,
5: ~27-
535 (1994)), CON193 was shown to contain seven transmembrane-spanning domains
corresponding to residues 30-49 (1TM), 61-81 (2TM), 103-122 (3TM), 146-165
(4TM), 199-222 (STM), 243-262 (6TM), and 270-295 (7TM) of SEQ ID NO: 2.
These transmembrane domains define first ("N-terminal," residues 1-29), second
("first EC loop," residues 82-102), third ("second EC loop," residues 166-
198), and
fourth ("third EC loop," residues 263-269) extracellular domains, as well as
first
("first IC loop," residues 50-60), second ("second IC loop," residues 123-
145), third

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("third IC loop," residues 223-242), and fourth ("C-terminal," residues 296-
321)
intracellular domains.
inspection of the CON193 amino acid sequence (SEQ ID NO: 2)
reveals that this GPCR contains a DRY sequence following the third
transmembrane
domain (3TM) and a PIVY sequence found in the sixth transmembrane domain
(TM6). In addition, the CON193 polynucleotide sequence was compared to
sequences of known genes. CON193 is 45% identical and 72% similar to the mouse
olfactory receptor gene S 19 [see Malnic et al., Cell 96:713-723 ( 1999)].
This level of
sequence similarity suggests that CON193 is a novel GPCR.
The CON193 cDNA clone (SEQ ID NO:I) was deposited with the
National Center for Agricultural Utilization Research at the United States
Department
of Agriculture 1815 North University Street, Peoria, Illinois 61604 in
accordance with
the Budapest Treaty on January 18, 2000. The clone was given accession no. B-
30250.
B. CON166
The DNA and deduced amino acid sequence for CON166 are set forth
in SEQ ID NOS: 3 and 4, respectively. Beginning with the initiation codon
(methionine), the CON166 genomic clone contains an open reading frame of 1,011
nucleotides encoding 337 amino acids, followed by a stop codon. Using a
FORTRAN computer program called "tmtrest.all" [Parodi et al., Comput. Appl.
Biosci., 5: 527-535 (1994)], CON166 was shown to contain seven transmembrane-
spanning domains corresponding to the following residues presented in SEQ ID
NO:
4: 1TM (30-49), 2TM (59-79), 3TM (99-119), 4TM (141-161), STM (191-215), 6TM
(231-251), and 7TM (277-296) . These transmembrane domains define first ("N-
terminal," residues 1-29), second ("first EC loop," residues 80-98), third
("second EC
loop," residues 162-190), and fourth ("third EC loop," residues 252-276),
extracellular domains as well as first ("first 1C loop," residues 50-58),
second
("second IC loop," residues 120-140), third ("third IC loop," residues 216-
230), and
fourth ("C-terminal," residues 297-337) intracellular domains.

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Inspection of the CON 166 amino acid sequence (SEQ ID N0:2)
reveals that this GPCR contains an FRC sequence following the third
transmemUrane
domain (3TM), which is typically occupied by a consensus DRY sequence in other
GPCRs; a P.LLY sequence is also found in the seventh transmembrane dOlnaln
(7'fM).
In addition, the CON 166 polynucleotide sequence was compared to sequences of
known genes. CON 166 is 44% identical and 62% similar to a T-cell-specific G
protein-coupled receptor of Gccllus gcillus found in the TREMBL database
(Accession
No. L06109). This level of sequence similarity suggests that CON166 is a novel
GPCR.
The CON166 cDNA clone (SEQ ID N0:3) was deposited with the
National Center for Agricultural Utilization Research at the United States
Department
of Agriculture 1815 North University Street, Peoria, Illinois 61604 in
accordance with
the Budapest Treaty on January 18, 2000. The clone was given accession no. B-
30248.
C. CON103
The DNA and deduced amino acid sequence for CON103 are set forth
in SEQ ID NOS: 5 and 6, respectively. Beginning with the initiation codon
(methionine), the CON103 genomic clone contains an open reading frame of 1,152
nucleotides encoding 384 amino acids, followed by a stop codon and a short
open
reading frame (SEQ ID NO: 5). Using a FORTRAN computer program called
"tmtrest.all" [Parodi et al., Comput. Appl. Biosci., 5: 527-535 (1994)],
CON103 was
shown to contain seven transmembrane-spanning domains corresponding to the
following residues in SEQ ID NO: 6: 54-77 (1TM), 89-108 (2TM), 134-149 (3TM),
167-188 (4TM), 216-240 (STM), 258-283 (6TM), and 301-320 (7TM). These
transmembrane domains define first ("N-terminal," residues 1-53), second
("first EC
loop," residues 109-133), third ("second EC loop," residues 189-215), and
fourth
("third EC loop," residues 284-300) extracellular domains, as well as first
("first IC
loop," residues 78-88), second ("second IC loop," residues 150-166), third
("third IC
loop," residues 241-257), and fourth ("C-terminal," residues 321-384)
intracellular
domains.

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Inspection of the CON l03 amino acid sequence (SEQ ID NO: 6)
reveals that this GPCR cOllta111S an NRY sequence following the third
transmembrane
domain (3TM), which is typically occupied by a consensus DRY sequence in other
GPCRs. In addition, the CON I 03 polynucleotide sequence was compared to
sequences of known genes. CON 103 is 36% identical to GPR31 (GenBank Accession
No. L165402) and 31% identical to the P2Y1 purinergic receptor (GenBank
Accession
No. 581950). This level of sequence similarity indicates that CON103 is a
novel
GPCR.
The CON103 cDNA clone (SEQ ID NO:S) was deposited with the
National Center for Agricultural Utilization Research at the United States
Department
of Agriculture 1815 North University Street, Peoria, Illinois 61604 in
accordance with
the Budapest Treaty on January 18, 2000. The clone was given accession no. B-
30247.
D. CON203
The DNA and deduced amino acid sequence for CON203 are set forth
in SEQ >D NOS: 7 and 8, respectively. Beginning with the initiation codon
(methionine), the CON203 genomic clone contains an open reading frame of 999
nucleotides encoding 333 amino acids, followed by a stop codon. Using a
FORTRAN
computer program called "tmtrest.all" [Parodi et al., Comput. Appl. Biosci.,
S: 527-
535 (1994)], CON203 was shown to contain seven transmembrane-spanning domains
corresponding to the following residues of SEQ ID NO: 7: nucleotides 29-53
(1TM),
63-82 (2TM), 97-118 (3TM), 136-160 (4TM), 189-211 (STM), 232-252 (6TM), and
281-300 (7TM). These transmembrane domains define first ("N-terminal,"
residues
1-28), second ("first EC loop," residues 83-96), third ("second EC loop,"
residues
161-188), and fourth ("third EC loop," residues 253-280) extracellular
domains, as
well as first ("first IC loop," residues 54-62), second ("second IC loop,"
residues 119-
135), third ("third IC loop," residues 212-231 ), and fourth ("C-terminal,"
residues
301-333) intracellular domains.
Inspection of the CON203 amino acid sequence (SEQ ID NO: 8)
reveals that this GPCR contains a DRF sequence following the third
transmembrane

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domain (3TM), which is typically occupied by a consensus DRY sequence in other
GPCRs; CON203 also exhibited a PLIY seduence in the seventh transmembrane
domain (7TM). (n addition, the CON203 polynucleotide sequence was compared to
sequences of known genes. CON203 is 33% identical to a platelet activating
receptor
(GenBanl< Accession No. AF002986. This level of sequence similarity suggests
that
CON203 is a novel GPCR.
The CON203 cDNA clone (SEQ ID NO: 7) was deposited with the
National Center for Agricultural Utilization Research at the United States
Department
of Agriculture 1815 North University Street, Peoria, Illinois 61604 in
accordance with
the Budapest Treaty on January 18, 2000. The clone was given accession no. B
30254.
E. CON198
The DNA and deduced amino acid sequence for CON198 are set forth
in SEQ >D NO: 9 and 10 respectively. Beginning with the initiator methionine,
the
CON198 genomic clone contains an open reading frame of 954 nucleotides
encoding
318 amino acids, followed by a stop codon. It will be appreciated that residue
2 of
SEQ ID NO: 10 also is a methionine. Amino-terminal sequencing of purified
native
or recombinant CON198 protein will provide an indication as to which
methionine
acts as an initiator codon in vivo. Using a FORTRAN computer program called
"tmtrest.all" [Parodi et al., Comput . Appl. Biosci., S: 527-535 (1994)],
CON198 was
deduced to contain seven transmembrane-spanning domains corresponding to
residues
28-52 (TM1), 61-80 (TM2), 104-123 (TM3), 147-167 (TM4), 200-226 (TMS), 239-
263 (TM6), and 274-295 (TM7) of SEQ ID NO: 10 . These transmembrane domains
define first ("N-terminal," residues 1-27 or 2-27), second ("first EC loop,"
residues
81-103), third ("second EC loop," residues 168-199), and fourth ("third EC
loop,"
residues 264-273) extracellular domains as well as first ("first IC loop,"
residues 53-
60), second ("second IC loop," residues 124-146), third ("third IC loop,"
residues
227-238), and fourth ("C-terminal," residues 296-318) intracellular domains.
CON198 contains a DRY sequence following the third transmembrane
domain (TM3), a feature that is conserved in most GPCR. The most similar
sequence

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in a public database, at the time of initial screening, was that of rat GPCR
RAIc,
which shared only 61 % identity at the amino acid level.
The CON198 cDNA clone (SEQ ID NO: 9) was deposited with the
National Center for Agricultural Utilization Research at the United States
Department
of Agriculture 1815 North University Street, Peoria, Illinois 61604 in
accordance with
the Budapest Treaty on January 18, 2000. The clone was given accession no. B-
30252.
F. CON197
The DNA and deduced amino acid sequence for CON 197 are set forth
in SEQ LD NO: 11 and 12, respectively. Beginning with the initiator
methionine, the
CON197 genomic clone contains an open reading frame of 921 nucleotides
encoding
307 amino acids, followed by a stop codon. Using a FORTRAN computer program
called "tmtrest.all" [Parodi et al., Comput. Appl. Biosci., 5: 527-535
(1994)], CON197
was deduced to contain seven transmembrane-spanning domains corresponding to
residues 23-47 (TM1), 58-78 (TM2), 99-120 (TM3), 142-164 (TM4), 195-219 (TMS),
237-258 (TM6), and 270-289 (TM7) of SEQ ID NO: 12. These transmembrane
domains define first ("N-terminal" residues 1-22), second ("first EC
loop"residues 79-
98), third ("second EC loop"residues 165-194), and fourth ("third EC
loop"residues
259-269) extracellular domains as well as first ("first IC loop" residues 48-
57), second
("second IC loop" residues 121-141), third ("third 1C loop" residues 220-236),
and
fourth ("C-terminal" residues 290-309) intracellular domains.
CON197 contains a DRY sequence following the third transmembrane
domain (TM3), a feature that is conserved in most GPCR. The most similar
sequence
in a public database, at the time of initial screening, was that of an
olfactory receptor,
which shared only 42% identity at the amino acid level.
The CON197 cDNA clone (SEQ ID NO: 11) was deposited with the
National Center for Agricultural Utilization Research at the United States
Department
of Agriculture 1815 North University Street, Peoria, Illinois 61604 in
accordance with
the Budapest Treaty on January 18, 2000. The clone was given accession no. B
30251.

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G. CON202
The DNA and deduced amino acid sequence for this phage insert,
tern~ed "CON202", are set forth in SEQ ID NO: 13 and 14, respectively. The
CON202 open reading frame, as depicted in SEQ ID NO: 14, begins with the
initiator
methionine and spans 1 1 10 nucleotides which encode 370 amino acids, followed
by a
stop codon. Since this gene was isolated from genomic DNA and there are no
apparent interruptions in the sequence, it is likely that CON202 contains no
introns
within the coding region. The full length clone of CON202 contained seven
transmembrane-spanning domains corresponding to residues, 24 to 46 (TM 1 ) ,
~7 to
77 (TM2), 96 to 1 17 (TM3), 135 to 159,(TM4) TMV comprises 184 to 202 (TMS),
286 to 308 (TM6), 316 to 339 (TM7) of SEQ >D NO: 14. TM2 terminates with PFVC
instead of the characteristic PXXY. TM3 is followed by the sequence TRY
instead of
the characteristic DRY. These transmembrane domains define first ("N-
terminal,"
residues 1-23), second ("first EC loop," residues 78-95), third ("second EC
loop,"
residues 160-183), and fourth ("third EC loop," residues 309-315)
extracellular
domains as well as first ("first IC loop," residues 47-56), second ("second 1C
loop,"
residues 118-134), third ("third IC loop," residues 203-285), and fourth ("C-
terminal,"
residues 340-370) intracellular domains.
The CON202 cDNA clone (SEQ 1D NO: 13) was deposited with the
National Center for Agricultural Utilization Research at the United States
Department
of Agriculture 1815 North University Street, Peoria, Illinois 61604 in
accordance with
the Budapest Treaty on January 18, 2000. The clone was given accession no. B-
30253.
H. CON222
The sequence of CON222 coding region deduced the DNA and amino
acid sequence set forth in SEQ ID NO: 15 and 16, respectively. The open
reading
frame that is depicted in SEQ ID NO: 16 begins with an initiator codon and
spans
1188 nucleotides which encode 396 amino acids, followed by a stop codon.
The full length clone of CON222 contains seven transmembrane-
spanning domains corresponding to residues 42-65 (TM1) 79-103, (TM2), 125-156,

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(TM3), 167-188 (TM4), 217-241 (TMS), 268-290 (TM6), 301-320 (TM7) of SEQ ID
NO: 16. TM2 is followed by a FRC sequence and TM7 contains a PILY sequence
within. These transmembrane dO111aInS define first ("N-tern~inal," residues 1-
41),
second ("first EC loop," residues 104-124), third ("second EC loop," residues
189-
216), and fourth ("third EC loop," residues 291-300) extracellular domains as
well as
first ("first IC loop," residues 66-78), second ("second IC loop," residues
157-166),
third ("third IC loop," residues 242-267), and fourth ("C-terminal," residues
321-396)
intracellular domains. A search of the public database indicated that CON222
is
about 35% identical to a unique GPCR found in the nervous system ofLymnae~c
stagnalis.
The CON222 cDNA clone (SEQ m NO: 15) was deposited with the
National Center for Agricultural Utilization Research at the United States
Department
of Agriculture 1815 North University Street, Peoria, Illinois 61604 in
accordance with
the Budapest Treaty on January 18, 2000. The clone was given accession no. B-
30257.
I. CON215
The DNA and deduced amino acid sequence for CON215 are set forth
in SEQ ID NO: 17 and 18, respectively. Beginning with the initiator
methionine, the
CON215 genomic clone contains an open reading frame of 1074 nucleotides
encoding
358 amino acids, followed by a stop codon. Using a FORTRAN computer program
called "tmtrest.all" [Parodi et al., Co~nput . Appl. Biosci., 5: 527-535
(1994)],
CON21 S was deduced to contain seven transmembrane-spanning domains
corresponding to residues 42-66 (TM1), 81-99 (TM2), 116-137 (TM3), 156-180
(TM4), 210-234 (TMS), 256-275 (TM6), and 308-328 (TM7) of SEQ 117 NO: 18.
These transmembrane domains define first ("N-terminal," residues I-41), second
("first EC loop," residues 100-115), third ("second EC loop," residues 181-
209), and
fourth ("third EC loop," residues 276-307) extracellular domains as well as
first ("first
IC loop," residues 67-80), second ("second IC loop," residues 138-155), third
("third
IC loop," residues 235-255), and fourth ("C-terminal," residues 329-358)
intracellular
domains.

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_O7_
CON215 contains a DRY sequence following the third transmembrane
domain (TM3), a feature that is conserved in most GPCR. CON215 also contains a
PIIY sequence within the seventh transmembrane domain (TM7).
The CON215 cDNA clone (SEQ LD NO: 17) was deposited with the
National Center for Agricultural Utilization Research at the United States
Department
of Agriculture 1815 North University Street, Peoria, Illinois 61604 in
accordance with
the Budapest Treaty on January 18, 2000. The clone was given accession no. B-
30255.
J. CON217
The DNA and deduced amino acid sequences of CON217 are set forth
as SEQ ID NO: 19 and 20, respectively. The open reading frame that is depicted
in
SEQ ID NO: 2 begins with an initiator methionine codon and spans 1116
nucleotides
which encode 372 amino acids, followed by a stop codon. In addition, the
nucleotide
sequence consists of 41 by in the S' untranslated region and 1323 by in the 3'
untranslated region.
The full length clone of CON217 contains seven transmembrane-
spanning domains as indicated by the FORTRAN computer program "tmtrest.all"
[Parodi et al., Comput. Appl. Biosci., 5: 527-535 (1994)] which corresponds to
29-50
(TMl), 57-75 (TM2), 96-117 (TM3), 137-160 (TM4), 188-210 (TMS), 235-258
(TM6), 277-297 (TM7). TM3 is followed by a DRY sequence and TM7 contains a
PLVY sequence within. These transmembrane domains define first ("N-terminal,"
residues 1-28), second ("first EC loop," residues 76-95), third ("second EC
loop,"
residues 161-187), and fourth ("third EC loop," residues 259-276)
extracellular
domains as well as first ("first IC loop," residues 51-56), second ("second IC
loop,"
residues 118-136), third ("third IC loop," residues 211-234), and fourth ("C-
terminal,"
residues 298-372) intracellular domains. A search of the public database
indicated
that CON217 is about 41 % identical to GPR23 (Genebank Accession No.: U66578)
and to a purinergic receptor P2Y9 (Genebank Accession No.: U90322).
The CON21 S cDNA clone (SEQ ID NO: 19) was deposited with the
National Center for Agricultural Utilization Research at the United States
Department

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of Agriculture 1815 North University Street, Peoria, Illinois 61604 in
accordance with
the Budapest Treaty on January 18, 2000. The clone was given accession no. B-
30256.
K. Summary of Deposits
The polynucleotides (SEQ )D NO: l, 3, 5, 7, 9, 1 1, 13, l5 and 17)
encoding the GPCR polypeptides of the invention were deposited with the
Agricultural Research Service Culture Collection (NRRL) at the National Center
Agricultural Utilization Research at the U.S. Department of the Agriculture
1815
North University Street, Peoria, Illinois 61604. These deposits were made in
accordance with the Budapest Treaty on the International Recognition of the
Deposit
of Microorganism for the Purposes of Patent Procedures. The table below lists
the
details of these deposits.
GPCR SEQ ID NO: NRRL No. DeRosit Date
CON193 1 B-30250 1/18/00
CON166 3 B-30248 1/18/00
CON103 5 B-30247 1/18/00
CON203 7 B-30254 1/18/00
CON 198 9 B-30252 1 / 18/00
CON 197 11 B-30251 1 / 18/00
CON202 13 B-30253 1 / 18/00
CON222 1 S B-30257 1 /18/00
CON215 17 B-30255 1/18/00
CON217 19 B-30256 1/18/00
EXAMPLE 3
Hybridization Analysis Demonstrates that the GPCRs are
Expressed in the Brain
The expression of GPCR polynucloetides in mammals, such as the rat,
was investigated by in situ hybridization histochemistry. Coronal and sagittal
rat

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brain cryosections (20 pm thick) were prepared using a Reichert-.lung
cryostat.
Individual sections were thaw-mounted onto silanizcd, nuclease-free slides
(CEL
Associates, lnc., f-louston, TX), and stored at -80°C. Sections were
processed starting
with post-fixation in cold 4% paraformaldehyde, rinsed in cold phosphate-
buffered
S saline (PBS), acetylated using acetic anhydride in triethanolamine buffer,
and
dehydrated through a series of alcohol washes in 70%, 95%, and 100% alcohol at
room temperature. Subsequently, sections were delipidated in chloroform,
followed
by rehydration through successive exposure to 100% and 95% alcohol at room
temperature. Microscope slides containing processed cryosections were allowed
to air
dry prior to hybridization.
A. CON193
A CON193-specific probe was generated using PCR. The probe
consisted of a 270 by fragment containing sequence at the 3' end of CON-193.
The
primers for PCR amplification were LW 1248 [5'-
GCATGAATTCCAATATACTTCCCCATACCTAC-3'; SEQ ID NO: 26) and LW
1249 [5'-GCATGGATCCGGAAAAGAAGGAGAAGAAAG-3'; SEQ ID NO: 27),
which introduced terminal EcoRI and BamHI restriction sites into the PCR
product.
Following PCR amplification, the fragment was digested with EcoRI and Bc~mHI
and
cloned into pBluescriptII cleaved with the same enzymes. For production of a
probe
specific for the sense strand of CON193, the CON193 Clone in pBluescriptII was
linearized with BamHI, which provided a substrate for labeled run-off
transcripts (i.e.,
cRNA riboprobes) using the vector-borne T7 promoter and commercially available
T7
RNA polymerase. A probe specific for the antisense strand of CON 193 was also
readily prepared using the CON193 Clone in pBluescriptIl by cleaving the
recombinant plasmid with EcoRI to generate a linearized substrate for the
production
of labeled run-off eRNA transcripts using the T3 promoter and cognate
polymerase.
The riboprobes were labeled with [35S]-UTP to yield a specific activity of
0.81 x 10~
cpm/pmol for antisense riboprobes and 0.55 x 106 cpm/pmol for sense-strand
riboprobes. Both riboprobes were subsequently denatured by incubating at
70°C for 3
minutes and added (2 pmol/ml) to hybridization buffer which contained 50%

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forn~amide, 10% dextrin, 0.3 M NaCI, 10 mM Tris (pH 8.0), 1 mM EDTA, 1X
Denhardt's Solution, and 10 mM dithiothreitol. Microscope slides containing
sequential brain cryosections were independently exposed to 45 p.1 of
hybridization
solution per slide and silanized cover slips were placed over the sections
being
exposed to hybridization solution. Sections were incubated overnight (15-18
hours) it
52°C to allow hybridization to occur. Equivalent series of cryosections
were exposed
to sense or antisense CON I 93-specific cRNA riboprobes.
Following the hybridization period, coverslips were washed off the
slides in 1X SSC. Slides were subjected to RNase A treatment by incubation in
a
buffer containing 20 pg/ml RNase A, 10 mM Tris (pH 8.0), 0.5 M NaCI and 1 mM
EDTA for 45 minutes at 37°C. The cryosections were then subjected to
three high-
stringency washes in 0.1 X SSC at 52°C for 20 minutes each. Following
the series of
washes, cryosections were dehydrated by consecutive exposure to 70%, 95%, and
100% ammonium acetate in alcohol, followed by air drying and exposure to Kodak
BioMax MR-1 film. After 13 days of exposure, the film was developed. Based on
these results, brain sections that gave rise to positive hybridization signals
were. coated
with Kodak NTB-2 nuclear track emulsion and the slides were stored in the dark
for
32 days The slides were then developed and counterstained with hematoxylin.
Emulsion-coated sections were analyzed microscopically to determine the
specificity
of labeling. The signal was determined to be specific if autoradiographic
grains
(generated by antisense probe hybridization) were clearly associated with
crystal
violet-stained cell bodies. Autoradiographic grains found between cell bodies
indicates non-specific binding.
Specific labeling with the antisense probe occurred at low levels in the
cortex and in the substantia nigra-pars compacta (SN-c). The specificity of
labeling
was confirmed by microscopic analysis of emulsion-coated cryosections, as
described
above. In contrast, hybridization using the riboprobe specific for the sense
strand of
CON193 did not result in specific tissue labeling. The observed regional
distribution
of CON 193 mRNA suggests that ligands for this GPCR may be involved in signal
transductions important for cellular processes underlying neurological
functioning. In
addition, expression of CON193 in the brain provides an indication that
modulators of

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CON193 activity have utility for treating neurological disorders, lIlCILIdIIlg
bllt not
limited to, schizophrenia, depression, anxiety, bipolar disease, epilepsy,
neuritis,
neurasthenia, neuropathy, neuroses, and the like. Use of CON193 modulators,
including CON 193 ligands and anti-CON I 93 antibodies, to treat individuals
having
such disease states is intended as an aspect of the invention.
B. CON166
A CON166-specific probe was generated using PCR as described
above for CON193 in Example 3A (but using CON166-specific primers). The probe
consisted of a 259 by fragment containing sequence at the 3' end of CON-166
(nucleotides 715-974 of SEQ ID NO:1 ) and containing terminal EcoRI and BamHI
restriction sites. The riboprobes were labeled with [35S]-UTP to yield a
specific
activity of 0.40 x 106 cpm/pmol for antisense riboprobes and 0.65 x 106
cpm/pmol for
sense-strand riboprobes Hybridization with the riboprobes and subsequent
washing
of the slides was carried out as described above for CON193 in Example 3A.
Specific labeling with the antisense probe occurred in cortical regions,
including the piriform complex, neostriatum, thalamus and hippocampus. The
specificity of labeling was confirmed by microscopic analysis of emulsion-
coated
cryosections. These sections revealed that the autoradiographic grains
resulting from
antisense riboprobe in situ hybridizations were distributed over cell bodies
rather than
trapped between cell bodies. In contrast, hybridization using the riboprobe
specific
for the sense strand of CON166 produced a faint signal in the hippocampus
only, but
even this signal was found to be non-specific upon microscopic examination.
The
observed regional distribution of CON166 mRNA suggests that ligands for this
GPCR
may be involved in signal transductions important for cellular processes
underlying
neurological functioning. In addition, expression of CON166 in the brain
provides an
indication that modulators of CON 166 activity have utility for treating
neurological
disorders, including but not limited to, schizophrenia, affective disorders,
ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/Attention Deficit
Disorder), and neural disorders such as Alzheimer's disease, Parkinson's
disease,
migraine, and senile dementia. Some other diseases for which modulators of

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CON 166 may have utility include depression, anxiety, bipolar disease,
epilepsy,
neuritis, neurasthenia, neuropathy, neuroses, and the like. Use of CON 166
modulators, including CON166 ligands and anti-CON 166 antibodies, to treat
individuals having such disease states is intended as an aspect of the
invention.
S
C. CON 103
A cocktail of two CON103-specific anti sense oligonucleotide probes
(CON 103a and CON103b) were used because of the relatively high GC content of
the
CON103 coding region. The CON103a sequence
(S'TTTATTAATATTGGAAGGGACAAACTGGAGAGCACAGAACAT3'; SEQ m
NO: 72) corresponds to the reverse complement of nucleotides 2196-2237 of SEQ
~
NO: 5 and CON103b sequence (5'AAAGCCACCATGGA
AGCCATGCCAAAGATGATGCTGGGCAAGAA 3'; SEQ 1D NO: 73) corresponds
to the reverse complement of nucleotides 195-1538 of SEQ ID NO: 5. Terminal
deoxynucleotidyltransferase and [a -33P]dATP were used to 3' end-label CON103a
(1.36 x 10' cpm/pmol) and CON103b (9.1 x 106 cpm/pmol). The probes were
denatured by incubation at 70°C for three minutes and added to
hybridization buffer
containing 50% formamide, 10% dextran, 0.3 M NaCI, 10 mM Tris (pH 8.0), 1 mM
EDTA, 1X Denhardt's Solution, and 200 mM dithiothreitol. The final
concentration
of each radiolabeled probe was 2 pW ol/ml of hybridization solution.
Microscope
slides containing sequential brain cryosections were independently exposed to
45 p.1
of hybridization solution (containing the antisense oligonucleotide probes
CON103a
and CON 103b) per slide and silanized cover slips were placed over the
sections being
exposed to hybridization solution. Sections were incubated overnight (15-18
hours) at
37°C to allow hybridization to occur.
Following the hybridization period, coverslips were washed off the
slides in 1X SSC. The cryosections were then subjected to three high-
stringency
washes in 1 X SSC at 65°C for 20 minutes each. Following two room-
temperature
washes, cryosections were dehydrated by consecutive exposure to 70%, 95%, and
100% ethanol (0.3 M ammonium acetate added to 70% and 95% ethanol solutions),
followed by air drying and exposure to Kodak BioMax MR-1 film. After 28 days
of

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exposure, the film was developed. Based on these results, brain sections that
showed
positive hybridization signals were coated with Kodak NTB-2 nuclear track
emulsion
and the slides were stored in the dark for four months. The slides were then
developed and counterstained with hematoxylin. Emulsion-coated sections were
analyzed microscopically to determine the specificity of labeling. l,he signal
was
determined to be specific if autoradiographic grains (generated by antisense
probe
hybridization) were present over cell bodies and not trapped between cell
bodies.
Specific labeling with the antisense probe occurred in all cortical
regions, including the piriform cortex and hippocampus. The specificity of
labeling
I 0 was confirmed by microscopic analysis of emulsion-coated cryosections.
These
sections revealed that the autoradiographic grains resulting from antisense
riboprobe
in situ hybridizations were distributed over cell bodies rather than trapped
between
cell bodies. The observed distribution of CON103 mRNA in the cortical and
paralimbic regions of the mammalian brain suggests that ligands for this GPCR
may
15 be involved in signal transductions important for cellular processes
underlying
neurological functioning. In addition, expression of CON103 in the brain
provides an
indication that modulators of CON103 activity have utility for treating
neurological
and neuropsychiatric disorders, including but not limited to, schizophrenia,
depression, anxiety, attention deficit disorder (with or without
hyperactivity), bipolar
20 disease, epilepsy, migraine, neuritis, neurasthenia, neuropathy, neuroses,
obesity,
Parkinson's disease, other demential, and the like. Use of CON103 modulators,
including CON103 ligands and anti-CON103 antibodies, to treat individuals
having
such disease states is intended as an aspect of the invention.
25 D. CON203
CON203-specific cRNA probes were prepared using conventional
techniques. Initially, a 293 by fragment of the CON203 coding region, with a
BamHl
site and an EcoRI site disposed on opposite ends, was prepared by PCR using
primers
LW 1314 (S'-GCATGAATTCCCACCTTCATCATC'rACCTC-3'; SEQ ID NO: 40)
30 and LW 1315 (5'-GCATGGATCCGAAGACCAAAAAGACCCAG-3'; SEQ ID NO:
41). LW1314 includes an EcoRT site and additional protective residues at its
5'

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tern~inus, with the rest of the sequence con-esponding to CON203 coding
nucleotides
164-183, which correspond to positions 309-328 of SEQ ID NO: 7. LW1315
includes
5' protective nucleotides and a BccmHI site, with the rest of the sequence
corresponding to the complement of CON203 coding nucleotides 438-456, which
con-espond to positions 583-601 of SEQ ID NO: 7. The PCR-amplified fragment
was
then digested with BacnHI and EcoR1 and ligated into the corresponding sites
of
pBluescript TI to yield pCon203 BS. The recombinant clone was then linearized
either
with BamHl or EcoRI. Linearization with BamHI provided a substrate for in
vitro
expression of a sense-strand cRNA probe using the vector-borne T7 'promoter.
Digestion with EcoRl was used to provide a substrate for in vitro
transcription using
the vector-borne T3 promoter to generate an anti-sense cRNA probe. In vitro
transcriptions were performed in the presence of [35S] UTP, thereby yielding
sense-
and anti-sense strand riboprobes having specific radioactivities of 5.38 x 10'
cpm/pmol and 5.34 x 10' cpm/pmol, respectively. Hybridization with the
riboprobes
1 S and subsequent washing of the slides was carried out as described above
for CON193
in Example 3A. Subsequently, the slides were exposed to Kodak BioMax MR-1
film.
After 9 days of exposure, the film was developed. Based on these results,
brain
sections that gave rise to positive hybridization signals were coated with
Kodak
NTB-2 nuclear track emulsion and the slides were stored in the dark for 25
days. The
slides were then developed as described above for CON193 in Example 3A.
Specific labeling with the antisense probe occurred in several limbic
and paralimbic regions, as well as areas thought to be involved in voluntary
motor
control. In particular, the probe hybridized to CON203 mRNAs in the following
regions of the brain: cortical regions, including the piriform cortex,
neostriatum,
lateral olfactory tract, hypothalamic nuclei, bed nucleus of the stria
terminalis,
amygdala, hippocampus, reticular thalamus and other thalamic regions,
subthalamic
nucleus, and the red nucleus. The specificity of labeling was confirmed by
microscopic analysis of emulsion-coated cryosections. These sections revealed
that
the autoradiographic grains resulting from antisense riboprobe ira .sitc.c
hybridizations
were distributed over cell bodies rather than trapped between cell bodies.
Confirming
expression of CON203 mRNA, the sense-strand riboprobe did not show specific

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hybridization. The observed distribution of CON203 mRNA in the cortical
(particularly, motor circuits) and paralimbic regions of the mammalian brain
suggests
that CON203 and the ligands for this GPCR may be involved in signal
transductions
important for cellular processes underlying neurological functioning. In
addition,
expression of CON203 in the brain provides an indication that modulators of
CON203 activity have utility for treating neurological disorders, including
but not
limited to, schizophrenia, depression, anxiety, bipolar disease, epilepsy,
migraine,
attention deficit disorder (with or without hyperactivity), neuritis,
neurasthenia,
neuropathy, neuroses, Parkinson's disease, dementia, obesity, and the like.
Use of
CON203 modulators, including CON203 ligands and anti-CON203 antibodies, to
treat individuals having such disease states is intended as an aspect of the
invention.
E. CON198
A 266 by fragment of CON198 containing EcoRI and BamHI
restriction sites was amplified from the full-length clone by PCR, using the
primers
LW 1308: S'-GCATGAATTCACTCACTTCTCATCTCCTTC-3' (SEQ >D NO: 46)
and LW1309:5'-GCATGGATCCAATCTCCTTTGTCTTCACTC-3' (SEQ ID NO:
47) Primer LW1308 contains an EcoRI site (underlined) followed by sequence
identical to nucleotides 638-657 of SEQ 117 NO: 9. Primer LW1309 contain a
BamHI
site (underlined) followed by sequence complementary to nucleotides 903-884 of
SEQ
ID NO: 9. The amplification product was digested with EcoRI and BamHI, and
then
subcloned into an EcoRI- and BamHI-digested pBluescript II vector
(Stratagene). The
266 amplified and subcloned basepairs correspond to nucleotides 638 to 903 of
SEQ
ID NO: 9.
The subcloned CON198-Bluescript construct was used to generate
strand-specific probes for the in situ hybridization experiments. The
construct was
linearized with BamHI, for labeling with T7 polymerase (sense), or EcoRI, for
T3
polymerase (antisense), and used as a template for in vitro transcription of
sense and
antisense cRNA riboprobes. The riboprobes were labeled with 3'S-UTP to yield a
specific activity of 0.45 x 10~ cpm/pmol for antisense and 0.732 x 10G
cpm/pmol for

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sense probe. Hybridization with the riboprobes and subsequent washing of the
slides was carried out as described above for CON I 93 in Example 3A.
Specific labeling with the antisense probe showed distribution of
CON198 mRNA in the rat brain in several limbic and paralimbic regions as well
as
areas thought to be involved in voluntary motor control. Labelled regions
included
cortical regions, piriform cortex, hypothalamic nuclei (paraventricular
nucleus,
supraoptic nucleus, suprachiasmatic nucleus), hippocampus, reticular thalmus,
substantia nigra-gars compacta (SN-C), ventral tegmental area, and the red
nucleus.
The specificity of labeling was confirmed by microscopic analysis of emulsion
coated
sections. These sections revealed that the autoradiographic grains generated
by the
antisense probe were distributed over cell bodies rather than trapped between
cell
bodies. Sense probe did not generate specific labeling.
The observed regional distribution of CON198 mRNA provides a
therapeutic indication for natural ligands for CON198 as well as modulators of
CON198 activity, such as anti-CON198 antibody substances or small molecules
that
agonize or antagonize ligand-mediated CON198 signalling. In particular, the
expression pattern provides an indication that such molecules will have
utility for
treating neurological and/or psychiatric diseases, including but not limited
to
schizophrenia, depression, anxiety, bipolar disease, affective disorders,
ADHD/ADD,
epilepsy, neuritis, neurasthenia, neuropathy, neuroses, Alzheimer's disease,
Parkinson's disease, migraine, senile dementia, and the like. Use of CON198
modulators, including CON198 ligands and anti-CON198 antibodies, to treat
individuals having such disease states is intended as an aspect of the
invention. Such
modulators are administered by any means effective to safely deliver the
modulators
to the CON198-expressing cells, including but not limited to oral
administration,
inhalation, or injection of compositions comprising the modulators in a
pharmaceutically acceptable diluent, adjuvant, or carrier. Efficacy of
treatment can
initially be determined in any accepted animal model that provides a
biochemical or
behavioral marker that correlates with disease severity or treatment efficacy.

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F. CON 197
A 261 by fragment of CON 197 COllta111117g EcoRl and f3ccnt H l
restriction sites was amplified from the full-length clone by PCR, using the
primers
LW 1306: 5'-GCATGAATTCTTCTACTTCATCATCCTCC-3' (SEQ 1D NO: 50) and
LW 1307: 5'-GCATGGATCCAAAGGCCATCACAACAAG-3' (SEQ ID NO: 51).
Primer LW 1306 includes sequence identical to nucleotides 100-1 18 of SEQ 1D
NO:
1 1 (underlined), preceded by an EcoRl site. Primer LW1307 includes sequence
complementary to nucleotides 361-343 of SEQ ~ NO: 11 (underlined), preceded by
a
BcemHl restriction site. The amplification product was digested with EcoRI and
BamHI, and then subcloned into an EcoRI- and BccmHI-digested pBluescript II
vector
(Stratagene). The 261 amplified and subcloned basepairs correspond to
nucleotides
100 to 361 of SEQ 1D NO: 11.
The subcloned CON197-Bluescript construct was used to generate
strand-specific probes for the in situ hybridization experiments. The
construct was
linearized with BamHI, for labeling with T7 polymerase (sense), or EcoRI, for
T3
polymerase (antisense), and used as a template for in vitro transcription of
sense and
antisense cRNA riboprobes. The riboprobes were labeled with 3SS-UTP to yield a
specific activity of 0.51 x 106 cpm/pmol for antisense and 0.432 x 10~'
cpm/pmol for
sense probe. Hybridization with the riboprobes and subsequent washing of the
slides
was carried out as described above for CON193 in Example 3A.
Specific labeling with the antisense probe showed wide spread
distribution of CON197 mRNA in the rat brain. Labelled regions included neo
and
allo cortex, piriform cortex, neostriatum, thalamic nuclei, hypothalamic
nuclei,
hippocampus, amygdala, cerebellum, and the olfactory bulb. The specificity of
labeling was confirmed by microscopic analysis of emulsion coated sections.
These
sections revealed that the autoradiographic grains generated by the antisense
probe
were distributed over cell bodies rather than trapped between cell bodies.
Sense probe
did not generate specific labeling.
The observed regional distribution of CON 197 mRNA provides a
therapeutic indication for natural ligands for CON197 as well as modulators of
CON 197 activity, such as anti-CON197 antibody substances or small molecules
that

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agonize or antagonize ligand-mediated CON197 signalling. In particular, the
expression pattern provides an indication that such molecules will have
utility for
treating neurological and/or psychiatric diseases, including but not limited
to
dementia, schizophrenia, depression, anxiety, bipolar disease, migraine,
Parkinson's
disease, affective disorders, Alzheimer's disease, senile dementia, attention
deficit
hyperactivity disorder/attention def cit disorder (ADHD/ADD), epilepsy,
neuritis,
neurasthenia, neuropathy, neuroses, and the like. Use of CON 197 modulators,
including CON 197 ligands and anti-CON197 antibodies, to treat individuals
having
such disease states is intended as an aspect of the invention. Such modulators
are
administered by any means effective to safely deliver the modulators to the
CON 197-
expressing cells, including but not limited to oral administration,
inhalation, or
injection of compositions comprising the modulators in a pharmaceutically
acceptable
diluent, adjuvant, or earner. Efficacy of treatment can initially be
determined in any
accepted animal model that provides a biochemical or behavioral marker that
1 S correlates with disease severity or treatment efficacy.
G. CON202
A 272 by fragment of CON202 containing EcoRI and BamHI
restriction sites was amplified from the full-length clone by PCR, using the
primers
LW 1310 GCATGAATTCGCAGAAGAAGGCTATTGG (SEQ ~ NO: 56) and
LW1311 GCATGGATCCGCAGTAAAGAAGGGTTGTG (SEQ ID NO: 57). The
amplification product was digested with EcoRI and BamHI, and then subcloned
into a
pBluescript II vector (Strategene) that was digested with EcoRI and BamHI. The
272
amplified and subcloned basepairs correspond to nucleotides 1065 to 1336 of
SEQ ID
NO: 13.
The subcloned CON202-Bluescript constn~ct was used to generate
strand-specific probes for the in situ hybridization experiments. The
construct was
linearized with BamHI, for labeling with T7 polymerase (sense), or EcoRI, for
T3
polymerase (antisense), and used as a template for in vitro transcription of
sense and
antisense cRNA riboprobes. The riboprobes were labeled with 35S-UTP to yield a
speciFc activity of 4.7 x 105 cpm/pmol for antisense and 4.3 x 10' cpm/pmol
for sense

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probe. Hybridization with the riboprobes and subsequent washing of the slides
was
can-ied out as described above for CON 193 in Example 3A.
Specific labeling with the antisense probe showed wide spread
distribution of CON202 mRNA in the rat brain. Labelled regions included the
cortical regions, lateral olfactory nuclei, hippocampus, subthalamic nucleus,
and at a
lower level, the nigra-pars compacta.
The observed regional distribution of CON202 mRNA provides a
therapeutic indication for natural ligands for CON202 as well as modulators of
CON202 activity, such as anti-CON202 antibody substances or small molecules
that
agonize or antagonize ligand-mediated CON202 signaling. In particular, the
expression pattern provides an indication that such molecules will have
utility for
treating neurological and/or psychiatric diseases, including but not limited
to
schizophrenia, affective disorders, attention deficit hyperactivity
disorder/attention
deficit disorder, depression, anxiety, bipolar disease, epilepsy, neuritis,
neurasthenia,
neuropathy, neuroses, Alzheimer's disease, Parkinson's disease, migraine,
senile
dementia and the like. Use of CON202 modulators, including CON202 ligands and
anti-CON202 antibodies, to treat individuals having such disease states is
intended as
an aspect of the invention. Such modulators are administered by any means
effective
to safely deliver the modulators to the CON202-expressing cells, including but
not
limited to oral administration, inhalation, or injection of compositions
comprising the
modulators in a pharmaceutically acceptable diluent, adjuvant, or carrier.
Efficacy of
treatment can initially be determined in any accepted animal model that
provides a
biochemical or behavioral marker that correlates with disease severity or
treatment
efficacy.
H. CON222
A 264 by fragment of CON222 containing EcoRI and BamHI
restriction sites was amplified from the full-length clone by PCR, using the
primers
LW 1472 (5'GCATGAATTCTGCCATGTCAATCATTTCTCTC3'; SEQ ID NO: 62,
EcoRI site is underlined) and LW 1473 (5'GCATGGATCCGTTCTGCATTTTCC-
AGGTCTC3'; SEQ ID NO: 63, BamHI site is underlined). The amplification product

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was digested with EcoRI and Baml-lI, and then subcloned into a predigested
pBluescript II vector (Stratagene). The 264 amplltied and subcloned basepaws
correspond to nucleotides 237 to 500 of SEQ ID NO: 15.
The subcloned CON222-Bluescript construct was used to generate
strand-specific probes for the in situ hybridization experiments. The
construct was
lincarized with BamHI, for labeling with T7 polymerase (sense), or EcoRI, for
T3
polylnerase (antisense), and used as a template .for in vitro transcription of
sense and
antisense cRNA riboprobes. The riboprobes were labeled with 35S-UTP to yield a
specific activity of 4.25 x 105 cpm/pmol for antisense and 3.9 x 105 cpm/pmol
for
sense probe. Hybridization with the riboprobes and subsequent washing of the
slides
was carried out as described above for CON 193 in Example 3A.
Specific labeling with the antisense probe showed wide spread
distribution of CON222 mRNA in the rat brain. Labelled regions included the
cortical regions, piriform cortex, striatum, hippocampus, thalamus,
hypothalamus,
dorsal raphe, and habenula.
The observed regional distribution of CON222 mRNA provides a
therapeutic indication for natural ligands for CON222 as well as modulators of
CON222 activity, such as anti-CON222 antibody substances or small molecules
that
agonize or antagonize ligand-mediated CON222 signaling. In particular, the
expression pattern provides an indication that such molecules will have
utility for
treating neurological and/or psychiatric diseases, including but not limited
to
schizophrenia, affective disorders, attention deficit hyperactivity
disorder/attention
deficit disorder, depression, anxiety, bipolar disease, epilepsy, neuritis,
neurasthenia,
neuropathy, neuroses, Alzhemeimer's disease, Parkinson's Disease, migraine,
senile
dementia, and the like. Use of CON222 modulators, including CON222 ligands and
anti-CON222 antibodies, to treat individuals having such disease states is
intended as
an aspect of the invention. Such modulators are administered by any means
effective
to safely deliver the modulators to the CON222-expressing cells, including but
not
limited to oral administration, inhalation, or injection of compositions
comprising the
modulators in a pharmaceutically acceptable diluent, adjuvant, or carrier.
Efficacy of
treatment can initially be determined in any accepted animal model that
provides a

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biochemical or behavioral marker that correlates with disease severity or
treatment
efficacy.
~nN~m
A 261 by fragment of CON215 containing EcoRI and BcamHl
restriction sites was amplified from the full-length clone by PCR, using the
primers
LW 1411: 5'-GCATGAATTCTGCCAAACATCATCCTGAC-3' (SEQ ID NO: 64)
and LW 1412: 5'-GCATGGATCCTACACAGCCACAACAACCC-3' (SEQ ID NO:
65). Primer LW1411 contains an EcoRI site (underlined) followed by sequence
identical to CON215 coding nucleotides 521-537, which correspond to positions
533-
549 of SEQ ID NO: 17. Primer LW1412 contain a BamH1 site (underlined) followed
by sequence complementary to CON215 coding nucleotides 764-781, which
correspond to positions 776-793 of SEQ ID NO: 17. The amplification product
was
digested with EcoRI and BamHI, and then subcloned into an EcoRI- and BamHI-
digested pBluescript II vector (Stratagene). The 261 amplified and subcloned
basepairs correspond to nucleotides 521 to 781 of SEQ ID NO: 17.
The subcloned CON215-Bluescript construct was used to generate
strand-specific probes for the in situ hybridization experiments. The
construct was
linearized with BamHI, for labeling with T7 polymerase (sense), or EcoRT, for
T3
polymerase (antisense), and used as a template for in vitro transcription of
sense and
antisense cRNA riboprobes. The riboprobes were labeled with 35S-UTP to yield a
specific activity of 48.03 x 10G cpm/pmol for antisense and 48.09 x 106
cpm/pmol for
sense probe. Hybridization with the riboprobes and subsequent washing of the
slides
was carried out as described above for CON193 in Example 3A.
Subsequently, the slides were exposed to Kodak BioMax MR-1 film.
After 9 days of exposure, the film was developed. Slides containing sections
that
showed a hybridization signal on film autoradiograms were coated with Kodak
NTB-2 nuclear track emulsion and stored in the dark for 25 days. The slides
were then
developed as described above for CON193 in Example 3A.
Specific labeling with the antisense probe showed distribution of
CON215 mRNA in the rat brain in limbic endocrine and motor circuits.
Specifically,

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CON215 mRNA was present in the cortex, hippocampus, and red nucleus. The
specificity of labeling was confirmed by microscopic analysis of emulsion
coated
sections. These sections revealed that the autoradiographic grains generated
by the
antisense probe were distributed over cell bodies rather than trapped between
cell
bodies. Sense probe did not generate specific labeling.
The observed regional distribution of CON21 S mRN~A provides a
therapeutic indication for natural ligands for CON21 S as well as modulators
of
CON215 activity, such as anti-CON215 antibody substances or small molecules
that
agonize or antagonize ligand-mediated CON1215 signaling. In particular, the
expression pattern provides an indication that such molecules will have
utility for
treating neurological and/or psychiatric diseases, including but not limited
to
schizophrenia, depression, anxiety, bipolar disease, epilepsy, migraine,
attention
deficit (with or without hyperactive disorder), neuritis, neuasthenia,
neuropathy,
neuroses, Parkinson's disease, dementia, obesity, and the like. Use of CON215
I S modulators, including CON215 ligands and anti-CON215 antibodies, to treat
individuals having such disease states is intended as an aspect of the
invention.
Such modulators are administered by any means effective to safely
deliver the modulators to the CON215-expressing cells, including but not
limited to
oral administration, inhalation, or injection of compositions comprising the
modulators in a pharmaceutically acceptable diluent, adjuvant, or carrier.
Efficacy of
treatment can initially be determined in any accepted animal model that
provides a
biochemical or behavioral marker that correlates with disease severity or
treatment
efficacy.
J. CON 217
Two oligonucleotides were designed based on SEQ ~ NO: 19 and
obtained from Sigma-Genosys (St. Louis, MO) to use as probes for ira situ
hybridization. The first oligonucleotide, designated 217A, has the sequence
5'TAGGTCGGTAGTCAGGACACGGGAGAACAGAACTGTTGGTTGA3' (SEQ
TD NO: 68) which is complementary to nucleotides 102 to 60 of SEQ TD NO: 19.
The
second oligonucleotide, designated 217B, has the sequence

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-Il3-
5'GCCCCTGTGGCGGTTTAGAfCCAGAATGCCCATTTTCTGTTCCATCTAAC
CA3' (SEQ ID NO: 69) which cowesponds to the complement of nucleotides I 530
to
1479 of SEQ 1D NO: 17. Both oligonucleotides, 217A and 217B, were
reconstituted
with lx TE buffer to a concentration of 20 pMol/ml and labeled with ;3P-dATP
to
yield a specific activity of 2.08 x 10~ and 1.53 x 10'' cpm/ml, respectively.
Hybridization was carried out at 37°C overnight as described above
for
CON193 in Example 3A. Following the hybridizations, the coverslips were washed
off the slides with 1 x SSC for 45 minutes. The slides were then washed for 20
minutes at room temperature in Ix SSC followed by three high stringency washes
in
lx SSC at 65°C. After washing, the slides were dehydrated with 70%,
95%, and
100% ethanol containing 0.3 mM NH40Ac, air-dried, and exposed to Kodak BioMax
MR-1 film. After 21 days of exposure, the film was developed. Based on these
results, sections that showed a hybridization signal on film autoradiography
were
coated with Kodak NTB-2 nuclear track emulsion and stored in the dark for 42
days.
The slides were then developed and counterstained with hematoxylin. Emulsion-
coated sections were analyzed microscopically to determine the specificity of
labeling.
The signal was judged to be specific if autoradiographic grains (generated by
antisense probe hybridization) were associated clearly with crystal violet
stained cell
bodies. Autoradiographic grains found between cell bodies were deemed non-
specific.
Specific labeling with the antisense probe showed wide spread
distribution of CON217 mRNA in the rat brain. Labelled regions included the
cortex,
piriform cortex, hippocampus, cerebellum, medulla, spinal cord, temporal lobe,
putamen, substantia nigra and thalamus.
The observed regional distribution of CON217 mRNAs provide a
therapeutic indication for natural ligands for these G protein-coupled
receptors as well
as modulators of their activity, such as anti-CON217 antibody substances or
small
molecules that mimic, agonize or antagonize ligand-mediated CON217 signaling.
In
particular, the expression patterns provide an indication that such molecules
will have
utility for treating neurological and/or psychiatric diseases, including but
not limited
to schizophrenia, affective disorders, attention deficit hyperactivity
disorder/attention

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deficit disorder, depression, anxiety, bipolar disease, epilepsy, neuritis,
neurasthenia,
neuropathy, neuroses, Alzhemeimer's disease, Parkinson's Disease, migraine,
senile
dementia, and the like. Use of CON217 polypeptide modulators, including CON217
ligands and anti-CON217 polypeptide antibodies, to treat individuals having
such
disease states is intended as an aspect of the invention. Such modulators are
administered by any means effective to safely deliver the modulators to the
GPCR
polypeptide-expressing cells, including but not limited to oral
administration,
inhalation, or injection of compositions comprising the modulators in a
pharmaceutically acceptable diluent, adjuvant, or carrier. Efficacy of
treatment can
initially be determined in any accepted animal model that provides a
biochemical or
behavioral marker that correlates with disease severity or treatment efficacy.
EXAMPLE 4
Recombinant Expression of GPCR Polypeptides in Eukaryotic Host Cells
To produce GPCR protein, a GPCR polypeptide-encoding
polynucleotide is expressed in a suitable host cell using a suitable
expression vector,
using standard genetic engineering techniques. For example, one of the GPCR
polypeptide-encoding sequences described in Example 1 (such as SEQ )D NOS: 1,
3,
5, 7, 9, 11, 13, 15, 17 or 19) is subcloned into the commercial expression
vector
pzeoSV2 (Invitrogen, San Diego, CA) and transfected into Chinese Hamster Ovary
(CHO) cells (ATCC CRL-1781) using the transfection reagent fuGENE 6
(Boehringer-Mannheim) and the transfection protocol provided in the product
insert.
Additional eukaryotic cell lines, such as African Green Monkey Kidney cells
(COS-
7, ATCC CRL-1651) or Human Kidney cells (HEK 293, ATCC CRL-1573), may be
used as well. Cells stably expressing a GPCR polypeptide (e.g., CON193,
CON166,
CON103, CON203, CON198, CON197, CON202, CON222, CON215, or CON217)
are selected by growth in the presence of 100 mg/ml zeocin (Stratagene,
LaJolla,
CA). Optionally, GPCR polypeptide is purified from the cells using standard
chromatographic techniques. To facilitate purification, antisera is raised
against one
or more synthetic peptide sequences that correspond to portions of the GPCR
amino
acid sequence, and the antisera is used to affinity purify GPCR polypeptides.
The

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GPCR gene also may be expressed in frame with a tag sequence (e.g.,
polyhistidine,
hcmaggluttinin, FLAG) to facilitate purification. Moreover, it will be
appreciated that
many of the uses for GPCR polypeptides, such as assays described below, do not
require purification of GPCR polypeptides from the host cell.
EXAMPLE 5
Antibodies to GPCR Polypeptides
Standard techniques are employed to generate polyclonal or
monoclonal antibodies to the GPCR receptors (e.g., CON 193, CON 166, CON i 03,
CON203, CON198, CON197, CON202, CON222, CON215, or CON217), and to
generate useful antigen-binding fragments thereof or variants thereof,
including
"humanized" variants. Such protocols can be found, for example, in Sambrook et
al.,
Molecular Cloning: a Laboratory Manual. Second Edition, Cold Spring Harbor,
New York: Cold Spring Harbor Laboratory (1989); Harlow et al. (Eds),
Antibodies A
Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor , NY
( 1988); and other documents cited below. In one embodiment, recombinant GPCR
polypeptides (or cells or cell membranes containing such polypeptides) of the
invention are used as an antigen to generate the antibodies. In another
embodiment,
one or more peptides having amino acid sequences corresponding to an
immunogenic
portion of a GPCR polypeptide (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19,
20, or more amino acids) are used as antigen. Peptides corresponding to
extracellular
portions of GPCR polypeptides, especially hydrophilic extracellular portions,
are
preferred. The antigen may be mixed with an adjuvant or linked to a hapten to
increase antibody production.
A. Polyclonal or Monoclonal antibodies
As one exemplary protocol, a recombinant GPCR polypeptide or
synthetic fragment thereof is used to immunize a mouse for generation of
monoclonal
antibodies (or larger mammal, such as a rabbit, for polyclonal antibodies). To
increase antigenicity, peptides are conjugated to Keyhole Lympet Hemocyanine
(Pierce), according to the manufacturer's recommendations. For an initial
injection,

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the antigen is emulsified with Freund's Complete Adjuvant and injected
subcutaneously. At intervals of two to three weeks, additional aliquots of
GPCR
antigen are emulsified with Freund's Lneomplete Adjuvant and injected
subcutaneously. Prior to the final booster injection, a serum sample is taken
from the
immunized mice and assayed by Western blot to confirm the presence of
antibodies
that immunoreact with GPCR polypeptide. Serum from the immunized animals may
be used as a polyclonal antisera or used to isolate polyclonal antibodies that
recognize
GPCR polypeptide. Alternatively, the mice are sacrificed and their spleen
removed
for generation of monoclonal antibodies.
To generate monoclonal antibodies, the spleens are placed in 10 ml
serum-free RPMI 1 G40, and single cell suspensions are formed by grinding the
spleens in serum-free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM
sodium pyruvate, 100 units/ml penicillin, and 100 p,g/ml streptomycin (RPMI)
(Gibco, Canada). The cell suspensions are filtered and washed by
centrifugation and
resuspended in serum-free RPMI. Thymocytes taken from three naive Balb/c mice
are prepared in a similar manner and used as a Feeder Layer. NS-1 myeloma
cells,
kept in log phase in RPMI with 10% fetal bovine serum (FBS) (Hyclone
Laboratories,
Inc., Logan, Utah) for three days prior to fusion, are centrifuged and washed
as well.
To produce hybridoma fusions, spleen cells from the immunized mice
are combined with NS-1 cells and centrifuged, and the supernatant is
aspirated. The
cell pellet is dislodged by tapping the tube, and 2 ml of 37°C PEG 1500
(50% in
75mM Hepes, pH 8.0) (Boehringer Mannheim) is stirred into the pellet, followed
by
the addition of serum-free RPMI. Thereafter, the cells are centrifuged and
resuspended in RPMl containing 15% FBS, 100 pM sodium hypoxanthine, 0.4 pM
aminopterin, 16 p,M thymidine (HAT) (Gibco), 25 units/ml of 1L-6 (Boehringer
Mannheim) and 1.5 x 106 thymocytes/ml and plated into 10 Corning flat-bottom
96-well tissue culture plates (Corning, Corning New York).
On days 2, 4, and 6, after the fusion, 100 p,1 of medium is removed
from the wells of the fusion plates and replaced with fresh medium. On day 8,
the
fusions are screened by ELISA, testing for the presence of mouse IgG that
binds to a

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GPCR polypeptidc. Selected fusion wells are further cloned by dilution until
monoclonal cultures producing anti-GPCR polypeptide antibodies are obtained.
B. Humanization of Anti-GPCR Monoclonal Antibodies
S The expression patterns of GPCR polypepties as reported herein and
the proven track record of GPCR's as targets for therapeutic intervention
suggest
therapeutic indications for GPCR polypeptide inhibitors (antagonists). GPCR
polypeptide-neutralizing antibodies comprise one class of therapeutics useful
as
antagonists. Following are protocols to improve the utility of anti-GPCR
polypeptide
monoclonal antibodies as therapeutics in humans, by "humanizing" the
monoclonal
antibodies to improve their serum half life and render them less immunogenic
in
human hosts (i.e., to prevent human antibody response to non-human anti-GPCR
polypeptide antibodies).
The principles of humanization have been described in the literature
and are facilitated by the modular arrangement of antibody proteins. To
minimize the
possibility of binding complement, a humanized antibody of the IgG4 isotype is
preferred.
For example, a level of humanization is achieved by generating
chimeric antibodies comprising the variable domains of non-human antibody
proteins
of interest with the constant domains of human antibody molecules. (See, e.g.,
Morrison and Oi, Adv. Immunol., 44:65-92 (1989). The variable domains of GPCR-
neutralizing anti-GPCR antibodies are cloned from the genomic DNA of a B-cell
hybridoma or from cDNA generated from mRNA isolated from the hybridoma of
interest. The V region gene fragments are linked to exons encoding human
antibody
constant domains, and the resultant construct is expressed in suitable
mammalian host
cells (e.g., myeloma or CHO cells).
To achieve an even greater level of humanization, only those portions
of the variable region gene fragments that encode antigen-binding
complementarity
determining regions ("CDR") of the non-human monoclonal antibody genes are
cloned into human antibody sequences. [See, e.g., Jones et al., Nature,
321:522-525
(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et o1.,
Science,

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239:1534-36 (1988); and Tempest et al.. BiolTechnologv. 9:266-71 (1991). If
necessary, the (3-sheet framework of the human antibody surrounding the CDR3
regions also is modified to more closely mirror the three dimensional
structure of the
antigen-binding domain of the original monoclonal antibody. (See Kettleborough
et al., Protein. Engin.. 4:773-783 ( 1991 ); and Foote et crl., J. Mol. Biol.,
224:487-499
( 1992).
In an alternative approach, the surface of a non-human monoclonal
antibody of interest is humanized by altering selected surface residues of the
non-human antibody, e.g., by site-directed mutagenesis, while retaining all of
the
interior and contacting residues of the non-human antibody. See Padlan,
Molecular
Immunol., 28(4/5):489-98 ( 1991 ).
The foregoing approaches are employed using GPCR-neutralizing
anti-GPCR monoclonal antibodies and the hybridomas that produce them to
generate
humanized GPCR-neutralizing antibodies useful as therapeutics to treat or
palliate
conditions wherein GPCR expression or ligand-mediated GPCR signaling is
detrimental.
C. Human GPCR-Neutralizing Antibodies from Phage Display
Human GPCR-neutralizing antibodies are generated by phage display
techniques such as those described in Aujame et al., Human Antibodies, 8(4):1
SS-168
(1997); Hoogenboom, TIBTECH. 15:62-70 (1997); and Rader et al., Curr. Opin.
Biotechnol., 8:503-508 ( 1997), all of which are incorporated by reference.
For
example, antibody variable regions in the form of Fab fragments or linked
single
chain Fv fragments are fused to the amino terminus of filamentous phage minor
coat
protein pIII. Expression of the fusion protein and incorporation thereof into
the
mature phage coat results in phage particles that present an antibody on their
surface
and contain the genetic material encoding the antibody. A phage library
comprising
such constructs is expressed in bacteria, and the library is panned (screened)
for
GPCR-specific phage-antibodies using labelled or immobilized GPCR polypeptide
as
antigen-probe.

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D. Human CPCR-Neutralizing Antibodies from Transgenic Mice
Human GPCR-neutralizing antibodies are generated in transgenic mice
essentially as described in Bruggemann and Neuberger, lmntunol. Today,
l7(8):391-97 (1996) and Bruggemann and Taussig, Curr. Opiu. Biotechnol., 8:455-
58
( 1997). Transgenic mice carrying human V-gene segments in germline
configuration
and that express these transgenes in their lymphoid tissue are immunized with
a
GPCR composition using conventional immunization protocols. Hybridomas are
generated using B cells from the immunized mice using conventional protocols
and
screened to identify hybridomas secreting anti-GPCR human antibodies (e.g., as
described above).
EXAMPLE 6
Assays to Identify Modulators of GPCR Poly~eptide Activitx
Set forth below are assays for identifying modulators (agonists and
antagonists) of GPCR polypeptide activity. Among the modulators that can be
identified by these assays include natural ligand compounds of the receptor;
synthetic
analogs and derivatives of natural ligands; antibodies, antibody fragments,
and/or
antibody-like compounds derived from natural antibodies or from antibody-like
combinatorial libraries; and/or synthetic compounds identified through high
throughput screening of libraries; and the like. All modulators that bind GPCR
polypeptide are useful for identifying GPCR polypeptide in tissue samples
(e.g., for
diagnostic purposes, pathological purposes, and the like). Agonist and
antagonist
modulators are useful for up-regulating and down-regulating GPCR polypeptide
activity, respectively, to treat disease states characterized by abnormal
levels of GPCR
polypeptide activity. GPCR polypeptide binding molecules also may be used to
deliver a therapeutic compound or a label to cells that express GPCR
polypeptide
(e.g., by attaching the compound or label to the binding molecule). The assays
may
be performed using single putative modulators, and/or may be performed using a
known agonist in combination with candidate antagonists (or visa versa).
Performance of the assays using any of the GPCR polypeptides of the invention
described herein (e.g., CON193, CON166, CON103, CON203, CON198, CON197,

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CON202, CON222, CON215, or CON217) is contemplated. It will be appreciated
that co-transfecting cells with two or more of the receptors for simultaneous
screening
also is possible.
S A. cAMP Assays
In one type of assay, levels of cyclic adenosine monophosphate
(CAMP) are measured in GPCR-transfected cells that have been exposed to
candidate
modulator compounds. Protocols for cAMP assays have been described in the
literature. [See, e.g., Sutherland et al., Circulation, 37: 279 (1968);
Frandsen, E.K.
and Krishna, G, Life Sciences, 18: 529-541 (1976); Dooley et crl., Journal of
Pharmacology and Experimental Therapeutics, 283 (2): 735-41 (1997); and George
et
crl., Journal of Biomolecular Screening, 2 (4): 235-40 (1997).] An exemplary
protocol for such an assay, using an Adenylyl Cyclase Activation FlashPlate~
Assay
from NENTM Life Science Products, is set forth below.
1 S Briefly, the GPCR coding sequence (e.g., a cDNA or intronless
genomic DNA) is subcloned into a commercial expression vector, such as pzeoSV2
(Invitrogen, San Diego, CA), and transiently transfected into Chinese Hamster
Ovary
(CHO) cells using known methods, such as the transfection reagent FuGENE 6
(Boehringer-Mannheim) and the transfection protocol provided in the product
insert.
The transfected CHO cells are seeded into the 96 well microplates
from the FlashPlate~ assay kit, which are coated with solid scintillant to
which
antisera to CAMP has been bound. For a control, some wells are seeded with
wild
type (untransfected) CHO cells. Other wells on the plate receive various
amounts of
cAMP standard solution for use in creating a standard curve.
One or more test compounds are added to the cells in each well, with
water and/or compound-free media/diluent serving as a control. After
treatment,
CAMP is allowed to accumulate in the cells for exactly 15 minutes at room
temperature. The assay is terminated by the addition of lysis buffer
containing [''S1]-
labelled cAMP, and the plate is counted using a Packard TopcountT"' 96-well
microplate scintillation counter. Unlabelled cAMP from the lysed cells (or
from
standards) competes with the fixed amounts of ['ZSI]-cAMP for antibody bound
to the

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plate. A standard curve is constructed, and cAMP values for the unknowns are
obtained by interpolation. Changes in intracellular cAMP level of the cells in
response to exposure to a test COll1pOLlild are indicative of GPC.R
polypeptide
modulating activity. Modulators that act as agonists at receptors which couple
to the
Gs subtype of G-proteins will stimulate production of cAMP, leading to a
measurable
3-10 fold increase. Receptor agonists which couple to the Gi/o subtype of G-
proteins
will inhibit forskolin-stimulated cAMP production, leading to a measurable
decrease
of 50-100%. Modulators that act as inverse agonists will reverse these effects
at
receptors that are either constitutively active or activated by known
agonists.
B. Aequorin Assay
In another assay cells (e.g., CHO cells) are transiently co-transfected
with both a GPCR expression construct and a construct that encodes the
photoprotein
apoaequorin. In the presence of the cofactor coelenterazine, apoaequorin will
emit a
measurable luminescence that is proportional to the amount of intracellular
(cytoplasmic) free calcium. [See generally Cobbold P.H. and Lee, J.A.C.
"Aequorin
measurements of cytoplasmic free calcium. In: McCormack J.G. and Cobbold P.H.,
eds., Cellular Calcium: A Practical Approach. Oxford:IRL Press (1991); Stables
et
al., Analytical Biochemistry, 252: 1 I 5-26 (1997); and Haugland, R.P.
Handbook of
Fluorescent Probes and Research Chemicals. Sixth edition. Eugene OR: Molecular
Probes (1996).]
In one exemplary assay, a GPCR-encoding polynucleotide is subcloned
into the commercial expression vector pzeoSV2 (Invitrogen, San Diego, CA) and
transiently co-transfected along with a construct that encodes the
photoprotein
apoaequorin (Molecular Probes, Eugene, OR) into CHO cells using the
transfection
reagent FuGENE 6 (Boehringer-Mannheim) and the transfection protocol provided
in
the product insert.
The cells are cultured for 24 hours at 37°C in aMEM (Gibco/BRL,
Gaithersburg, MD) supplemented with 10% FBS, 2 mM glutamine, 10 U/ml of
penicillin and 10 p,g/ml of streptomycin. Subsequently, the media is changed
to
serum-free aMEM containing 5 pM coelenterazine (Molecular Probes, Eugene, OR),

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and the cells are cultured for two additional hours at 37°C. Cells are
then detached
from the plate using VE.RSEN (Gibco/BRL), washed and resuspended at 2 x 10'
cells/ml in serum-free aMEM. .
Dilutions oi~candidate GPCR modulator drugs are prepared in serum-
s free aMEM and dispensed into wells of an opaque 96-well assay plate, 50
pl/well.
Plates are loaded onto an MLX microtiter plate luminometer (Dynex
Technologies,
Inc., Chantilly, VA). The instrument is programmed to dispense 50 p.1 of cell
suspension into each well, one well at a time, and immediately read
luminescence for
15 seconds. Dose-response curves for the modulator candidates are constructed
using
the area under the curve for each light signal peak. Data are analyzed with
SlideWrite, using the equation For 1-site ligand, and ECso values are
obtained.
Changes in luminescence caused by the drugs are considered indicative of
modulatory
activity. Modulators that act as receptor agonists which couple to the Gq
subtype of
G-proteins give an increase in luminescence of up to 100 fold. Modulators that
act as
inverse agonists will reverse this effect at receptors that are either
constitutively active
or activated by known agonists.
C. Luciferase Reporter Gene Assay
The photoprotein luciferase provides another useful tool for assaying
for modulators of GPCR activity. Cells (e.g., CHO cells or COS 7 cells) are
transiently co-transfected with both a GPCR expression construct (e.g., GPCR-
encoding sequence in pzeoSV2 (Invitrogen, San Diego, CA)) and a reporter
construct
which includes a gene for the luciferase protein downstream from a
transcription
factor, either cAMP-response element (CRE), AP-l, or NF kappa B. Agonist
binding
to receptors coupled to the Gs subtype of G-proteins leads to increases in
CAMP,
activating the CRE transcription factor and resulting in expression of the
luciferase
gene. Agonist binding to receptors coupled to the Gq subtype of G-protein
leads to
production of diacylglycerol that activates protein kinase C. As a result, the
AP-1 or
NF kappa B transcription factors are activated which stimulate expression of
the
luciferase gene. Expression levels of luciferase reflect the activation status
of the
signaling events. [See generally George et al., Journal of Biomolecular
Screening,

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- 12; _
2(4): 235-40 (1997); and Stratowa et n1., C'~n-refrt Uninion in Biotechnoloy.
6: 574-81
(1995).] Luciferase activity may be quantitatively measured using, e.g.,
luciferase
assay reagents that are commercially available from Promega (Madison, WI).
In one exemplary assay, CHO cells are plated in 24-well culture dishes
at a density of 100,000 cells/well one day prior to transfection and cultured
at 37°C in
aMEM (Gibco/BRL, Gaithersburg, MD) supplemented with 10% FBS, 2 mM
glutamine, 10 U/ml penicillin and 10 p.g/ml streptomycin. Cells are
transiently
co-transfected with both a GPCR expression construct and a reporter construct
containing the luciferase gene. The reporter plasmids CRE-luciferase, AP-1-
luci.ferase
and NF kappa B-luciferase may be purchased from Stratagene (LaJolla, CA).
Transfections are performed using FuGENE 6 transfection reagent
(Boehringer-Mannheim), and the protocol provided in the product insert. Cells
transfected with the reporter construct alone are used as a control. Twenty-
four hours
after transfection, cells are washed once with phosphate buffered saline (PBS)
pre-warmed to 37°C. Serum-free aMEM is then added to the cells either
alone
(control) or with one or more candidate modulators and the cells are incubated
at
37°C for five hours. Thereafter, cells are washed once with ice cold
PBS and lysed by
the addition of 100 p,1 of lysis buffer/well (from luciferase assay kit,
Promega,
Madison, WI). After incubation for 15 minutes at room temperature, 1 S p,1 of
the
lysate is mixed with 50 p1 substrate solution (Promega) in an opaque white 96-
well
plate, and the luminescence is read immediately on a Wallace model 1450
MicroBeta
scintillation and luminescence counter (Wallace Instruments, Gaithersburg,
MD).
Differences in luminescence in the presence versus the absence of a
candidate modulator compound are indicative of modulatory activity. Receptors
that
are either constitutively active or activated by agonists give a 3-20 fold
stimulation of
luminescence compared to cells transfected with the reporter gene alone.
Modulators
that act as inverse agonists will reverse this effect.
D. Intracellular Calcium Measurement usin~a FLIPR
Changes in intracellular calcium levels are another recognized
indicator of G protein-coupled receptor activity, and such assays can be
employed to

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evaluate modulators of GPCR activity. For example, CHO cells stably
transfected
with a GPCR expression vector are plated at a density of 4 x I Oa cells/well
in Packard
black-walled 96-well plates specially designed to isolate fluorescent signal
to
individual wells. The cells are incubated for 60 minutes at 37°C in
modified
Dulbecco's PBS (D-PBS) containing 36 mg/L of pyruvate and 1 g/L of glucose
with
the addition of 1% FBS and one of four calcium indicator dyes (Fluo-3TM AM,
Fluo-
4TN' AM, Calcium GreenTM-1 AM, or Oregon GreenT"'' 488 BAPTA-1 AM) at a
concentration of 4 p.M. Plates are washed once with modified D-PBS without 1%
FBS and incubated for 10 minutes at 37°C to remove residual dye from
the cellular
membrane. In addition, a series of washes with modified D-PBS without 1% FBS
is
performed immediately prior to activation of the calcium response.
Calcium response is initiated by the addition of one or more candidate
receptor agonist compounds, calcium ionophore A23187 ( 10 p,M), or ATP (4 pM).
Fluorescence is measured by Molecular Device's FLIPR with an argon laser,
excitation at 488 nm. [See, e.g., Kuntzweiler et al.. Dratg Development
Research,
44(I): 14-20 (1998).] The F-stop for the detector camera was set at 2.5 and
the length
of exposure was 0.4 milliseconds. Basal fluorescence of cells was measured for
20
seconds prior to addition of agonist, ATP, or A23187, and was subtracted from
the
response signal. The calcium signal is measured for approximately 200 seconds,
taking readings every two seconds. Calcium ionophore and ATP increase the
calcium
signal 200% above baseline levels. In general, activated orphan GPCRs increase
the
calcium signal approximately 10-15% above baseline signal.
E. Mitogenesis AssaX
In mitogenesis assays, the ability of candidate modulators to induce or
inhibit GPCR-mediated cell growth is determined. [See, e.g., Lajiness et cal.,
Journal
of Pharmacology and Experimental Therapeutics, 267(3): 1573-81 (1993).]
For example, CHO cells stably expressing a GPCR are seeded into 96-
well plates at a density of 5000 cells/well and grown at 37°C in aMEM
supplemented
with 10% fetal calf serum. After 48 hours, the cells are rinsed twice with
serum-free
aMEM and 80 p.1 of fresh aMEM, or aMEM containing a known mitogen, is added

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along with 20 p1 aMEM containing varying concentrations of one or more test
compounds diluted in serum free media. As controls, some wells on each plate
receive serum-free media alone, and some receive media containing 10% FBS.
Untransfected cells or cells transfected with vector alone also may serve as
controls.
After culture for 16-18 hours, 1 ~,Ci/well of [3H]-thymidine (2
Ci/mmol; cpm) is added to the wells and cells are incubated for an additional
2 hours
at 37°C. The cells are trypsinized and harvested onto filter mats with
a cell harvester
(Tomtec) and the filters are counted in a Betaplate counter. The incorporation
of 3H-
thylnidine in semm-free test wells is compared to the results achieved in
cells
stimulated with serum. Use of multiple concentrations of test compounds
perniits
creation and analysis of dose-response curves using the non-linear, least
squares fit
equation: A = B x [C/ (D + C)] + G where A is the percent of serum
stimulation; B is
the maximal effect minus baseline; C is the ECSO; D is the concentration of
the
compound; and G is the maximal effect. Parameters B, C and G are determined by
Simplex optimization.
Agonists that bind to the receptor are expected to increase
[3H]-thymidine incorporation into cells, showing up to 80% of the response to
serum.
Antagonists that bind to the receptor will inhibit the stimulation seen with a
known
agonist by up to 100%.
F. [35S]GTPyS Binding Assay
Because G protein-coupled receptors signal through intracellular "G
proteins" whose activity involves GTP/GDP binding and hydrolysis. Another
indicator of GPCR modulator activity is measuring binding of the non-
hydrolyzable
GTP analog [35S]GTPyS in the presence and absence of putative modulators.
[See,
e.g., Kowal, et czl., Neuropharmacology, 37: 179-87 (1998).]
Tn one exemplary assay, cells stably transfected with a GPCR
expression vector are grown in 10 cm dishes to subconfluence, rinsed once with
S ml
of ice cold Ca'-"/MgZ+ free PBS, and scraped into 5 ml of the same buffer.
Cells are
pelleted by centrifugation (500 x g, 5 minutes), resuspended in TEE buffer (25
mM
Tris, S mM EDTA, 5 mM EGTA, pH 7.5) and frozen in liquid nitrogen. After

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thawing, the cells are homogenized using a dounce (one ml TEE per plate of
cells),
and centrifuged at 1,000 x g for 5 minutes to remove nuclei and unbroken
cells.
The homogenate supernatant is centrifuged at 20,000 x g for 20
minutes to isolate the membrane fraction. The membrane pellet is then washed
once
with TEE and resuspended in binding buffer (20 mM HEPES, pH 7.5, 150 mM NaCI,
mM MgCIZ, 1 mM EDTA). The resuspended membranes can be frozen in liquid
nitrogen and stored at -70°C until use.
Aliquots of cell membranes prepared as described above and stored at
-70°C are thawed, homogenized, and diluted to a concentration of 10-50
~,g/ml in
10 buffer containing 20 mM HEPES, 10 mM MgCl2, 1 mM EDTA, 120 mM NaCI, l0
p,M GDP, and 0.2 mM ascorbate. In a final volume of 90 p.1, homogenates are
incubated with varying concentrations of putative modulator compounds or 100
p,M
GTP for 30 minutes at 30°C and then placed on ice. To each sample, 10
p,1 guanosine
5'-O-(3[35S]thio) triphosphate (NEN, 1200 Ci/mmol), ([35S]-GTPyS), was added
to a
final concentration of 100-200 pM. Samples are incubated at 30°C for an
additional
30 minutes. The reaction is then stopped by the addition of 1 ml of 10 mM
HEPES,
and 10 mM MgCl2 (pH 7.4), at 4°C, and filtration.
Samples are filtered over Whatman GF/B filters. These filters are
washed with 20 ml ice-cold 10 mM HEPES (pH 7.4) and 10 mM MgClz and counted
by liquid scintillation spectroscopy. Nonspecific binding of [35S]-GTPyS is
measured
in the presence of 100 p,M GTP and subtracted from the total. Compounds are
selected that modulate the amount of [35S)-GTPyS binding in the cells,
compared to
untransfected control cells. Activation of receptors by agonists gives up to a
five-fold
increase in [35S]GTPyS binding. This response is blocked by antagonists.
G. MAP Kinase Activity Assay
Evaluation of MAP Kinase activity in cells expressing a GPCR provide
another assay to identify modulators of GPCR activity. [See, e.g., Lajiness et
al.,
Journal ofPharmacology and Experimental Therapeutics, 267(3): 1573-81 (1993);
and Boulton et czl., Cell, 65: 663-75 (1991).]

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In one embodiment, CHO cells stably transfected with a GPCR-
encoding polynucleotide are seeded into G well plates at a density of 70,000
cells/well
48 hours prior to the assay. During this time, the cells are cultured at
37°C in aMEM
media supplemented with 10% FBS, 2 mM glutamine, 10 U/ml penicillin and 70
~.g/ml streptomycin. The cells are serum starved for 1-2 hours prior to the
addition of
stimulants.
For the assay, the cells are treated with media alone or media
containing a putative agonist or phorbal ester-myistoyl acetate (PMA) as a
positive
control. After treatment, cells are incubated at 37°C for varying
times. To stop the
reaction, the plates are placed on ice, the media is aspirated, and the cells
are rinsed
with 1 ml of ice-cold PBS containing 1 mM EDTA. Thereafter, 200 ~,1 cell lysis
buffer (12.5 mM MOPS (pH 7.3), 12.5 mM ~3-glycerophosphate, 7.5 mM MgClz, 0.5
mM EGTA, 0.5 mM sodium vanadate, 1 mM benzamidine, 1 mM dithiothreitol, 10
~,g/ml leupeptin, 10 ~,g/ml aprotinin, 2 ~,g/ml pepstatin A, and 1 ~,M okadaic
acid) is
added to the cells. The cells are scraped from the plates and homogenized by
10
passages through a 23 3/4 gauge needle. The cytosol fraction is prepared by
centrifugation at 20,000 x g for 15 minutes.
Aliquots (S-10 p1 containing 1-S ~.g protein) of cytosols are mixed with
1 mM MAPK Substrate Peptide (APRTPGGRR; SEQ ~ NO: 25); Upstate
Biotechnology, Inc., N.Y.) and 50 ~,M (y_3zP~ATP, (NEN, 3000 Ci/mmol) diluted
to a
final specific activity of 2000 cpm/pmol in a total volume of 25 P1. The
samples are
incubated for 5 minutes at 30°C, and reactions are stopped by spotting
20 ~.l on 2 cmz
of Whatman P81 phosphocellulose paper. The filter squares are washed in 4
changes
of 1% H3P04, and the squares are counted by liquid scintillation spectroscopy.
Equivalent cytosolic extracts are incubated without MAPK substrate peptide,
and the
cpm from these samples are subtracted from the matched samples with the
substrate
peptide. The cytosolic extract from each well is used as a separate point.
Protein
concentrations are determined by a dye binding protein assay (Bio-Rad).
Agonist
activation of the receptor is expected to result in up to a five fold increase
in MAPK
enzyme activity. This increase is blocked by antagonists.

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Fl. 1'H~Arachidonic Acid Release
The activation of GPCR's also has been observed to potentiate
arachidonic acid release in cells, providing yet another useful assay for
modulators of
the activity of GPC:R's of the present invention. [See, e.g., Kantenoan et
cd.,
Molecular Pharmacology, 39: 364-9 (1991 ).] For example, CHO cells that are
stably
transfected with a GPCR expression vector are plated in 24-well plates at a
density of
15,000 cells/well and grown in aMEM media supplemented with 10% FBS, 2 mM
glutamine, 10 U/ml penicillin and 10 pg/ml streptomycin for 48 hours at
37°C before
use. Cells of each well are labeled by incubation with [3H]arachidonic acid
(Amersham Corp., 210 Ci/mmol) at 0.5 p,Ci/ml in 1 ml aMEM supplemented with 10
mM HEPES (pH 7.5), and 0.5% fatty-acid-free bovine serum albumin for 2 hours
at
37°C. The cells are then washed twice with 1 ml of the same buffer.
Candidate modulator compounds are added in 1 ml of the same buffer,
either alone or containing 10 p,M ATP (Adenosine 5'-triphosphate) and the
cells are
incubated at 37°C for 30 minutes. Buffer alone and mock transfected
cells are used as
controls. Samples (0.5 ml) from each well are counted by liquid scintillation
spectroscopy. Agonists which activate the receptor will lead to potentiation
of the
ATP-stimulated release of [3H]-arachidonic acid. This potentiation is blocked
by
antagonists.
I. Extracellular Acidification Rate
In yet another assay, the effects of putative modulators of GPCR
activity are assayed by monitoring extracellular changes in pH induced by the
putative
modulators. [See, e.g., Dunlop et al., Journal of Pharmacological and
Toxicological
Methods, 40(I): 47-55 (1998).]
CHO cells transfected with a GPCR expression vector are seeded into
12-mm capsule cups (Molecular Devices Corp.) at 4 x 105 cells/cup in aMEM
supplemented with 10% FBS, 2 mM 1-glutamine, 10 units/ml penicillin, and 10
~g/ml
streptomycin. The cells are incubated in this media at 37°C in 5% COZ
for 24 hours.
Extracellular acidification rates are measured using a Cytosensor
microphysiometer (Molecular Devices Corp.). The capsule cups are loaded into
the

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sensor chambers of the microphysiometer and the chambers are perfilscd with
running
buffer (bicarbonate free aMEM supplemented with 4 mM 1-gllltalllille, 10
unlts/ml
penicillin, 10 p.g/ml streptomycin, 26 mM NaCI) at a flow rate of 1 U0
~,I/min.
Agonists or other agents are diluted into the running buffer and perfilsed
through a
second fluid path. During each 60 second pump cycle, the pump is run for 38
seconds
and is off for the remaining 22 seconds. The pH of the running buffer in the
sensor
chamber is recorded during the cycle from 43-58 seconds, and the pump is re-
started
at 60 seconds to start the next cycle. The rate of acidification of the
running buffer
during the recording time is calculated by the Cytosoft program. Changes in
the rates
of acidification are calculated by subtracting the baseline value (the average
of 4 rate
measurements immediately before addition of modulator candidates) from the
highest
rate measurement obtained after addition of a modulator candidate. The
selected
instrument detects 61 mV/pH unit. Modulators that act as agonists at the
receptor
result in an increase in the rate of extracellular acidification as compared
to the rate in
1 S the absence of agonist. This response is blocked by modulators which act
as
antagonists at the receptor.
EXAMPLE 7
Luciferase Reporter Gene Assays
Luciferase reporter gene assays (essentially as described in Example 6)
were carried out to measure signaling activity of the GPCR receptors when
coupled to
Gs, Gi or Gq G-proteins. Activation of Gs coupled receptors results in
stimulation of
intracellualar cAMP production which leads to activation of the transcription
factor
cyclic AMP response element (CRE). Therefore activation of Gs coupled
receptors
can be detected by measuring transcription and translation of the reporter
gene CRE-
luciferase. The level of expression of the CRE reporter gene is dependent on
the
intracellular level of cAMP. Similarily, activation of Gs, Gi or Gq coupled
receptors
will result in activation of the AP-1 transcription factor. Expression of the
AP-1
transcription factor can be attributed to changes in cAMP levels and/or
increases in
the levels of intracellular calcium and therefore can be an indication of G-
protein
coupled receptor activation.

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CHO I 0001 A cells (Gottesman et al., S'onurtic Cell Genetics 6: 45-61,
1980) were maintained in Minimal Essential Medium (MEM) supplemented with
10% FBS (Hyclone Laboratories, Inc., Logan, Utah) at 37°C in an
atmosphere of 5%
CO,. The cells were split 1:5 twice a week for maintence. Plasmids used in the
experiments were propogated in 6.coli strain DHS (Gibco BRL) and purified
using
the Qiagen Maxi-prep plasmid purification system according to the
manufacturer's
instructions.
One day prior to transfection, 1x105 CHO cells/well were plated on 24
well culture plates and allowed to adhere overnight. Each well on the plate
was
transfected with 0.5 p,g of either AP-1 luciferase (Stratagene" LaJolla, CA)
or CRE
luciferase plasmid alone or in combination with 0.125 pg of a GPCR plasmid
(GPCR
DNA inserted into the pCDNA3 vector form Invitrogen). Cell were transiently
transfected with the commercially available transfection reagent FUGENE-6
according the manufacturer's instructions (Boehringer Mannheim, Indianapolis,
IN).
Twenty-four hours after transfection, the cells were washed in PBS
pre-warmed to 37°C. Agonists and antagonists were diluted in pre-warmed
serum-
free MEM, added to the transfected cells and incubated at 37°C, 5% COz
for 5 hours.
Subsequently, the cells were washed once in ice cold PBS and lysed with the
addition
of 100 p,1 of lysis buffer (Promega) to each well. fter a 15 minute incubation
at room
temperature, luciferase reporter gene activation was analyzed with the
Luciferase
Assay Reagents commercially available from Promega (Madison. WI). An alloquot
of
lysate (15 w1) was mixed with 50 ~l of substrate solution in an opaque white
96 well
plate. The luminescence from the plate was read in a Wallance 1450 MicroBeta
scintillation and luminscence counter (Wallac Instruments, Gaithersburg, MD).
Constitutive GPCR activity was calculated as activity measured in GPCR
transfected
cells divided by activity measured in control cells (control cells= luciferase-
transfected cells in the absence of GPCR plasmid). The measurements of GPCR
constitutive activity (as a percentage of control measurements) are summarized
in the
table below:

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GPCR CRE flctivity AP-1 Activity
CON 193 I 28% 100%
CON 197 I 65% 100%
CON 198 I 78% 146%
CON203 100% 468%
CON215 173% 307%
CON222 I 00% 100%
CON202 135% 336%
CON166 115% 100%
CON217 211 % 100%
These results provide useful information for designing screening assays
to identify molecules (natural or artificial) that activate or inhibit the
GPCR's of the
invention. For example, compound libraries can be screened using the AP-1
luciferase (for CON198, CON203, CON215, or CON202) or the CRE-luciferase assay
(for CON193, CON197, CON198, CON215, CON202, and CON166) to identify
compounds which increase the signaling activity in GPCR polypeptide expressing
cells as compared to receptor negative cells. The identified compounds may be
useful
for predicting endogenous ligands for the GPCR polypeptides, for measuring the
physiological effects of GPCR activation in animal models, and for designing
therapeutics to modulate GPCR activity to treat disease states.
EXAMPLE 8
Chromosomal Localization of GPCR
The following example pertains to chromosomal localization of GPCR
genes of the present invention (e.g., CON193, CON166, CON103, CON203,
CON198, CON197, CON202, CON222, CON215, or CON217). The chromosomal
localization permits use of the GPCR polynucleotide sequences (including
fragments
thereof) as chromosomal markers to assist with genome mapping and to provide
markers for disease states. Chromosomal localization also permits correlation
of the

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- I 32 -
GPCR's of the invention with disease states in which aben-ant activity of the
GPCR is
implicated, especially disease states that have previously linked (or will be
linked)
with mutations, polymorphisms, chromosomal rearrangements, and other
chromosomal changes near the locus of the GPCR gene.
A. CON197
Chomosomal localization of the gene encoding CON197 (SEQ ID NO:
11) was determined using the Standford G3 Radiation Hybrid Panel (Research
Genetics, Inc. Huntsville, AL). This panel contains 83 radiation hybrid clones
of the
entire human genome as created by the Stanford Human Gemone Center (Stanford,
California). PCR was carried out with each clone within the Hybrid Panel and
the
results were submitted to the Standford Human Genomic Center via e-mail for
analysis (http://www.shgc.standford.edu/RH/rhserverformnew.html).
PCR reactions were carried out with the Expand Hi-Fi PCR SystemTM
according the manufacturer's instructions (Roche Molecular Biochemicals,
Indianapolis, IN). Primers, synthesized by Genosys Corp. (The Woodlands, TX),
were designed to generate a 10 base pair fragment of CON197-encoding DNA in
the
presence of the appropriate genomic DNA. The forward primer, denoted as LW1332
(TCCTACTGTCATGAACCC; SEQ ~ NO: 74), corresponded to nuceotides 396
through 413 of SEQ m NO: 11. The reverse primer, denoted as LW 1333
(CAGAAGAAGTTGTCCAGC; SEQ ID NO: 75), corresponded to the complement
of nucleotides 519 through 536 of SEQ ID NO: 11. Each reaction contained 25 ng
of
DNA from a hybrid clone, 60 ng of Primer LW 1332, and 60 ng of Primer LW 1333
resulting in a final volume of 15 p1. The PCR reactions were carried our in a
GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems) under the
following conditions: 94°C for 3 minutes followed by 35 cycles of
94°C for 30
seconds, 52°C for 1 minute, and 72°C for 2 minutes. The PCR
reactions were then
analyzed on a 2.0% agarose gel and stained with ethidium bromide. The lanes
were
scored for the presence of the 140 base pair PCR product.
The G3 Hybrid Panal analysis revealed that the CON197 gene (SEQ ID
NO: 11 ) was localized to chromosome 14, most nearly linked to Standford
marker

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SHGC-10764 with a LOD score of 9.10. The SHGC-10764 marker lies at position
lql I.I.
B. CON202
Chomosomal localization of the gene encoding CON202 (SEQ ID NO:
13) was determined using the Standford G3 Radiation Hybrid Panel (Research
Genetics, Inc. Huntsville, AL). This panel contains 83 radiation hybrid clones
of the
entire human genome as created by the Stanford Human Gemone Center (Stanford,
California). PCR was carried out with each clone within the Hybrid Panel and
the
results were submitted to the Standford Human Genomic Center via e-mail (or
analysis (http://www.shgc.standford.edu/RH/rhserverformnew.html).
PCR reactions were carried out with the Expand Hi-Fi PCR SystemTM
according the manufacturer's instructions (Roche Molecular Biochemicals,
Indianapolis, IN). Primers, synthesized by Genosys Corp. (The Woodlands, TX),
were designed to generate a 250 base pair fragment of CON202-encoding DNA in
the
presence of the appropriate genomic DNA. The forward primer, denoted as LW
1480
(GGTTCTACCTGGACTTATGG; SEQ ID NO: 70), corresponded to nuceotides 515
through 534 of SEQ ID NO: 13. The reverse primer, denoted as LW 1481
(TAATGAATGAGTAAGTGCCC; SEQ ID NO: 71 ), corresponded to the
complement of nucleotides 745 through 764 of SEQ ID NO: 13. Each reaction
contained 25 ng of DNA from a hybrid clone, 60 ng of Primer LW 1480, and 60 ng
of
Primer LW 1481 resulting in a final volume of 15 p.1. The PCR reactions were
carried
our in a GeneAmp 9700 PCR thermocycler (Perkin Elmer Applied Biosystems) under
the following conditions: 94°C for 3 minutes followed by 35 cycles of
94°C for 30
seconds, 52°C for 1 minute, and 72°C for 2 minutes. The PCR
reactions were then
analyzed on a 2.0% agarose gel and stained with ethidium bromide. The lanes
were
scored for the presence of the 250 base pair PCR product.
The G3 Hybrid Panal analysis revealed that the CON202 gene (SEQ ID
NO: 13) was localized to chromosome 7, most nearly linked to Standford marker
SHGC-12021 with a LOD score of 10.36. The SHGC-12021 marker lies at position
7q21. There is evidence that schizophrenia is linked to chromosome 7q22, and

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- 134 -
therefor any genes localized to this region are candidates for disease
involvement or
susceptibility. [See Ekelund et al., Ilrrnmln Mol. Genetics 9(7): 1049-IlJ_57
(2000);
Faraone et crl.. flm. J. Med. Genet. 8l: 290-295 (September, 1998); and Blouin
et crl.,
Ncrt. Genet., 20: 70-73 (1998)]. The SHGC-12021 marker is proximal to 7q22 (~l
cM) and therefore may be associated with schizophrenia susceptibility.
In particular, G protein-coupled receptors, such as CON202
polypeptide, have the biochemical and functional potential to play a role in
the disease
process of schizophenia. CON202 is an attractive target for screening for
ligands
(natural and synthetic) that are useful in modulating cellular processes
involved in
schizophrenia. In addition, the chromosomal localization data (especially
coupled
with CON202 expression patterns in the brain) identifies CON202 as a candidate
for
screening healthy and affected (schizophrenia) individuals for CON202 allelic
variants, mutations, duplications, rearrangements, and other chromosomal
variations
that correlate with the disesase state. Variations that correlate with disease
state are
useful for diagnosis of disease or disease susceptibility. CON202 constnicts
containing the variations are useful for designing targeted therapeutics for
treatment
of the disease (e.g., by using the assays for modulators described in
preceding
examples.
C. High throughout Analysis
The EMBL High Throughput Genome database (provided by the
European Bioinformics Institute) was searched with GPCR nucleotide sequences
to
determine chromosomal localization for CON193, CON166, CON103, CON203,
CON198, and CON215 genes. The results are summarized in the table below:
30

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GPCR SECT ID NO: C1101110SOtlle Based on Genbank
Localization Accession No.
CONl 93 1 11 AC026090
CON 166 3 X AC021992
CONI 03 5 2 AC013396
CON203 7 3 AC024886
CON198 9 11 AC025249
CON215 17 3 AC024886
While the present
invention has
been described
in terms of
specific
embodiments,
it is understood
that variations
and modifications
will occur
to those in
the art, all h are intended as aspects of the present invention.
of whic Accordingly,
only such limitations
as appear in
the claims
should be placed
on the invention.
Summary of Sequences:
SEQ ID NO. Description
1 CON 193 DNA
2 CON 193 protein
3 CON 166 DNA
4 CON 166 protein
5 CON 103 DNA
6 CON 103 protein
7 CON 203 DNA
8 CON 203 protein
9 CON 198 DNA
10 CON 198 protein
11 CON 197 DNA
12 CON 197 protein
13 CON 202 DNA
14 CON 202 protein
15 CON 222 DNA
16 CON 222 protein
17 CON 215 DNA

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SEQ ID NO. Description
18 CON 215 protein
19 CON 217 DNA
20 CON 217 protein
21 PCR primer LW 1282 for CON 193
22 PCR primer LW 1283 for CON 193
23 PCR primer LW 1372 for CON 193
24 PCR primer LW 1374 for CON 193
25 MAPK Substrate Peptide
26 Primer LW 1248 .for CON 193 to generate insitu
hybridization probe
27 Primer LW 1249 for CON 193 to generate insitu
hybridization probe
28 PCR primer LW 1278 for CON 166
29 PCR primer LW 1279 for CON 166
30 PCR primer LW 1405 for CON 166
31 PCR primer LW 1406 for CON 166
32 PCR primer LW 1280 for CON 103
33 PCR primer LW 1281 for CON 103
34 PCR primer LW 1385 for CON 103
35 PCR primer LW 1386 for CON 103
36 PCR primer LW 1329 for CON 203
37 PCR primer LW 1377 for CON 203
38 PCR primer LW 1387 for CON 203
39 PCR primer LW 1388 for CON 203
40 Primer LW 1314 for CON 203 to generate insitu
hybridization probe
l
41 Primer LW 1315 for CON 203 to generate
insitu hybridization probe
42 PCR primer LW 1326 for CON 198
43 PCR primer LW 1327 for CON 198
44 PCR primer LW 1415 for CON 198
45 PCR primer LW 1416 for CON 198
46 Primer LW 1308 for CON 198 to generate insitu
hybridization probe
47 Primer LW 1309 for CON 198 to generate insitu
hybridization probe
48 PCR primer LW 1324 for CON 197
49 PCR primer LW 1325 for CON 197
50 Primer LW 1306 for CON 197 to generate insitu
hybridization probe
51 Primer LW 1307 for CON 197 to generate insitu
hybridization probe

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S~ 1 D NO. Description
52 PCR primer GV 599 for CON 202
53 PCR primer GV 600 for CON 202
54 PCR primer LW 1482 for CON 202
55 PCR primer LW 148 for CON 202
56 Primer LW 1310 for CON 202 to generate insitu
hybridization probe
57 Primer LW 1311 .for CON 202 to generate insitu
hybridization probe
58 PCR primer LW 1442 for CON 222
59 PCR primer LW 1443 for CON 222
60 PCR primer LW 1440 for CON 222
61 PCR primer LW 1441 for CON 222
62 Primer LW 1472 for CON 222 to generate insitu
hybridization probe
63 Primer LW 1473 for CON 222 to generate insitu
hybridization probe
64 Primer LW 1411 For CON 215 to generate insitu
hybridization probe
65 Primer LW 1412 for CON 21 S to generate insitu
hybridization probe
66 PCR primer LW 1448 for CON 217
67 PCR primer LW 1449 for CON 217
68 Primer LW 217A for CON 217 to generate insitu
hybridization probe
69 Primer LW 218B for CON 217 to generate insitu
hybridization probe
70 Primer LW 1480 for CON 202 chromosomal localization
71 Primer LW 1481 for CON 202 chromosomal localization
72 Primer CON103a for CON 103 to generate insitu
hybridization probe
73 Primer CON103b for CON 103 to generate insitu
hybridization probe
74 Primer LW 1332 for CON 197 chromosomal localization
75 Primer LW 1333 for CON 197 chromosomal localization
30

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INDICATIONS ARE MADE (ijthe indications
are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the genernl
nature ojthe indications e.g.,
"Accession Number ojDeposit')
For receiving Office use only For International Bureau use only
This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
Authorize officer ~ 'r~~ ( ~ Authorized officer
Form PCT/RO/134 (July 1992) LegaIStar ~ss~, Form PCTMS

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-147
Applicant's or agent's file ' ' ~ International application N. -
refercnce number 283411b~tbr To Be Determ
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
( PCT Rule 136is)
A. The indications made below relate
to the microorganism referred
to in the description
on page 94; 98 , line 3-7 T 19
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
Agricultural Research Service Culture
Collection
Address of depositary institution
(including portal code and country)
National Center for Agricultural
Utilization Research
Agricultural Research Service,
U.S. Department of Agriculture
1815 North University Street, Peoria,
Illinois 61604 U.S.A.
Date of deposit Accession Number
18 January 2000 B-30252
C. ADDITIONAL INDICATIONS (leave
blank i~not applicable) This information
is continued on an additional
sheet
In respect of those designations
in which a European patent or
a patent in Norway is sought,
a sample of the
deposited microorganism will be
made available until the publication
of the mention of the grant of
the European patent or
the corresponding information concerning
the patent in Norway or until
the date on which the application
has been refused
or withdrawn or is deemed to be
withdrawn, only by the issue of
such a sample to an expert nominated
by the person
requesting the sample (Rule 28
(4) EPC and the corresponding
regulations in Norway).
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (ifthe indications
are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the genera!
nature of the indications e.g.,
"Accession Number of Deposit')
For receiving Office use only For International Bureau use only
This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
oriz offic ~ ~ ~ Authorized officer
Form PCT/RO1134 (July 1992) ~e9aistar ~ss~. Form PcrMs

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-148
Applicant's or agent's file ~ ~ Inter~~~~~~~ ~~~''~°"~~ "~
reference number 2834OOZtor ~ ~ To E
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136~s)
A. The indications made below
relate to the microorganism referred
to in the description
on page 94 ; 98 , line 27-31;
20
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
Agricultural Research Service
Culture Collection
Address of depositary institution
(including postal code and country)
National Center for Agricultural
Utilization Research
Agricultural Research Service,
U.S. Department of Agriculture
1815 North University Street,
Peoria, Illinois 61604 U.S. A.
Date of deposit Accession Number
18 January 2000 B-30251
C. ADDITIONAL INDICATIONS (leave
blank if not applicable) This
information is continued on an
additional sheet
When designating Australia, in
accordance with regulation 3.25
of the Patents Regulations (Australia
Statutory
Rules 1991 No. 71 ), samples of
materials deposited in accordance
with the Budapest Treaty in relation
to this Patent
Request are only to be provided
before: the patent is granted
on the application; or the application
has lapsed or been
withdrawn or refused; to a person
who is: a skilled addressee without
an interest in the invention;
and nominated by a
person who makes a request for
the furnishing of those samples.
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (ifthe indications
are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank ifnot applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the general
nature of the indications e.g.,
"Accession Number of Deposit')
For receiving Office use only For International Bureau use only
~ This sheeW vas received with the international application ~ This sheet was
received by the International Bureau on:
Authorize officer ~ ~ ~ Authorized officer
Form PCT/RO/I 34 (July 1992) ~e9aiscar ~ss~, Form PCTMS

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-149
Applicant's or agent's file ' - ~ ~ Internation°' ~~~'~~~"~~'~l
reference number 283411b~tbN To Be Dei
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis)
A. The indications made below
relate to the microorganism referred
to in the description
on page 94 ~ 98 , line 27-31;
20
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
Agricultural Research Service
Culture Collection
Address of depositary institution
(including postal code and country)
National Center for Agricultural
Utilization Research
Agricultural Research Service,
U.S. Department of Agriculture
1815 North University Street,
Peoria, Illinois 61604 U.S.A.
Date of deposit Accession Number
18 January 2000 B-30251
C. ADDITIONAL INDICATIONS (leave
blank if not applicable) This
information is continued on an
additional sheet
In respect of those designations
in which a European patent or
a patent in Norway is sought,
a sample of the
deposited microorganism will be
made available until the publication
of the mention of the grant of
the European patent or
the corresponding information
concerning the patent in Norway
or until the date on which the
application has been refused
or withdrawn or is deemed to be
withdrawn, only by the issue
of such a sample to an expert
nominated by the person
requesting the sample (Rule 28
(4) EPC and the corresponding
regulations in Norway).
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if the
indications are not for all designated
States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the general
nature of the indications e.g.,
'Accession NumberofDeposit')
For receiving Office use only For International Bureau use only
This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
Authoriz d offic Authorized officer
Form PCT/R0/134 (July 1992) legalStar tssz, Form PCTMS

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-150
Applicant's or agent's file , International a~, i~ on 1~'
reference number 2834' ' ~ ~ To Be Determi~t~~~~~°
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is)
A. The indications made below
relate to the microorganism referred
to in the description
on page 95 ; 98 , line f 9-23
; 21
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of dcpositary institution
Agricultural Research Service
Culture Collection
Address of depositary institution
(including postal code and country)
National Center for Agricultural
Utilization Research
Agricultural Research Service,
U.S. Department of Agriculture
1815 North University Street,
Peoria, Illinois 61604 U.S. A.
Date of deposit Accession Number
18 January 2000 B-30253
C. ADDITIONAL INDICATIONS (leave
blank if not applicable) This
information is continued on an
additional sheet
When designating Australia, in
accordance with regulation 3.25
of the Patents Regulations (Australia
Statutory
Rules 1991 No. 71 ), samples of
materials deposited in accordance
with the Budapest Treaty in relation
to this Patent
Request are only to be provided
before: the patent is granted
on the application; or the application
has lapsed or been
withdrawn or refused; to a person
who is: a skilled addressee without
an interest in the invention;
and nominated by a
person who makes a request for
the furnishing of those samples.
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (ijthe indications
are nor for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the genernl
nature of the indications e.g.,
"Accession Number of Deposit')
For receiving Office use only For International Bureau use only
,~ This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
Authorized officer
Form PCT/120/134 (July 1992) legalStar 1997, Form PCTMS

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-151
Applicant's or agent's file ' . International a
reference number 28341",~. ", ITo Be Detern
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is)
A. The indications made below relate
to the microorganism referred
to in the description
on page 95; 98 , line 19-23' 21
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
Agricultural Research Service Culture
Collection
Address of depositary institution
(including postal code and country)
National Center for Agricultural
Utilization Research
Agricultural Research Service,
U.S. Department of Agriculture
1815 North University Street, Peoria,
Illinois 616D4 U.S.A.
Date of deposit Accession Number
18 January 2000 B-30253
C. ADDITIONAL INDICATIONS (leave
blank i~not applicable) This information
is continued on an additional
sheet
In respect of those designations
in which a European patent or
a patent in Norway is sought,
a sample of the
deposited microorganism will be
made available until the publication
of the mention of the grant of
the European patent or
the corresponding information concerning
the patent in Norway or until
the date on which the application
has been refused
or withdrawn or is deemed to be
withdrawn, only by the issue of
such a sample to an expert nominated
by the person
requesting the sample (Rule 28
(4) EPC and the corresponding
regulations in Norway).
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if the indications
are not jor all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank ifnot applicable)
The indications listed below will
be submitted to the International
Bureau later (specify tke general
nature of the indications e.g.,
..Accession NumberofDeposit')
For receiving Office use only For International Bureau use only
This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
Author' ed offi -~'~ ~ ~ Authorized officer
Form PCT/R0I134 (July 1992) LegalStar ~ss~. Form Pcrrns

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-152
Applicant's or agent's file . ~ Internahional appl~rarinn T
reference number 28341ItiCtbr To Be Determin
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is)
A. The indications made below
relate to the microorganism referred
to in the description
on page 96; 98 , line 1115; 22
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
Agricultural Research Service
Culture Collection
Address of depositary institution
(including postal code and country)
National Center for Agricultural
Utilization Research
Agricultural Research Service,
U.S. Department of Agriculture
1815 North University Street,
Peoria, Illinois 61604 U.S. A.
Date of deposit Accession Number
18 January 2000 B-30257
C. ADDITIONAL INDICATIONS (leave
blank ifnot applicable) This
information is continued on an
additional sheet
When designating Australia, in
accordance with regulation 3.25
of the Patents Regulations (Australia
Statutory
Rules 1991 No. 71), samples of
materials deposited in accordance
with the Budapest Treaty in relation
to this Patent
Request are only to be provided
before: the patent is granted
on the application; or the application
has lapsed or been
withdrawn or refused; to a person
who is: a skilled addressee without
an interest in the invention;
and nominated by a
person who makes a request for
the furnishing of those samples.
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if the
indications are not for all designated
States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the general
nature of the indications e.g..
"Accession Number of Deposit')
For receiving Office use only For International Bureau use only
This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
Authorized officer
Form PCT/RO/I 34 (July 1 J92) LegaIStar ~ss~, Form PCTM5

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-153
Applicant's or agent's file ~ . Internatior
reference number 28341m« ~~ I To Be De
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3his)
A. The indications made below
relate to the microorganism referred
to in the description
on page 96; 98 , line 11-15; 22
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
Agricultural Research Service
Culture Collection
Address of depositary institution
(including po.s~n/ code and country)
National Center for Agricultural
Utilization Research
Agricultural Research Service,
U.S. Department of Agriculture
,
1815 North University Street,
Peoria, Illinois 61604 U.S.A.
Date of deposit Accession Number
18 January 2000 B-30257
C. ADDITIONAL INDICATIONS (leave
blank ijnot applicable) This
information is continued on an
additional sheet
In respect of those designations
in which a European patent or
a patent in Norway is sought,
a sample of the
deposited microorganism will be
made available until the publication
of the mention of the grant of
the European patent or
the corresponding information
concerning the patent in Norway
or until the date on which the
application has been refused
or withdrawn or is deemed to be
withdrawn, only by the issue
of such a sample to an expert
nominated by the person
requesting the sample (Rule 28
(4) EPC and the corresponding
regulations in Norway).
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (ifthe indications
are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the general
nature of the indications e.g.,
"Accession Number of Deposit')
For receiving Office use only For International Bureau use only
~ This sheet was received with the international application a This sheet was
received by the International Bureau on:
Author ed offi r Authorized officer
Form PCTlRO/134 (July 1992) LegatStar 1997.FOrmPCTMS

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-154
Applicant's or agent's file ' . Internatronal aonlication Na
reference number 283411vcr ~r To Be Detei
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is)
A. The indications made below relate
to the microorganism referred
to in the description
on page 97; 98 , line 4-8; 23
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depository institution
Agricultural Research Service Culture
Collection
Address of depository institution
(including postal code and country)
National Center for Agricultural
Utilization Research
Agricultural Research Service,
U.S. Department of Agriculture
1815 North University Street, Peoria,
Illinois 61604 U.S. A.
Date of deposit Accession Number
18 January 2000 B-30255
C. ADDITIONAL INDICATIONS (leave
blank if not applicable) This
information is continued on an
additional sheet
When designating Australia, in
accordance with regulation 3.25
of the Patents Regulations (Australia
Statutory
Rules 1991 No. 71 ), samples of
materials deposited in accordance
with the Budapest Treaty in relation
to this Patent
Request are only to be provided
before: the patent is granted
on the application; or the application
has lapsed or been
withdrawn or refused; to a person
who is: a skilled addressee without
an interest in the invention;
and nominated by a
person who makes a request for
the furnishing of those samples.
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if the indications
are not for all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank ifnot applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the general
nature of the indications e.g.,
"Accession Nanrber ofDepasit')
For receiving Office use only For International Bureau use only
This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
Authoriz offic . ~G~~ Authorized officer
Form PCT/RO/134 (July 1992) LegaIStartss7, FormPCTMS

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-155
Applicant's or agent's file ' Internation,
refcrcnccnumbcr 2834116~76P ITo Be Det
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is)
A. The indications made below relate
to the microorganism referred
to in the description
on page 97 9$ , line 4-R ~ 2'~
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depository institution
Agricultural Research Service Culture
Collection
Address of depository institution
(including postal code and country)
National Center for Agricultural
Utilization Research
Agricultural Research Service,
U.S. Department of Agriculture
1815 North University Street, Peoria,
Illinois 61604 U.S.A.
Date of deposit Accession Number
18 January 2000 B-30255
C. ADDITIONAL INDICATIONS (lenve
blank if not applicable) This
information is continued on an
additional sheet
In respect of those designations
in which a European patent or
a patent in Norway is sought,
a sample of the
deposited microorganism will be
made available until the publication
of the mention of the grant of
the European patent or
the corresponding information concerning
the patent in Norway or until
the date on which the application
has been refused
or withdrawn or is deemed to be
withdrawn, only by the issue of
such a sample to an expert nominated
by the person
requesting the sample (Rule 28
(4) EPC and the corresponding
regulations in Norway).
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if the indications
are not for all designated Stares)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blankifnot applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the general
nature ojthe indications e.g.,
"Accession Number of Deposit')
For receiving Office use only For International Bureau use only
This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
Authori d offic Authorized officer
Form PCT/RO/134 (July 1992) ~esaistar iss~. FormPCrtns

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-156
Applicant's or agent's file ~ International api
reference number 28341~~« ~~ ,I To Be Determi
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136ts)
A. The indications made below
relate to the microorganism referred
to in the description
on page 98 , line 1-3; 24
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
Agricultural Research Service
Culture Collection
Address of dcpositary institution
(including postal code and country)
National Center for Agricultural
Utilization Research
Agricultural Research Service,
U.S. Department of Agriculture
1815 North University Street,
Peoria, Illinois 61604 U.S. A.
Date of deposit Accession Number
18 January 2000 B-30256
C. ADDITIONAL INDICATIONS (leave
blank if not applicable) This
information is continued on an
additional sheet
When designating Australia, in
accordance with regulation 3.25
of the Patents Regulations (Australia
Statutory
Rules 1991 No. 71 ), samples of
materials deposited in accordance
with the Budapest Treaty in relation
to this Patent
Request are only to be provided
before: the patent is granted
on the application; or the application
has lapsed or been
withdrawn or refused; to a person
who is: a skilled addressee without
an interest in the invention;
and nominated by a
person who makes a request for
the furnishing of those samples.
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if the
indications are not for all designated
States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank ifnot applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the general
nature of tire indications e.g.,
"Accession Number of Deposit')
For receiving Office use only For International Bureau use only
This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
Authorized officer
r
Form PCT/RO/134 (July 1992) LegaIStar ~ss~. FormPCrnts

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-157
Applicant's or agent's file ~ International a
reference number 28341~~« ~~ '' I To Be Detern -
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is)
A. The indications made below
relate to the microorganism referred
to in the description
on page 98 , line 1-3; 24
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depository institution
Agricultural Research Service
Culture Collection
Address of depository institution
(including postal code and country)
National Center for Agricultural
Utilization Research
Agricultural Research Service,
U.S. Department of Agriculture
1815 North University Street,
Peoria, Illinois 61604 U.S.A.
Date of deposit Accession Number
18 January 2000 B-30256
C. ADDITIONAL INDICATIONS (leave
blank ijnot applicable) This
information is continued on an
additional sheet
In respect of those designations
in which a European patent or
a patent in Norway is sought,
a sample of the
deposited microorganism will be
made available until the publication
of the mention of the grant of
the European patent or
the corresponding information
concerning the patent in Norway
or until the date on which the
application has been refused
or withdrawn or is deemed to be
withdrawn, only by the issue
of such a sample to an expert
nominated by the person
requesting the sample (Rule 28
(4) EPC and the corresponding
regulations in Norvvay).
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (if the
indications are not for all designated
States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the general
nature of the indications e.g.,
'Accession Number of Deposit')
For receiving Office use only For International Bureau use only
This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
Authori ed offi r Authorized officer
Form PCT/RO/134 (July 1992) LegaIStar issi. FormPCTMS

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
SEQUENCE LISTING
<110> Pharmacia & Upjohn Company
<120> G PROTEIN-COUPLED RECEPTORS EXPRESSED IN BRAIN
<130> 28341/6276P
<140>
<141>
<150> US 09/481,794
<151> 2000-O1-12
<150> US 09/454,399
<151> 1999-12-03
<150> US 09/429,517
<151> 1999-10-28
<150> US 09/429,555
<151> 1999-10-28
<150> US 09/429,676
<151> 1999-10-28
<150> US 09/429,695
<151> 1999-10-28
<150> US 09/428,114
<151> 1999-10-27
<150> US 09/428,020
<151> 1999-10-27
<150> US 09/427,859
<151> 1999-10-27
<150> US 09/427,653
<151> 1999-10-27
<160> 75
<170> PatentIn Ver. 2.0
<210> 1
<211> 1308
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (157)..(1122)
<220>
<221> misc_feature
<222> (1)
<223> N = A or C or G or T
<220>
<221> misc feature

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
_7_
<222> (1237)
<223> N = A or C or G or T
<220>
<221> misc_feature
<222> (1274)
<223> N = A or C or G or T
<400> 1
ntggttgttg gaccattaaa atgcattatg gaatttttaa aagttggggg agagggagac 60
agtaaaaata acctatattt tctcttgttt tttttttttt aactctagga aagcccagac 120
aaattttgag ctatttcata acctaccaga cttatc atg cta aca ctg aat aaa 174
Met Leu Thr Leu Asn Lys
1 5
aca gac cta ata cca get tca ttt att ctg aat gga gtc cca gga ctg 222
Thr Asp Leu Ile Pro Ala Ser Phe Ile Leu Asn Gly Val Pro Gly Leu
15 20
gaa gac aca caa ctc tgg att tcc ttc cca ttc tgc tct atg tat gtt 270
Glu Asp Thr Gln Leu Trp Ile Ser Phe Pro Phe Cys Ser Met Tyr Val
25 30 35
gtg get atg gta ggg aat tgt gga ctc ctc tac ctc att cac tat gag 318
Val Ala Met Val Gly Asn Cys Gly Leu Leu Tyr Leu Ile His Tyr Glu
40 45 50
gat gcc ctg cac aaa ccc atg tac tac ttc ttg gcc atg ctt tcc ttt 366
Asp Ala Leu His Lys Pro Met Tyr Tyr Phe Leu Ala Met Leu Ser Phe
55 60 65 70
act gac ctt gtt atg tgc tct agt aca atc cct aaa gcc ctc tgc atc 414
Thr Asp Leu Val Met Cys Ser Ser Thr Ile Pro Lys Ala Leu Cys Ile
75 80 85
ttc tgg ttt cat ctc aag gac att gga ttt gat gaa tgc ctt gtc cag 462
Phe Trp Phe His Leu Lys Asp Ile Gly Phe Asp Glu Cys Leu Val Gln
90 95 100
atg ttc ttc atc cac acc ttc aca ggg atg gag tct ggg gtg ctt atg 510
Met Phe Phe Ile His Thr Phe Thr Gly Met Glu Ser Gly Val Leu Met
105 110 115
ctt atg gcc ctg gat cgc tat gtg gcc atc tgc tac ccc tta cgc tat 558
Leu Met Ala Leu Asp Arg Tyr Val Ala Ile Cys Tyr Pro Leu Arg Tyr
120 125 130
tca act atc ctc acc aat cct gta att gca aag gtt ggg act gcc acc 606
Ser Thr Ile Leu Thr Asn Pro Val Ile Ala Lys Val Gly Thr Ala Thr
135 140 145 150
ttc ctg aga ggg gta tta ctc att att ccc ttt act ttc ctc acc aag 654
Phe Leu Arg Gly Val Leu Leu Ile Ile Pro Phe Thr Phe Leu Thr Lys
155 160 165
cgc ctg ccc tcc tgc aga ggc aat ata ctt ccc cat acc tac tgt gac 702
Arg Leu Pro Ser Cys Arg Gly Asn Ile Leu Pro His Thr Tyr Cys Asp
170 175 180

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-3-
cacatg tctgtagcc aaattgtcc tgtggt aatgtcaag gtcaatgcc 750
HisMet SerValAla LysLeuSer CysGly AsnValLys ValAsnAla
185 190 195
atctat ggtctgatg gttgccctc ctgatt gggggcttt gacatactg 798
IleTyr GlyLeuMet ValAlaLeu LeuIle GlyGlyPhe AspIleLeu
200 205 210
tgtatc accatctcc tataccatg attctc cgggcagtg gtcagcctc 846
CysIle ThrIleSer TyrThrMet IleLeu ArgAlaVal ValSerLeu
215 220 225 230
tcctca gcagatget cggcagaag gccttt aatacctgc actgcccac 894
SerSer AlaAspAla ArgGlnLys AlaPhe AsnThrCys ThrAlaHis
235 240 245
atttgt gccattgtt ttctcctat actcca getttcttc tccttcttt 942
IleCys AlaIleVal PheSerTyr ThrPro AlaPhePhe SerPhePhe
250 255 260
tcccac cgctttggg gaacacata atcccc ccttcttgc cacatcatt 990
SerHis ArgPheGly GluHisIle IlePro ProSerCys HisIleIle
265 270 275
gtagcc aatatttat ctgctccta ccaccc actatgaac cctattgtc 1038
ValAla AsnIleTyr LeuLeuLeu ProPro ThrMetAsn ProIleVal
280 285 290
tatggg gtgaaaacc aaacagata cgagac tgtgtcata aggatcctt 1086
TyrGly ValLysThr LysGlnIle ArgAsp CysValIle ArgIleLeu
295 300 305 310
tcaggt tctaaggat accaaatcc tacagc atgtgaatgaacactt 1132
SerGly SerLysAsp ThrLysSer TyrSer Met
315 320
gccaggagtg agaagagaag gaaagaatta cttctatttg cctcttatgc aggagttcat 1192
aaaatctttc tggaagtact gtattgatca caaaatggag tttgntgact ggtgcattct 1252
caataagtac cttgggaatc tnacatcact ggaaggccca ccacatttct ataaat 1308
<210> 2
<211> 321
<212> PRT
<213> Homo sapiens
<400> 2
Met Leu Thr Leu Asn Lys Thr Asp Leu Ile Pro Ala Ser Phe Ile Leu
1 5 10 15
Asn Gly Val Pro Gly Leu Glu Asp Thr Gln Leu Trp Ile Ser Phe Pro
20 25 30
Phe Cys Ser Met Tyr Val Val Ala Met Val Gly Asn Cys Gly Leu Leu
35 40 45
Tyr Leu Ile His Tyr Glu Asp Ala Leu His Lys Pro Met Tyr Tyr Phe
50 55 60
Leu Ala Met Leu Ser Phe Thr Asp Leu Val Met Cys Ser Ser Thr Ile
65 70 75 80

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-4-
Pro Lys Ala Leu Cys Ile Phe Trp Phe His Leu Lys Asp Ile Gly Phe
85 90 95
Asp Glu Cys Leu Val Gln Met Phe Phe Ile His Thr Phe Thr Gly Met
100 105 110
Glu Ser Gly Val Leu Met Leu Met Ala Leu Asp Arg Tyr Val Ala Ile
115 120 125
Cys Tyr Pro Leu Arg Tyr Ser Thr Ile Leu Thr Asn Pro Val Ile Ala
130 135 140
Lys Val Gly Thr Ala Thr Phe Leu Arg Gly Val Leu Leu Ile Ile Pro
145 150 155 160
Phe Thr Phe Leu Thr Lys Arg Leu Pro Ser Cys Arg Gly Asn Ile Leu
165 170 175
Pro His Thr Tyr Cys Asp His Met Ser Val Ala Lys Leu Ser Cys Gly
180 185 190
Asn Val Lys Val Asn Ala Ile Tyr Gly Leu Met Val Ala Leu Leu Ile
195 200 205
Gly Gly Phe Asp Ile Leu Cys Ile Thr Ile Ser Tyr Thr Met Ile Leu
210 215 220
Arg Ala Val Val Ser Leu Ser Ser Ala Asp Ala Arg Gln Lys Ala Phe
225 230 235 240
Asn Thr Cys Thr Ala His Ile Cys Ala Ile Val Phe Ser Tyr Thr Pro
245 250 255
Ala Phe Phe Ser Phe Phe Ser His Arg Phe Gly Glu His Ile Ile Pro
260 265 270
Pro Ser Cys His Ile Ile Val Ala Asn Ile Tyr Leu Leu Leu Pro Pro
275 280 285
Thr Met Asn Pro Ile Val Tyr Gly Val Lys Thr Lys Gln Ile Arg Asp
290 295 300
Cys Val Ile Arg Ile Leu Ser Gly Ser Lys Asp Thr Lys Ser Tyr Ser
305 310 315 320
Met
<210> 3
<211> 1014
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (1)..(1014)
<400> 3
atg gat gaa aca gga aat ctg aca gta tct tct gcc aca tgc cat gac 48
Met Asp Glu Thr Gly Asn Leu Thr Val Ser Ser Ala Thr Cys His Asp
1 5 10 15

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-5-
actatt gatgacttc cgcaatcaa gtgtattcc accttg tactctatg 96
ThrIle AspAspPhe ArgAsnGln ValTyrSer ThrLeu TyrSerMet
20 25 30
atctct gttgtaggc ttctttggc aatggcttt gtgctc tatgtcctc 144
IleSer ValValGly PhePheGly AsnGlyPhe ValLeu TyrValLeu
35 40 45
ataaaa acctatcac aagaagtca gccttccaa gtatac atgattaat 192
IleLys ThrTyrHis LysLysSer AlaPheGln ValTyr MetIleAsn
50 55 60
ttagca gtagcagat ctactttgt gtgtgcaca ctgcct ctccgtgtg 240
LeuAla ValAlaAsp LeuLeuCys ValCysThr LeuPro LeuArgVal
65 70 75 80
gtctat tatgttcac aaaggcatt tggctcttt ggtgac ttcttgtgc 288
ValTyr TyrValHis LysGlyIle TrpLeuPhe GlyAsp PheLeuCys
85 90 95
cgcctc agcacctat getttgtat gtcaacctc tattgt agcatcttc 336
ArgLeu SerThrTyr AlaLeuTyr ValAsnLeu TyrCys SerIlePhe
100 105 110
tttatg acagccatg agctttttc cggtgcatt gcaatt gtttttcca 384
PheMet ThrAlaMet SerPhePhe ArgCysIle AlaIle ValPhePro
115 120 125
gtccag aacattaat ttggttaca cagaaaaaa gccagg tttgtgtgt 432
ValGln AsnIleAsn LeuValThr GlnLysLys AlaArg PheValCys
130 135 140
gtaggt atttggatt tttgtgatt ttgaccagt tctcca tttctaatg 480
ValGly IleTrpIle PheValIle LeuThrSer SerPro PheLeuMet
145 150 155 160
gccaaa ccacaaaaa gatgagaaa aataatacc aagtgc tttgagccc 528
AlaLys ProGlnLys AspGluLys AsnAsnThr LysCys PheGluPro
165 170 175
ccacaa gacaatcaa actaaaaat catgttttg gtcttg cattatgtg 576
ProGln AspAsnGln ThrLysAsn HisValLeu ValLeu HisTyrVal
180 185 190
tcattg tttgttggc tttatcatc ccttttgtt attata attgtctgt 624
SerLeu PheValGly PheIleIle ProPheVal IleIle IleValCys
195 200 205
tacaca atgatcatt ttgacctta ctaaaaaaa tcaatg aaaaaaaat 672
TyrThr MetIleIle LeuThrLeu LeuLysLys SerMet LysLysAsn
210 215 220
ctgtca agtcataaa aaggetata ggaatgatc atggtc gtgaccget 720
LeuSer SerHisLys LysAlaIle GlyMetIle MetVal ValThrAla
225 230 235 240
gccttt ttagtcagt ttcatgcca tatcatatt caacgt accattcac 768
AlaPhe LeuValSer PheMetPro TyrHisIle GlnArg ThrIleHis
245 250 255
cttcat tttttacac aatgaaact aaaccctgt gattct gtccttaga 816
LeuHis PheLeuHis AsnGluThr LysProCys AspSer ValLeuArg
260 265 270

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-G-
atgcag aagtccgtg gtcataacc ttgtct ctggetgca tccaattgt 864
MetGln LysSerVal ValIleThr LeuSer LeuAlaAla SerAsnCys
275 280 285
tgcttt gaccctctc ctatatttc ttttct gggggtaac tttaggaaa 912
CysPhe AspProLeu LeuTyrPhe PheSer GlyGlyAsn PheArgLys
290 295 300
aggctg tctacattt agaaagcat tctttg tccagcgtg acttatgta 960
ArgLeu SerThrPhe ArgLysHis SerLeu SerSerVal ThrTyrVal
305 310 315 320
cccaga aagaaggcc tctttgcca gaaaaa ggagaagaa atatgtaaa 1008
ProArg LysLysAla SerLeuPro GluLys GlyGluGlu IleCysLys
325 330 335
gtatag 1014
Val
<210> 4
<211> 337
<212> PRT
<213> Homo sapiens
<400> 2
Met Asp Glu Thr Gly Asn Leu Thr Val Ser Ser Ala Thr Cys His Asp
1 5 10 15
Thr Ile Asp Asp Phe Arg Asn Gln Val Tyr Ser Thr Leu Tyr Ser Met
20 25 30
Ile Ser Val Val Gly Phe Phe Gly Asn Gly Phe Val Leu Tyr Val Leu
35 40 45
Ile Lys Thr Tyr His Lys Lys Ser Ala Phe Gln Val Tyr Met Ile Asn
50 55 60
Leu Ala Val Ala Asp Leu Leu Cys Val Cys Thr Leu Pro Leu Arg Val
65 70 75 80
Val Tyr Tyr Val His Lys Gly Ile Trp Leu Phe Gly Asp Phe Leu Cys
85 90 95
Arg Leu Ser Thr Tyr Ala Leu Tyr Val Asn Leu Tyr Cys Ser Ile Phe
100 105 110
Phe Met Thr Ala Met Ser Phe Phe Arg Cys Ile Ala Ile Val Phe Pro
115 120 125
Val Gln Asn Ile Asn Leu Val Thr Gln Lys Lys Ala Arg Phe Val Cys
130 135 140
Val Gly Ile Trp Ile Phe Val Ile Leu Thr Ser Ser Pro Phe Leu Met
145 150 155 160
Ala Lys Pro Gln Lys Asp Glu Lys Asn Asn Thr Lys Cys Phe Glu Pro
165 170 175
Pro Gln Asp Asn Gln Thr Lys Asn His Val Leu Val Leu His Tyr Val
180 185 190

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
Ser Leu Phe Val Gly Phe Ile Ile Pro Phe Val Ile Ile Ile Val Cys
195 200 205
Tyr Thr Met Ile Ile Leu Thr Leu Leu Lys Lys Ser Met Lys Lys Asn
210 215 220
Leu Ser Ser His Lys Lys Ala Ile Gly Met Ile Met Val Val Thr Ala
225 230 235 240
Ala Phe Leu Val Ser Phe Met Pro Tyr His Ile Gln Arg Thr Ile His
245 250 255
Leu His Phe Leu His Asn Glu Thr Lys Pro Cys Asp Ser Val Leu Arg
260 265 270
Met Gln Lys Ser Val Val Ile Thr Leu Ser Leu Ala Ala Ser Asn Cys
275 280 285
Cys Phe Asp Pro Leu Leu Tyr Phe Phe Ser Gly Gly Asn Phe Arg Lys
290 295 300
Arg Leu Ser Thr Phe Arg Lys His Ser Leu Ser Ser Val Thr Tyr Val
305 310 315 320
Pro Arg Lys Lys Ala Ser Leu Pro Glu Lys Gly Glu Glu Ile Cys Lys
325 330 335
Val
<210> 5
<211> 2429
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (691)..(1845)
<400> 5
ggggcctact tcaccgtgta cccggacttg ggaccatcac agacttcaga accatcagga 60
acctgggagc aactgaaagc tgaactacag tgggctttca gacacacagc aggctgcgga 120
gcacaaatag gactggttcc ctccaggcca ccagcagggc ggtggaggtc ttcactgact 180
ccctgcctac ctctcaggac aatgtccttt tggctccaca gtccctgaag ccagagctgg 240
tgggggcagg gaggcagcca ccagcctcta tatgtagtgg aggagggggt gtccagggag 300
ggctgcatga tcctgagagc ccccacctca cccggctgga ctatcctccc acttcagggt 360
ttctctgggc ttccatcttg cccctgctga gccctgcttc ctcctctacc agcagcacaa 420
cccccaggct gggctcagag acctcatgtg gtgggatcac tcagtacccc gaggcggagg 480
gaaggaggga gggctgcagg gttccccttg gcctgcaaac aggaacacag ggtgtttctc 540
agtggctgcg agaatgctga tgaaaacccc aggatgttgt gtcaccgtgg tggccagctg 600
atagtgccaa tcatcccact ttgccctgag cactcctgca ggggtagaag actccagaac 660

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
_g_
cttctctcag gcccatggcc caagcagccc atg gaa ctt cat aac ctg agc tct 714
Met Glu Leu His Asn Leu Ser Ser
1 5
CCa tCt CCC tCt CtC tCC tCC tCt gtt CtC CCt CCC tCC ttC tCt CCC 762
Pro Ser Pro Ser Leu Ser Ser Ser Val Leu Pro Pro Ser Phe Ser Pro
15 20
tca ccc tcc tct get ccc tct gcc ttt acc act gtg ggg ggg tcc tct 810
Ser Pro Ser Ser Ala Pro Ser Ala Phe Thr Thr Val Gly Gly Ser Ser
25 30 35 40
gga ggg ccc tgc cac ccc acc tct tcc tcg ctg gtg tct gcc ttc ctg 858
Gly Gly Pro Cys His Pro Thr Ser Ser Ser Leu Val Ser Ala Phe Leu
45 50 55
gca cca atc ctg gcc ctg gag ttt gtc ctg ggc ctg gtg ggg aac agt 906
Ala Pro Ile Leu Ala Leu Glu Phe Val Leu Gly Leu Val Gly Asn Ser
60 65 70
ttg gcc ctc ttc atc ttc tgc atc cac acg cgg ccc tgg acc tcc aac 954
Leu Ala Leu Phe Ile Phe Cys Ile His Thr Arg Pro Trp Thr Ser Asn
75 80 85
acg gtg ttc ctg gtc agc ctg gtg gcc get gac ttc ctc ctg atc agc 1002
Thr Val Phe Leu Val Ser Leu Val Ala Ala Asp Phe Leu Leu Ile Ser
90 95 100
aac ctg ccc ctc cgc gtg gac tac tac ctc ctc cat gag acc tgg cgc 1050
Asn Leu Pro Leu Arg Val Asp Tyr Tyr Leu Leu His Glu Thr Trp Arg
105 110 115 120
ttt ggg get get gcc tgc aaa gtc aac ctc ttc atg ctg tcc acc aac 1098
Phe Gly Ala Ala Ala Cys Lys Val Asn Leu Phe Met Leu Ser Thr Asn
125 130 135
cgc acg gcc agc gtt gtc ttc ctc aca gcc atc gca ctc aac cgc tac 1146
Arg Thr Ala Ser Val Val Phe Leu Thr Ala Ile Ala Leu Asn Arg Tyr
140 145 150
ctg aag gtg gtg cag ccc cac cac gtg ctg agc cgt get tcc gtg ggg 1194
Leu Lys Val Val Gln Pro His His Val Leu Ser Arg Ala Ser Val Gly
155 160 165
gca get gcc cgg gtg gcc ggg gga ctc tgg gtg ggc atc ctg ctc ctc 1242
Ala Ala Ala Arg Val Ala Gly Gly Leu Trp Val Gly Ile Leu Leu Leu
170 175 180
aac ggg cac ctg ctc ctg agc acc ttc tcc ggc ccc tcc tgc ctc agc 1290
Asn Gly His Leu Leu Leu Ser Thr Phe Ser Gly Pro Ser Cys Leu Ser
185 190 195 200
tac agg gtg ggc acg aag ccc tcg gcc tcg ctc cgc tgg cac cag gca 1338
Tyr Arg Val Gly Thr Lys Pro Ser Ala Ser Leu Arg Trp His Gln Ala
205 210 215
ctg tac ctg ctg gag ttc ttc ctg cca ctg gcg ctc atc ctc ttt get 1386
Leu Tyr Leu Leu Glu Phe Phe Leu Pro Leu Ala Leu Ile Leu Phe Ala
220 225 230

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-9-
attgtg agcattggg ctcaccatc cggaaccgt ggtctg ggcgggcag 1434
IleVal SerIleGly LeuThrIle ArgAsnArg GlyLeu GlyGlyGln
235 240 245
gcaggc ccgcagagg gccatgcgt gtgctggcc atggtg gtggccgtc 1482
AlaGly ProGlnArg AlaMetArg ValLeuAla MetVal ValAlaVal
250 255 260
tacacc atctgcttc ttgcccagc atcatcttt ggcatg gettccatg 1530
TyrThr IleCysPhe LeuProSer IleIlePhe GlyMet AlaSerMet
265 270 275 280
gtgget ttctggctg tccgcctgc cgatccctg gacctc tgcacacag 1578
ValAla PheTrpLeu SerAlaCys ArgSerLeu AspLeu CysThrGln
285 290 295
ctcttc catggctcc ctggccttc acctacctc aacagt gtcctggac 1626
LeuPhe HisGlySer LeuAlaPhe ThrTyrLeu AsnSer ValLeuAsp
300 305 310
cccgtg ctctactgc ttctctagc cccaacttc ctccac cagagccgg 1674
ProVal LeuTyrCys PheSerSer ProAsnPhe LeuHis GlnSerArg
315 320 325
gccttg ctgggcctc acgcggggc cggcagggc ccagtg agcgacgag 1722
AlaLeu LeuGlyLeu ThrArgGly ArgGlnGly ProVal SerAspGlu
330 335 340
agctcc taccaaccc tccaggcag tggcgctac cgggag gcctctagg 1770
SerSer TyrGlnPro SerArgGln TrpArgTyr ArgGlu AlaSerArg
345 350 355 360
aaggcg gaggccata gggaagctg aaagtgcag ggcgag gtctctctg 1818
LysAla GluAlaIle GlyLysLeu LysValGln GlyGlu ValSerLeu
365 370 375
gaaaag gaaggctcc tcccagggc tgagggccagctg cagggctgca 1865
GluLys GluGlySer SerGlnGly
380 385
gcgctgtggg ggtaagggct gccgcgctct ggcctggagg gacaaggcca gcacacggtg 1925
cctcaaccaa ctggacaagg gatggcggca gaccaggggc caggccaaag cactggcagg 1985
actcatgtgg gtggcaggga gagaaaccca cctaggcctc tcagtgtgtc caggatggca 2045
ttcccagaat gcaggggaga gcaggatgcc gggtggagga gacaggcaag gtgccgttgg 2105
cacaccagct cagacagggg cctgcgcagc tgcaggggac agacgccaat cactgtcaca 2165
gcagagtcac cttagaaatt ggacagctgc atgttctgtg ctctccagtt tgtcccttcc 2225
aatattaata aacttccctt ttaaatatat ttatttgcag accaatatct gtctttaatt 2285
ctaacctggg actgtcagta ggcgtcaaag tgagcgcccc agtgaaggaa ccttggagag 2345
agtgggagca ttcccagcct tccaggggga ctcgtcttcc agactttgga gcccgcatgt 2405
ctgaagcaga ctctttcttg gtag 2429

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
- 10-
<210> 6
<211> 384
<212> PRT
<213> Homo sapiens
<400> 6
Met Glu Leu His Asn Leu Ser Ser Pro Ser Pro Ser Leu Ser Ser Ser
1 5 10 15
Val Leu Pro Pro Ser Phe Ser Pro Ser Pro Ser Ser Ala Pro Ser Ala
20 25 30
Phe Thr Thr Val Gly Gly Ser Ser Gly Gly Pro Cys His Pro Thr Ser
35 40 45
Ser Ser Leu Val Ser Ala Phe Leu Ala Pro Ile Leu Ala Leu Glu Phe
50 55 60
Val Leu Gly Leu Val Gly Asn Ser Leu Ala Leu Phe Ile Phe Cys Ile
65 70 75 80
His Thr Arg Pro Trp Thr Ser Asn Thr Val Phe Leu Val Ser Leu Val
85 90 95
Ala Ala Asp Phe Leu Leu Ile Ser Asn Leu Pro Leu Arg Val Asp Tyr
100 105 110
Tyr Leu Leu His Glu Thr Trp Arg Phe Gly Ala Ala Ala Cys Lys Val
115 120 125
Asn Leu Phe Met Leu Ser Thr Asn Arg Thr Ala Ser Val Val Phe Leu
130 135 140
Thr Ala Ile Ala Leu Asn Arg Tyr Leu Lys Val Val Gln Pro His His
145 150 155 160
Val Leu Ser Arg Ala Ser Val Gly Ala Ala Ala Arg Val Ala Gly Gly
165 170 175
Leu Trp Val Gly Ile Leu Leu Leu Asn Gly His Leu Leu Leu Ser Thr
180 185 190
Phe Ser Gly Pro Ser Cys Leu Ser Tyr Arg Val Gly Thr Lys Pro Ser
195 200 205
Ala Ser Leu Arg Trp His Gln Ala Leu Tyr Leu Leu Glu Phe Phe Leu
210 215 220
Pro Leu Ala Leu Ile Leu Phe Ala Ile Val Ser Ile Gly Leu Thr Ile
225 230 235 240
Arg Asn Arg Gly Leu Gly Gly Gln Ala Gly Pro Gln Arg Ala Met Arg
245 250 255
Val Leu Ala Met Val Val Ala Val Tyr Thr Ile Cys Phe Leu Pro Ser
260 265 270
Ile Ile Phe Gly Met Ala Ser Met Val Ala Phe Trp Leu Ser Ala Cys
275 280 285
Arg Ser Leu Asp Leu Cys Thr Gln Leu Phe His Gly Ser Leu Ala Phe
290 295 300

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
Thr LeuAsnSer ValLeuAsp ProVal LeuTyrCys PheSerSer
Tyr
305 310 315 320
Pro PheLeuHis GlnSerArg AlaLeu LeuGlyLeu ThrArgGly
Asn
325 330 335
Arg GlyProVal SerAspGlu SerSer TyrGlnPro SerArgGln
Gln
340 345 350
Trp TyrArgGlu AlaSerArg LysAla GluAlaIle GlyLysLeu
Arg
355 360 365
Lys GlnGlyGlu ValSerLeu GluLys GluGlySer SerGlnGly
Val
370 375 380
<210>
7
<211>
1484
<212>
DNA
<213> Sapiens
Homo
<220>
<221>
CDS
<222> ..(1147)
(146)
<400>
7
ttgaatttag gtgacactat tgcacgcgta cgtaagctcg
60
agaagagcta
tgacgtcgca
gaattcggct cgagctgaac agacagagag aactgagtat
120
taatgactgc
cgccataaga
cctcccaaag gtgacactgg atg aacacc acagtgatg caaggcttc 172
aagca
Met AsnThr ThrValMet GlnGlyPhe
1 5
aac tctgagcgg tgccccaga gacact cggatagta cagctggta 220
aga
Asn SerGluArg CysProArg AspThr ArgIleVal GlnLeuVal
Arg
15 20 25
ttc gccctctac acagtggtt ttcttg accggcatc ctgctgaat 268
cca
Phe AlaLeuTyr ThrValVal PheLeu ThrGlyIle LeuLeuAsn
Pro
30 35 40
act getctgtgg gtgtttgtt cacatc cccagctcc tccaccttc 316
ttg
Thr AlaLeuTrp ValPheVal HisIle ProSerSer SerThrPhe
Leu
45 50 55
atc tacctcaaa aacactttg gtggcc gacttgata atgacactc 364
atc
Ile TyrLeuLys AsnThrLeu ValAla AspLeuIle MetThrLeu
Ile
60 65 70
atg cctttcaaa atcctctct gactca cacctggca ccctggcag 412
ctt
Met ProPheLys IleLeuSer AspSer HisLeuAla ProTrpGln
Leu
75 80 85
ctc gettttgtg tgtcgtttt tcttcg gtgatattt tatgagacc 460
aga
Leu AlaPheVal CysArgPhe SerSer ValIlePhe TyrGluThr
Arg
90 95 100 105
atg tat gtg ggc atc gtg ctg tta ggg ctc ata gcc ttt gac aga ttc 508
Met Tyr Val Gly Ile Val Leu Leu Gly Leu Ile Ala Phe Asp Arg Phe
110 115 120

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
- 12-
ctcaag atcatcaga cctttgaga aatattttt ctaaaa aaacctgtt 556
LeuLys IleIleArg ProLeuArg AsnIlePhe LeuLys LysProVal
125 130 135
tttgca aaaacggtc tcaatcttc atctgggtc tttttg gtcttcatc 604
PheAla LysThrVal SerIlePhe IleTrpVal PheLeu ValPheIle
140 145 150
tccctg ccaaatatg atcttgagc aacaaggaa gcaaca ccatcgtct 652
SerLeu ProAsnMet IleLeuSer AsnLysGlu AlaThr ProSerSer
155 160 165
gtgaaa aagtgtget tccttaaag gggcctctg gggctg aaatggcat 700
ValLys LysCysAla SerLeuLys GlyProLeu GlyLeu LysTrpHis
170 175 180 185
caaatg gtaaataac atatgccag tttattttc tggact ggttttatc 748
GlnMet ValAsnAsn IleCysGln PheIlePhe TrpThr GlyPheIle
190 195 200
ctaatg cttgtgttt tatgtggtt attgcaaaa aaagta tatgattct 796
LeuMet LeuValPhe TyrValVal IleAlaLys LysVal TyrAspSer
205 210 215
tataga aagtccaaa agtaaggac agaaaaaac aacaaa aagctggaa 844
TyrArg LysSerLys SerLysAsp ArgLysAsn AsnLys LysLeuGlu
220 225 230
ggcaaa gtatttgtt gtcgtgget gtcttcttt gtgtgt tttgetcca 892
GlyLys ValPheVal ValValAla ValPhePhe ValCys PheAlaPro
235 240 245
tttcat tttgccaga gttccatat actcacagt caaacc aacaataag 940
PheHis PheAlaArg ValProTyr ThrHisSer GlnThr AsnAsnLys
250 255 260 265
actgac tgtagactg caaaatcaa ctgtttatt getaaa gaaacaact 988
ThrAsp CysArgLeu GlnAsnGln LeuPheIle AlaLys GluThrThr
270 275 280
ctcttt ttggcagca actaacatt tgtatggat ccctta atatacata 1036
LeuPhe LeuAlaAla ThrAsnIle CysMetAsp ProLeu IleTyrIle
285 290 295
ttctta tgtaaaaaa ttcacagaa aagctacca tgtatg caagggaga 1084
PheLeu CysLysLys PheThrGlu LysLeuPro CysMet GlnGlyArg
300 305 310
aagacc acagcatca agccaagaa aatcatagc agtcag acagacaac 1132
LysThr ThrAlaSer SerGlnGlu AsnHisSer SerGln ThrAspAsn
315 320 325
ataacc ttaggctga caactgtaca tagggttaac tgatgagact
1187
ttctatttat
IleThr LeuGly
330
tccgtagata atgtggaaat caaatttaac caagaaaaaa agattggaac aaatgctctc 1247
ttacatttta tttatcctgg tgtccaggaa aagattatat taaatttaaa tccacataga 1307
tctattcata agctgaatga accattacct aagagaatgc aacaggatac caatggccac 1367
tagaggcata ttccttcttc tttttttttt gttaaatttc aagagcattc actttacatt 1427

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-13-
tggaaagact aaggggaacg gttatcctac aaacctccct tcaacacctt ttacatt 1484
<210> 8
<211> 333
<212> PRT
<213> Homo sapiens
<400> 8
Met Asn Thr Thr Val Met Gln Gly Phe Asn Arg Ser Glu Arg Cys Pro
1 5 10 15
Arg Asp Thr Arg Ile Val Gln Leu Val Phe Pro Ala Leu Tyr Thr Val
20 25 30
Val Phe Leu Thr Gly Ile Leu Leu Asn Thr Leu Ala Leu Trp Val Phe
35 40 45
Val His Ile Pro Ser Ser Ser Thr Phe Ile Ile Tyr Leu Lys Asn Thr
50 55 60
Leu Val Ala Asp Leu Ile Met Thr Leu Met Leu Pro Phe Lys Ile Leu
65 70 75 80
Ser Asp Ser His Leu Ala Pro Trp Gln Leu Arg Ala Phe Val Cys Arg
85 90 95
Phe Ser Ser Val Ile Phe Tyr Glu Thr Met Tyr Val Gly Ile Val Leu
100 105 110
Leu Gly Leu Ile Ala Phe Asp Arg Phe Leu Lys Ile Ile Arg Pro Leu
115 120 125
Arg Asn Ile Phe Leu Lys Lys Pro Val Phe Ala Lys Thr Val Ser Ile
130 135 140
Phe Ile Trp Val Phe Leu Val Phe Ile Ser Leu Pro Asn Met Ile Leu
145 150 155 160
Ser Asn Lys Glu Ala Thr Pro Ser Ser Val Lys Lys Cys Ala Ser Leu
165 170 175
Lys Gly Pro Leu Gly Leu Lys Trp His Gln Met Val Asn Asn Ile Cys
180 185 190
Gln Phe Ile Phe Trp Thr Gly Phe Ile Leu Met Leu Val Phe Tyr Val
195 200 205
Val Ile Ala Lys Lys Val Tyr Asp Ser Tyr Arg Lys Ser Lys Ser Lys
210 215 220
Asp Arg Lys Asn Asn Lys Lys Leu Glu Gly Lys Val Phe Val Val Val
225 230 235 240
Ala Val Phe Phe Val Cys Phe Ala Pro Phe His Phe Ala Arg Val Pro
245 250 255
Tyr Thr His Ser Gln Thr Asn Asn Lys Thr Asp Cys Arg Leu Gln Asn
260 265 270
Gln Leu Phe Ile Ala Lys Glu Thr Thr Leu Phe Leu Ala Ala Thr Asn
275 280 285

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
- 14-
Ile Cys Met Asp Pro Leu Ile Tyr Ile Phe Leu Cys Lys Lys Phe Thr
290 295 300
Glu Lys Leu Pro Cys Met Gln Gly Arg Lys Thr Thr Ala Ser Ser Gln
305 310 315 320
Glu Asn His Ser Ser Gln Thr Asp Asn Ile Thr Leu Gly
325 330
<210>
9
<211>
957
<212>
DNA
<213> ns
Homo
sapie
<220>
<221>
CDS
<222> )..(954)
(1
<400>
9
atgatg gtggatcccaat ggcaatgaa tccagtgetaca tacttcatc 48
MetMet ValAspProAsn GlyAsnGlu SerSerAlaThr TyrPheIle
1 5 10 15
ctaata ggcctccctggt ttagaagag getcagttctgg ttggccttc 96
LeuIle GlyLeuProGly LeuGluGlu AlaGlnPheTrp LeuAlaPhe
20 25 30
ccattg tgctccctctac cttattget gtgctaggtaac ttgacaatc 144
ProLeu CysSerLeuTyr LeuIleAla ValLeuGlyAsn LeuThrIle
35 40 45
atctac attgtgcggact gagcacagc ctgcatgagccc atgtatata 192
IleTyr IleValArgThr GluHisSer LeuHisGluPro MetTyrIle
50 55 60
tttctt tgcatgctttca ggcattgac atcctcatctcc acctcatcc 240
PheLeu CysMetLeuSer GlyIleAsp IleLeuIleSer ThrSerSer
65 70 75 80
atgccc aaaatgctggcc atcttctgg ttcaattccact accatccag 288
MetPro LysMetLeuAla IlePheTrp PheAsnSerThr ThrIleGln
85 90 95
tttgat gettgtctgcta cagatgttt gccatccactcc ttatctggc 336
PheAsp AlaCysLeuLeu GlnMetPhe AlaIleHisSer LeuSerGly
100 105 110
atggaa tccacagtgctg ctggccatg gettttgaccgc tatgtggcc 384
MetGlu SerThrValLeu LeuAlaMet AlaPheAspArg TyrValAla
115 120 125
atctgt cacccactgcgc catgccaca gtacttacgttg cctcgtgtc 432
IleCys HisProLeuArg HisAlaThr ValLeuThrLeu ProArgVal
130 135 140
accaaa attggtgtgget getgtggtg cggggggetgca ctgatggca 480
ThrLys IleGlyValAla AlaValVal ArgGlyAlaAla LeuMetAla
145 150 155 160
cccctt cctgtcttcatc aagcagctg cccttctgccgc tccaatatc 528
ProLeu ProValPheIle LysGlnLeu ProPheCysArg SerAsnIle
165 170 175

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-15-
ctttcc cattcctactgC Ctacaccaa gatgtcatg aagctggcctgt 576
LeuSer HisSerTyrCys LeuHisGln AspValMet LysLeuAlaCys
180 185 190
gatgat atccgggtcaat gtcgtctat ggccttatc gtcatcatctcc 624
AspAsp IleArgValAsn ValValTyr GlyLeuIle ValIleIleSer
195 200 205
gccatt ggcctggactca cttctcatc tccttctca tatctgcttatt 672
AlaIle GlyLeuAspSer LeuLeuIle SerPheSer TyrLeuLeuIle
210 215 220
cttaag actgtgttgggc ttgacacgt gaagcccag gccaaggcattt 720
LeuLys ThrValLeuGly LeuThrArg GluAlaGln AlaLysAlaPhe
225 230 235 240
ggcact tgcgtctctcat gtgtgtget gtgttCata ttctatgtacct 768
GlyThr CysValSerHis ValCysAla ValPheIle PheTyrValPro
245 250 255
ttcatt ggattgtccatg gtgcatcgc tttagcaag cggcgtgactct 816
PheIle GlyLeuSerMet ValHisArg PheSerLys ArgArgAspSer
260 265 270
ccgctg cccgtcatcttg gccaatatc tatctgctg gttcctcctgtg 864
ProLeu ProValIleLeu AlaAsnIle TyrLeuLeu ValProProVal
275 280 285
ctcaac ccaattgtctat ggagtgaag acaaaggag attcgacagcgc 912
LeuAsn ProIleValTyr GlyValLys ThrLysGlu IleArgGlnArg
290 295 300
atcctt cgacttttccat gtggccaca cacgettca gagccctag 957
IleLeu ArgLeuPheHis ValAlaThr HisAlaSer GluPro
305 310 315
<210>
<211>
318
<212>
PRT
<213> Sapiens
Homo
<400>
10
Met Met AspProAsn GlyAsnGlu SerSerAlaThr TyrPheIle
Val
1 5 10 15
Leu Ile LeuProGly LeuGluGlu AlaGlnPheTrp LeuAlaPhe
Gly
20 25 30
Pro Leu SerLeuTyr LeuIleAla ValLeuGlyAsn LeuThrIle
Cys
35 40 45
Ile Tyr ValArgThr GluHisSer LeuHisGluPro MetTyrIle
Ile
50 55 60
Phe Leu MetLeuSer GlyIleAsp IleLeuIleSer ThrSerSer
Cys
65 70 75 80
Met Pro MetLeuAla IlePheTrp PheAsnSerThr ThrIleGln
Lys
85 90 95
Phe Asp CysLeuLeu GlnMetPhe AlaIleHisSer LeuSerGly
Ala
100 105 110

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
- 1G-
MetGlu SerThrValLeu LeuAlaMet AlaPheAspArg TyrValAla
115 120 125
IleCys HisProLeuArg HisAlaThr ValLeuThrLeu ProArgVal
130 135 140
ThrLys IleGlyValAla AlaValVal ArgGlyAlaAla LeuMetAla
145 150 155 160
ProLeu ProValPheIle LysGlnLeu ProPheCysArg SerAsnIle
165 170 175
LeuSer HisSerTyrCys LeuHisGln AspValMetLys LeuAlaCys
180 185 190
AspAsp IleArgValAsn ValValTyr GlyLeuIleVal IleIleSer
195 200 205
AlaIle GlyLeuAspSer LeuLeuIle SerPheSerTyr LeuLeuIle
210 215 220
LeuLys ThrValLeuGly LeuThrArg GluAlaGlnAla LysAlaPhe
225 230 235 240
GlyThr CysValSerHis ValCysAla ValPheIlePhe TyrValPro
245 250 255
PheIle GlyLeuSerMet ValHisArg PheSerLysArg ArgAspSer
260 265 270
ProLeu ProValIleLeu AlaAsnIle TyrLeuLeuVal ProProVal
275 280 285
LeuAsn ProIleValTyr GlyValLys ThrLysGluIle ArgGlnArg
290 295 300
IleLeu ArgLeuPheHis ValAlaThr HisAlaSerGlu Pro
305 310 315
<210>
11
<211>
995
<212>
DNA
<213>
Homo
sapiens
<220>
<221>
CDS
<222> (921)
(1)..
<400>
11
atggaa agcgagaacaga agagtgata agagaattcatc ctccttggt 48
MetGlu SerGluAsnArg ArgValIle ArgGluPheIle LeuLeuGly
1 5 10 15
ctgacc cagtctcaagat attcagctc ctggtctttgtg ctagtttta 96
LeuThr GlnSerGlnAsp IleGlnLeu LeuValPheVal LeuValLeu
20 25 30
atattc tacttcatcatc ctccctgga aattttctcatt attttcacc 144
IlePhe TyrPheIleIle LeuProGly AsnPheLeuIle IlePheThr
35 40 45
ataaag tcagaccctggg ctcacagcc cccctctatttc tttctgggc 192
IleLys SerAspProGly LeuThrAla ProLeuTyrPhe PheLeuGly
50 55 60

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
- 17-
aacttg gccttcctggat gcatcctac tccttcattgtg getccccgg 240
AsnLeu AlaPheLeuAsp AlaSerTyr SerPheIleVal AlaProArg
65 70 75 80
atgttg gtggacttcctc tctgcgaag aagataatctcc tacagaggc 288
MetLeu ValAspPheLeu SerAlaLys LysIleIleSer TyrArgGly
85 90 95
tgcatc_actcagctcttt ttcttgcac ttccttggagga ggggaggga 336
CysIle ThrGlnLeuPhe PheLeuHis PheLeuGlyGly GlyGluGly
100 105 110
ttactc cttgttgtgatg gcctttgac cgctacatcgcc atctgccgg 384
LeuLeu LeuValValMet AlaPheAsp ArgTyrIleAla IleCysArg
115 120 125
cctctg cactatcctact gtcatgaac cctagaacctgc tatgcaatg 432
ProLeu HisTyrProThr ValMetAsn ProArgThrCys TyrAlaMet
130 135 140
atgttg getctgtggctt gggggtttt gtccactccatt atccaggtg 480
MetLeu AlaLeuTrpLeu GlyGlyPhe ValHisSerIle IleGlnVal
145 150 155 160
gtc.ctc atcctccgcttg cctttttgt ggcccaaaccag ctggacaac 528
ValLeu IleLeuArgLeu ProPheCys GlyProAsnGln LeuAspAsn
165 170 175
ttcttc tgtgatgtccca caggtcatc aagctggcctgc accgacaca 576
PhePhe CysAspValPro GlnValIle LysLeuAlaCys ThrAspThr
180 185 190
tttgtg gtggagcttctg atggtcttc aacagtggcctg atgacactc 624
PheVal ValGluLeuLeu MetVal~Phe AsnSerGlyLeu MetThrLeu
195 200 205
ctgtgc tttctggggctt ctggcctcc tatgcagtcatt ctttgtcgc 672
LeuCys PheLeuGlyLeu LeuAlaSer TyrAlaValIle LeuCysArg
210 215 220
atacga gggtcttcttct gaggcaaaa aacaaggccatg tccacgtgo 720
IleArg GlySerSerSer GluAlaLys AsnLysAlaMet SerThrCys
225 230 235 240
atcacc catatcattgtt atattcttc atgtttggacct ggcatcttc 768
IleThr HisIleIleVal IlePhePhe MetPheGlyPro GlyIlePhe
245 250 255
atctac acgcgccccttc agggetttc ccagetgacaag gtggtttct 816
IleTyr ThrArgProPhe ArgAlaPhe ProAlaAspLys ValValSer
260 265 270
ctcttc cacacagtgatt tttcctttg ttgaatcctgtc atttatacc 864
LeuPhe HisThrValIle PheProLeu LeuAsnProVal IleTyrThr
275 280 285
cttcgc aaccaggaagtg aaagettcc atgaaaaaggtg tttaataag 912
LeuArg AsnGlnGluVal LysAlaSer MetLysLysVal PheAsnLys
290 295 300
cacata gcctgaaaaaggg cgcaaaaaaa 961
aaaagaataa
aaatagactg
HisIle Ala
305

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-18-
tagaattttt aaaaaaaaaa aaaaaaaaaa aaaa 995
<210> 12
<211> 307
<212> PRT
<213> Homo Sapiens
<400> 12
Met Glu Ser Glu Asn Arg Arg Val Ile Arg Glu Phe Ile Leu Leu Gly
1 5 10 15
Leu Thr Gln Ser Gln Asp Ile Gln Leu Leu Val Phe Val Leu Val Leu
20 25 30
Ile Phe Tyr Phe Ile Ile Leu Pro Gly Asn Phe Leu Ile Ile Phe Thr
35 40 45
Ile Lys Ser Asp Pro Gly Leu Thr Ala Pro Leu Tyr Phe Phe Leu Gly
50 55 60
Asn Leu Ala Phe Leu Asp Ala Ser Tyr Ser Phe Ile Val Ala Pro Arg
65 70 75 80
Met Leu Val Asp Phe Leu Ser Ala Lys Lys Ile Ile Ser Tyr Arg Gly
85 90 95
Cys Ile Thr Gln Leu Phe Phe Leu His Phe Leu Gly Gly Gly Glu Gly
100 105 110
Leu Leu Leu Val Val Met Ala Phe Asp Arg Tyr Ile Ala Ile Cys Arg
115 120 125
Pro Leu His Tyr Pro Thr Val Met Asn Pro Arg Thr Cys Tyr Ala Met
130 135 140
Met Leu Ala Leu Trp Leu Gly Gly Phe Val His Ser Ile Ile Gln Val
145 150 155 160
Val Leu Ile Leu Arg Leu Pro Phe Cys Gly Pro Asn Gln Leu Asp Asn
165 170 175
Phe Phe Cys Asp Val Pro Gln Val Ile Lys Leu Ala Cys Thr Asp Thr
180 185 190
Phe Val Val Glu Leu Leu Met Val Phe Asn Ser Gly Leu Met Thr Leu
195 200 205
Leu Cys Phe Leu Gly Leu Leu Ala Ser Tyr Ala Val Ile Leu Cys Arg
210 215 220
Ile Arg Gly Ser Ser Ser Glu Ala Lys Asn Lys Ala Met Ser Thr Cys
225 230 235 240
Ile Thr His Ile Ile Val Ile Phe Phe Met Phe Gly Pro Gly Ile Phe
245 250 255
Ile Tyr Thr Arg Pro Phe Arg Ala Phe Pro Ala Asp Lys Val Val Ser
260 265 270
Leu Phe His Thr Val Ile Phe Pro Leu Leu Asn Pro Val Ile Tyr Thr
275 280 285

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
- 19-
Leu Arg Asn Gln Glu Val Lys Ala Ser Met Lys Lys Val Phe Asn Lys
290 295 300
His Ile Ala
305
<210> 13
<211> 1380
<212> DNA
<213> Homo Sapiens
<220>
<221> CDS
<222> (266)..(1375)
<220>
<221> misc_feature
<222> (32)
<223> n = A or C or G or T
<220>
<221> misc_feature
<222> (55)
<223> n = A or C or G or T
<220>
<221> misc_feature
<222> (74)
<223> n = A or C or G or T
<400> 13
tgcttcccca taaggtaaca gctttgttag cnctgtctga catcattgct tgttnactta 60
agaactgata ggtntttttt tttttttttt ttcagatatt ctgatggcaa aacaagtgga 120
agaaaagagg aagcatgact gcagatcaga tcagttctct ttgtggatta tattttcagt 180
aaaatgtatg gatctatctt ttccttgttc ttatatctag atcatgagac ttgactgagg 240
ctgtatcctt atcctccatc catct atg gcg aac tat agc cat gca get gac 292
Met Ala Asn Tyr Ser His Ala Ala Asp
1 5
aacatt ttgcaaaat ctctcgcct ctaacagcc tttctgaaa ctgact 340
AsnIle LeuGlnAsn LeuSerPro LeuThrAla PheLeuLys LeuThr
15 20 25
tccttg ggtttcata ataggagtc agcgtggtg ggcaacctc ctgatc 388
SerLeu GlyPheIle IleGlyVal SerValVal GlyAsnLeu LeuIle
30 35 40
tccatt ttgctagtg aaagataag accttgcat agagcacct tactac 436
SerIle LeuLeuVal LysAspLys ThrLeuHis ArgAlaPro TyrTyr
45 50 55
ttcctg ttggatctt tgctgttca gatatcctc agatctgca atttgt 484
PheLeu LeuAspLeu CysCysSer AspIleLeu ArgSerAla IleCys
60 65 70
ttccca tttgtgttc aactctgtc aaaaatggt tctacctgg acttat 532
PhePro PheValPhe AsnSerVal LysAsnGly SerThrTrp ThrTyr
75 80 85

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-20-
gggact ctgacttgc aaagtgatt gccttt ctgggggtt ttgtcc tgt 580
GlyThr LeuThrCys LysVallle AlaPhe LeuGlyVal LeuSer Cys
90 95 100 105
ttccac actgetttc atgctcttc tgcatc agtgtcacc agatat tta 628
PheHis ThrAlaPhe MetLeuPhe CysIle SerValThr ArgTyr Leu
110 115 120
getatc gcccatcac cgcttctat acaaag aggctgacc ttttgg acg 676
AlaIle AlaHisHis ArgPheTyr ThrLys ArgLeuThr PheTrp Thr
125 130 135
tgtctg getgtgatc tgtatggtg tggact ctgtctgtg gccatg gca 724
CysLeu AlaValIle CysMetVal TrpThr LeuSerVal AlaMet A1a
140 145 150
tttccc ccggtttta gacgtgggc acttac tcattcatt agggag gaa 772
PhePro ProValLeu AspValGly ThrTyr SerPheIle ArgGlu Glu
155 160 165
gatcaa tgcaccttc caacaccgc tccttc agggetaat gattcc tta 820
AspGln CysThrPhe GlnHisArg SerPhe ArgAlaAsn AspSer Leu
170 175 180 185
ggattt atgctgctt cttgetctc atcctc ctagccaca cagctt gtc 868
GlyPhe MetLeuLeu LeuAlaLeu IleLeu LeuAlaThr GlnLeu Val
190 195 200
tacctc aagctgata tttttcgtc cacgat cgaagaaaa atgaag cca 916
TyrLeu LysLeuIle PhePheVal HisAsp ArgArgLys MetLys Pro
205 210 215
gtccag tttgtagca gcagtcagc cagaac tggactttt catggt cct 964
ValGln PheValAla AlaValSer GlnAsn TrpThrPhe HisGly Pro
220 225 230
gga gcc agt ggc cag gca get gcc aat tgg cta gca gga ttt gga agg 1012
Gly Ala Ser Gly Gln Ala Ala Ala Asn Trp Leu Ala Gly Phe Gly Arg
235 240 245
ggtcccacacca cccaccttg ctgggcatcagg caaaat gcaaacacc 1060
GlyProThrPro ProThrLeu LeuGlyIleArg GlnAsn AlaAsnThr
250 255 260 265
acaggcagaaga aggctattg gtcttagacgag ttcaaa atggagaaa 1108
ThrGlyArgArg ArgLeuLeu ValLeuAspGlu PheLys MetGluLys
270 275 280
agaatcagcaga atgttctat ataatgactttt ctgttt ctaaccttg 1156
ArgIleSerArg MetPheTyr IleMetThrPhe LeuPhe LeuThrLeu
285 290 295
tggggcccctac ctggtggcc tgttattggaga gttttt gcaagaggg 1204
TrpGlyProTyr LeuValAla CysTyrTrpArg ValPhe AlaArgGly
300 305 310
cctgtagtacca gggggattt ctaacagetget gtctgg atgagtttt 1252
ProValValPro GlyGlyPhe LeuThrAlaAla ValTrp MetSerPhe
315 320 325

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-21 -
gcc caa gca gga atc aat cct ttt gtc tgc att ttc tca aac agg gag 1300
Ala Gln Ala Gly Ile Asn Pro Phe Val Cys Il.e Phe Ser Asn Arg Glu
330 335 340 345
ctg agg cgc tgt ttc agc aca acc ctt ctt tac tgc aga aaa tcc agg 1348
Leu Arg Arg Cys Phe Ser Thr Thr Leu Leu Tyr Cys Arg Lys Ser Arg
350 355 360
tta cca agg gaa cct tac tgt gtt ata tgagg 1380
Leu Pro Arg Glu Pro Tyr Cys Val Ile
365 370
<210>
14
<211>
370
<212>
PRT
<213>
Homo
Sapiens
c400>
14
MetAla AsnTyrSer HisAlaAla AspAsnIle LeuGlnAsn LeuSer
1 5 10 15
ProLeu ThrAlaPhe LeuLysLeu ThrSerLeu GlyPheIle IleGly
20 25 30
ValSer ValValGly AsnLeuLeu IleSerIle LeuLeuVal LysAsp
35 40 45
LysThr LeuHisArg AlaProTyr TyrPheLeu LeuAspLeu CysCys
50 55 60
SerAsp IleLeuArg SerAlaIle CysPhePro PheValPhe AsnSer
65 70 75 80
ValLys AsnGlySer ThrTrpThr TyrGlyThr LeuThrCys LysVal
85 90 95
IleAla PheLeuGly ValLeuSer CysPheHis ThrAlaPhe MetLeu
100 105 110
PheCys IleSerVal ThrArgTyr LeuAlaIle AlaHisHis ArgPhe
115 120 125
TyrThr LysArgLeu ThrPheTrp ThrCysLeu AlaValIle CysMet
130 135 140
ValTrp ThrLeuSer ValAlaMet AlaPhePro ProValLeu AspVal
145 150 155 160
GlyThr TyrSerPhe IleArgGlu GluAspGln CysThrPhe GlnHis
165 170 175
ArgSer PheArgAla AsnAspSer LeuGlyPhe MetLeuLeu LeuAla
180 185 190
LeuIle LeuLeuAla ThrGlnLeu ValTyrLeu LysLeuIle PhePhe
195 200 205
ValHis AspArgArg LysMetLys ProValGln PheValAla AlaVal
210 215 220
SerGln AsnTrpThr PheHisGly ProGlyAla SerGlyGln AlaAla
225 230 235 240

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-22-
Ala Asn Trp Leu Ala Gly Phe Gly Arg Gly Pro Thr Pro Pro Thr Leu
245 250 255
Leu Gly Ile Arg G.Ln Asn Ala Asn Thr Thr Gly Arg Arg Arg Leu Leu
260 265 270
Val Leu Asp Glu Phe Lys Met Glu Lys Arg Ile Ser Arg Met Phe Tyr
275 280 285
Ile Met Thr Phe Leu Phe Leu Thr Leu Trp Gly Pro Tyr Leu Val Ala
290 295 300
Cys Tyr Trp Arg Val Phe Ala Arg Gly Pro Val Val Pro Gly Gly Phe
305 310 315 320
Leu Thr Ala Ala Val Trp Met Ser Phe Ala Gln Ala Gly Ile Asn Pro
325 330 335
Phe Val Cys Ile Phe Ser Asn Arg Glu Leu Arg Arg Cys Phe Ser Thr
340 345 350
Thr Leu Leu Tyr Cys Arg Lys Ser Arg Leu Pro Arg Glu Pro Tyr Cys
355 360 365
Val Ile
370
<210> 15
<211> 1191
<212> DNA
<213> Homosapiens
<220>
<221> CDS
<222> (1)..(1188)
<400> 15
atg ttt cctctt gtgaatctc tctcac atatatttt aagaaattc 48
aga
Met Phe ProLeu ValAsnLeu SerHis IleTyrPhe LysLysPhe
Arg
1 5 10 15
cag tac gggtat gcaccacat gttcgc agctgtaaa ccaaacact 96
tgt
Gln Tyr GlyTyr AlaProHis ValArg SerCysLys ProAsnThr
Cys
20 25 30
gat gga tcatct ctagagaat ctcttg gcaagcatt attcagaga 144
att
Asp Gly SerSer LeuGluAsn LeuLeu AlaSerIle IleGlnArg
Ile
35 40 45
gta ttt tgggtt gtatctgca gttacc tgctttgga aacattttt 192
gtc
Val Phe TrpVal ValSerAla ValThr CysPheGly AsnIlePhe
Val
50 55 60
gtc att atgcga ccttatatc aggtct gagaacaag ctgtatgcc 240
tgc
Val Ile MetArg ProTyrIle ArgSer GluAsnLys LeuTyrAla
Cys
65 70 75 80
atg tca atttct ctctgctgt gccgac tgcttaatg ggaatatat 288
atc
Met Ser IleSer LeuCysCys AlaAsp CysLeuMet GlyIleTyr
Ile
85 90 95

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-23-
ttattc gtgatcgga ggctttgac ctaaagttt cgtgga gaatacaat 336
LeuPhe ValIleGly GlyPheAsp LeuLysPhe ArgGly GluTyrAsn
100 105 110
aagcat gcgcagctg tggatggag agtactcat tgtcag cttgtagga 384
LysHis AlaGlnLeu TrpMetGlu SerThrHis CysGln I~euValGly
115 120 125
tctttg gccattctg tccacagaa gtatcagtt ttactg ttaacattt 432
SerLeu AlaIleLeu SerThrGlu ValSerVal LeuLeu LeuThrPhe
130 135 140
ctgaca ttggaaaaa tacatctgc attgtctat cctttt agatgtgtg 480
LeuThr LeuGluLys TyrIleCys IleValTyr ProPhe ArgCysVal
145 150 155 160
agacct ggaaaatgc agaacaatt acagttctg attctc atttggatt 528
ArgPro GlyLysCys ArgThrIle ThrValLeu IleLeu IleTrpTle
165 170 175
actggt tttatagtg getttcatt ccattgagc aataag gaatttttc 576
ThrGly PheIleVal AlaPheIle ProLeuSer AsnLys GluPhePhe
180 185 190
aaaaac tactatggc accaatgga gtatgcttc cctctt cattcagaa 624
LysAsn TyrTyrGly ThrAsnGly ValCysPhe ProLeu HisSerGlu
195 200 205
gataca gaaagtatt ggagcccag atttattca gtggca atttttctt 672
AspThr GluSerIle GlyAlaGln IleTyrSer ValAla I7.ePheLeu
210 215 220
ggtatt aatttggcc gcatttatc atcatagtt ttttcc tatggaagc 720
GlyIle AsnLeuAla AlaPheIle IleIleVal PheSer TyrGlySer
225 230 235 240
atgttt tatagtgtt catcaaagt gccataaca gcaact gaaatacgg 768
MetPhe TyrSerVal HisGlnSer AlaIleThr AlaThr GluIleArg
245 250 255
aatcaa gttaaaaaa gagatgatc cttgccaaa cgtttt ttctttata 816
AsnGln ValLysLys GluMetIle LeuAlaLys ArgPhe PhePheIle
260 265 270
gtattt actgatgca ttatgctgg atacccatt tttgta gtgaaattt 864
ValPhe ThrAspAla LeuCysTrp IleProIle PheVal ValLysPhe
275 280 285
ctttca ctgcttcag gtagaaata ccaggtacc ataacc tcttgggta 912
LeuSer LeuLeuGln ValGluIle ProGlyThr IleThr SerTrpVal
290 295 300
gtgatt tttattctg cccattaac agtgetttg aaccca attctctat 960
ValIle PheIleLeu ProIleAsn SerAlaLeu AsnPro IleLeuTyr
305 310 315 320
actctg accacaaga ccatttaaa gaaatgatt catcgg ttttggtat 1008
ThrLeu ThrThrArg ProPheLys GluMetIle HisArg PheTrpTyr
325 330 335
aactac agacaaaga aaatctatg gacagcaaa ggtcag aaaacatat 1056
AsnTyr ArgGlnArg LysSerMet AspSerLys GlyGln LysThrTyr
340 345 350

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-24-
get cca tca ttc atc tgg gtg gaa atg tgg cca ctg cag gag atg cca 1104
Ala Pro Ser Phe Ile Trp Val Glu Met Trp Pro Leu Gln Glu Met Pro
355 360 365
cct gag tta atg aag ccg gac ctt ttc aca taC CCC tgt gaa atg tca 1152
Pro Glu Leu Met Lys Pro Asp Leu Phe Thr Tyr Pro Cys Glu Met Ser
370 375 380
ctg att tct caa tca acg aga ctc aat tcc tat tca tga 1191
Leu Ile Ser Gln Ser Thr Arg Leu Asn Ser Tyr Ser
385 390 395
<210> 16
<211> 396
<212> PRT
<213> Homo Sapiens
<400> 16
Met Phe Arg Pro Leu Val Asn Leu Ser His Ile Tyr Phe Lys Lys Phe
1 5 10 15
Gln Tyr Cys Gly Tyr Ala Pro His Val Arg Ser Cys Lys Pro Asn Thr
20 25 30
Asp Gly Ile Ser Ser Leu Glu Asn Leu Leu Ala Ser Ile Ile Gln Arg
35 40 45
Val Phe Val Trp Val Val Ser Ala Val Thr Cys Phe Gly Asn Ile Phe
50 55 60
Val Ile Cys Met Arg Pro Tyr Ile Arg Ser Glu Asn Lys Leu Tyr Ala
65 70 75 80
Met Ser Ile Ile Ser Leu Cys Cys Ala Asp Cys Leu Met Gly Ile Tyr
85 90 95
Leu Phe Val Ile Gly Gly Phe Asp Leu Lys Phe Arg Gly Glu Tyr Asn
100 105 110
Lys His Ala Gln Leu Trp Met Glu Ser Thr His Cys Gln Leu Val Gly
115 120 125
Ser Leu Ala Ile Leu Ser Thr Glu Val Ser Val Leu Leu Leu Thr Phe
130 135 140
Leu Thr Leu Glu Lys Tyr Ile Cys Ile Val Tyr Pro Phe Arg Cys Val
145 150 155 160
Arg Pro Gly Lys Cys Arg Thr Ile Thr Val Leu Ile Leu Ile Trp Ile
165 170 175
Thr Gly Phe Ile Val Ala Phe Ile Pro Leu Ser Asn Lys Glu Phe Phe
180 185 190
Lys Asn Tyr Tyr Gly Thr Asn Gly Val Cys Phe Pro Leu His Ser Glu
195 200 205
Asp Thr Glu Ser Ile Gly Ala Gln Ile Tyr Ser Val Ala Ile Phe Leu
210 215 220
Gly Ile Asn Leu Ala Ala Phe Ile Ile Ile Val Phe Ser Tyr Gly Ser
225 230 235 240

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-25-
MetPhe TyrSerVal HisGlnSer AlaIle ThrAlaThrGlu IleArg
245 250 255
AsnGln ValLysLys GluMetIle LeuAla LysArgPhePhe PheIle
260 265 270
ValPhe ThrAspAla LeuCysTrp IlePro IlePheValVal LysPhe
275 280 285
LeuSer LeuLeuGln ValGluIle ProGly ThrIleThrSer TrpVal
290 295 300
ValIle PheIleLeu ProIleAsn SerAla LeuAsnProIle LeuTyr
305 310 315 320
ThrLeu ThrThrArg ProPheLys GluMet IleHisArgPhe TrpTyr
325 330 335
AsnTyr ArgGlnArg LysSerMet AspSer LysGlyGlnLys ThrTyr
340 345 350
AlaPro SerPheIle TrpValGlu MetTrp ProLeuGlnGlu MetPro
355 360 365
ProGlu LeuMetLys ProAspLeu PheThr TyrProCysGlu MetSer
370 375 380
LeuIle SerGlnSer ThrArgLeu AsnSer TyrSer
385 390 395
<210>
17
<211> 64
11
<212>
DNA
<213> Sapiens
Homo
<220>
<221>
CDS
<222> 3)..(1089)
(1
<400>
17
cacaactgaa ga 51
atg
ggg
ttc
aac
ttg
acg
ctt
gca
aaa
tta
cca
aat
aac
Met he
Gly Asn
P Leu
Thr
Leu
Ala
Lys
Leu
Pro
Asn
Asn
1 5 10
gagctg cacggccaa gagagtcac aattca ggcaacaggagc gacggg 99
GluLeu HisGlyGln GluSerHis AsnSer GlyAsnArgSer AspGly
15 20 25
ccagga aagaacacc acccttcac aatgaa tttgacacaatt gtcttg 147
ProGly LysAsnThr ThrLeuHis AsnGlu PheAspThrIle ValLeu
30 35 40 45
ccagtg ctttatctc attatattt gtggca agcatcttgctg aatggt 195
ProVal LeuTyrLeu IleIlePhe ValAla SerIleLeuLeu AsnGly
50 55 60
ttagca gtgtggatc ttcttccac attagg aataaaaccagc ttcata 243
LeuAla ValTrpIle PhePheHis IleArg AsnLysThrSer PheIle
65 70 75
ttctat ctcaaaaac atagtggtt gcagac ctcataatgacg ctgaca 291
PheTyr LeuLysAsn IleValVal AlaAsp LeuIleMetThr LeuThr
80 85 90

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-2G-
tttcca tttcgaata gtccatgat gcagga tttggacct tggtacttc 339
PhePro PheArgIle ValHisAsp AlaGly PheGlyPro TrpTyrPhe
95 100 105
aagttt attctctgc agatacact tcagtt ttgttttat gcaaacatg 387
LysPhe IleLeuCys ArgTyrThr SerVal LeuPheTyr AlaAsnMet
110 115 120 125
tatact tccatcgtg ttccttggg ctgata agcattgat cgctatctg 435
TyrThr SerIleVal PheLeuGly LeuIle SerIleAsp ArgTyrLeu
130 135 140
aaggtg gtcaagcca tttggggac tctcgg atgtacagc ataaccttc 483
LysVal ValLysPro PheGlyAsp SerArg MetTyrSer IleThrPhe
145 150 155
acgaag gttttatct gtttgtgtt tgggtg atcatgget gttttgtct 531
ThrLys ValLeuSer ValCysVal TrpVal IleMetAla ValLeuSer
160 165 170
ttgcca aacatcatc ctgacaaat ggtcag ccaacagag gacaatatc 579
LeuPro AsnIleIle LeuThrAsn GlyGln ProThrGlu AspAsnIle
175 180 185
catgac tgctcaaaa cttaaaagt Cctttg ggggtcaaa tggcatacg 627
HisAsp CysSerLys LeuLysSer ProLeu GlyValLys TrpHisThr
190 195 200 205
gcagtc acctatgtg aacagctgc ttgttt gtggccgtg ctggtgatt 675
AlaVal ThrTyrVal AsnSerCys LeuPhe ValAlaVal LeuValI.Le
210 215 220
ctgatc ggatgttac atagccata tccagg tacatccac aaatccagc 723
LeuIle GlyCysTyr IleAlaIle SerArg TyrIleHis LysSerSer
225 230 235
aggcaa ttcataagt cagtcaagc cgaaag cgaaaacat aaccagagc 771
ArgGln PheIleSer GlnSerSer ArgLys ArgLysHis AsnGlnSer
240 245 250
atcagg gttgttgtg getgtgttt tttacc tgctttcta ccatatcac 819
IleArg ValValVal AlaValPhe PheThr CysPheLeu ProTyrHis
255 260 265
ttgtgc agaattcct tttactttt agtcac ttagacagg cttttagat 867
LeuCys ArgIlePro PheThrPhe SerHis LeuAspArg LeuLeuAsp
270 275 280 285
gaatct gcacaaaaa atcctatat tactgc aaagaaatt acacttttc 915
GluSer AlaGlnLys IleLeuTyr TyrCys LysGluIle ThrLeuPhe
290 295 300
ttgtct gcgtgtaat gtttgcctg gatcca ataatttac tttttcatg 963
LeuSer AlaCysAsn ValCysLeu AspPro IleIleTyr PhePheMet
305 310 315
tgtagg tcattttca agaaggctg ttcaaa aaatcaaat atcagaacc 1011
CysArg SerPheSer ArgArgLeu PheLys LysSerAsn IleArgThr
320 325 330
aggagt gaaagcatc agatcactg caaagt gtgagaaga tcggaagtt 1059
ArgSer GluSerIle ArgSerLeu GlnSer ValArgArg SerGluVal
335 340 345

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-27-
ctc ata tat tat gat tat act gat gtg tag gccttttatt gtttgttgga 1109
Leu Ile Tyr Tyr Asp Tyr Thr Asp Val
350 355
atcgatatgt acaaagtgta aataaatgtt tcttttcatt aaaaaaaaaa aaaaa 1164
<210> 18
<211> 358
<212> PRT
<213> Homo sapiens
<400> 18
Met Gly Phe Asn Leu Thr Leu Ala Lys Leu Pro Asn Asn Glu Leu His
1 5 10 15
Gly Gln Glu Ser His Asn Ser Gly Asn Arg Ser Asp Gly Pro Gly Lys
20 25 30
Asn Thr Thr Leu His Asn Glu Phe Asp Thr Ile Val Leu Pro Val Leu
35 40 45
Tyr Leu Ile Ile Phe Val Ala Ser Ile Leu Leu Asn Gly Leu Ala Val
50 55 60
Trp Ile Phe Phe His Ile Arg Asn Lys Thr Ser Phe Ile Phe Tyr Leu
65 70 75 80
Lys Asn Ile Val Val Ala Asp Leu Ile Met Thr Leu Thr Phe Pro Phe
85 90 95
Arg Ile Val His Asp Ala Gly Phe Gly Pro Trp Tyr Phe Lys Phe Ile
100 105 110
Leu Cys Arg Tyr Thr Ser Val Leu Phe Tyr Ala Asn Met Tyr Thr Ser
115 120 125
Ile Val Phe Leu Gly Leu Ile Ser Ile Asp Arg Tyr Leu Lys Val Val
130 135 140
Lys Pro Phe Gly Asp Ser Arg Met Tyr Ser Ile Thr Phe Thr Lys Val
145 150 155 160
Leu Ser Val Cys Val Trp Val Ile Met Ala Val Leu Ser Leu Pro Asn
165 170 175
Ile Ile Leu Thr Asn Gly Gln Pro Thr Glu Asp Asn Ile His Asp Cys
180 185 190
Ser Lys Leu Lys Ser Pro Leu Gly Val Lys Trp His Thr Ala Val Thr
195 200 205
Tyr Val Asn Ser Cys Leu Phe Val Ala Val Leu Val Ile Leu Ile Gly
210 215 220
Cys Tyr Ile Ala Ile Ser Arg Tyr Ile His Lys Ser Ser Arg Gln Phe
225 230 235 240
Ile Ser Gln Ser Ser Arg Lys Arg Lys His Asn Gln Ser Ile Arg Val
245 250 255
Val Val Ala Val Phe Phe Thr Cys Phe Leu Pro Tyr His Leu Cys Arg
260 265 270

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-28-
IlePro PheThrPhe SerHisLeu AspArg LeuLeuAsp GluSerAla
275 280 285
GlnLys IleLeuTyr TyrCysLys GluIle ThrLeuPhe LeuSerAla
290 295 300
CysAsn ValCysLeu AspProIle IleTyr PhePheMet CysArgSer
305 310 315 320
PheSer ArgArgLeu PheLysLys SerAsn IleArgThr ArgSerGlu
325 330 335
SerIle ArgSerLeu GlnSerVal ArgArg SerGluVal LeuIleTyr
340 345 350
TyrAsp TyrThrAsp Val
355
<210>
19
<211>
2480
<212>
DNA
<213> sapiens
Homo
<220>
<221>
CDS
<222> .(1157)
(42).
<400>
19
catggcatcc ccagcctagc actttggcac g 56
tcccaatccc atg
tta
gcc
aac
agc
Met a
Leu Asn
Al Ser
1 5
tcctca accaacagt tctgttctc ccgtgt cctgactac cgacctacc 104
SerSer ThrAsnSer SerValLeu ProCys ProAspTyr ArgProThr
10 15 20
caccgc ctgcacttg gtggtctac agcttg gtgctgget gccgggctc 152
HisArg LeuHisLeu ValValTyr SerLeu ValLeuAla AlaGlyLeu
25 30 35
cccctc aacgcgcta gccctctgg gtcttc ctgcgcgcg ctgcgcgtg 200
ProLeu AsnAlaLeu AlaLeuTrp ValPhe LeuArgAla LeuArgVal
40 45 50
cactcg gtggtgagc gtgtacatg tgtaac ctggcggcc agcgacctg 248
HisSer ValValSer ValTyrMet CysAsn LeuAlaAla SerAspLeu
55 60 65
ctcttc accctctcg ctgcccgtt cgtctc tcctactac gcactgcac 296
LeuPhe ThrLeuSer LeuProVal ArgLeu SerTyrTyr AlaLeuHis
70 75 80 85
cactgg cccttcccc gacctcctg tgccag acgacgggc gccatcttc 344
HisTrp ProPhePro AspLeuLeu CysGln ThrThrGly AlaIlePhe
90 95 100
cagatg aacatgtac ggcagctgc atcttc ctgatgctc atcaacgtg 392
GlnMet AsnMetTyr GlySerCys IlePhe LeuMetLeu IleAsnVal
105 110 115
gaccgc tacgccgcc atcgtgcac ccgctg cgactgcgc cacctgcgg 440
AspArg TyrAlaAla IleValHis ProLeu ArgLeuArg HisLeuArg
120 125 130

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-29-
cggccccgcgtggcg cggctgctc tgcctg ggcgtgtgg gcgctcatc 488
ArgProArgValAla ArgLeuLeu CysLeu GlyValTrp AlaLeuIle
135 140 145
ctggtgtttgccgtg cccgccgcc cgcgtg cacaggccc tcgcgttgc 536
LeuValPheAlaVal ProA.LaAla ArgVal HisArgPro SerArgCys
150 155 160 165
cgctaccgggacctc gaggtgcgc ctatgc ttcgagagc ttcagcgac 584
ArgTyrArgAspLeu GluValArg LeuCys PheGluSer PheSerAsp
170 175 180
gagctgtggaaaggc aggctgctg cccctc gtgctgctg gccgaggcg 632
GluLeuTrpLysGly ArgLeuLeu ProLeu ValLeuLeu AlaGluAla
185 190 195
ctgggcttcctgctg cccctggcg gcggtg gtctactcg tcgggccga 680
LeuGlyPheLeuLeu ProLeuAla AlaVal ValTyrSer SerGlyArg
200 205 210
gtcttctggacgctg gcgcgcccc gacgcc acgcagagc cagcggcgg 728
ValPheTrpThrLeu AlaArgPro AspAla ThrGlnSer GlnArgArg
215 220 225
cggaagaccgtgcgc ctcctgctg getaac ctcgtcatc ttcctgctg 776
ArgLysThrValArg LeuLeuLeu AlaAsn LeuValIle PheLeuLeu
230 235 240 245
tgcttcgtgccctac aacagcacg ctggcg gtctacggg ctgctgcgg 824
CysPheValProTyr AsnSerThr LeuAla ValTyrGly LeuLeuArg
250 255 260
agcaagctggtggcg gccagcgtg cctgcc cgcgatcgc gtgcgcggg 872
SerLysLeuValAla AlaSerVal ProAla ArgAspArg ValArgGly
265 270 275
gtgctgatggtgatg gtgctgctg gccggc gccaactgc gtgctggac 920
ValLeuMetValMet ValLeuLeu AlaGly AlaAsnCys ValLeuAsp
280 285 290
ccgctggtgtactac tttagcgcc gagggc ttccgcaac accctgcgc 968
ProLeuValTyrTyr PheSerAla GluGly PheArgAsn ThrLeuArg
295 300 305
ggcctgggcactccg caccgggcc aggacc tcggccacc aacgggacg 1016
GlyLeuGlyThrPro HisArgAla ArgThr SerAlaThr AsnGlyThr
310 315 320 325
cgggcggcgctcgcg caatccgaa aggtcc gccgtcacc accgacgcc 1064
ArgAlaAlaLeuAla GlnSerGlu ArgSer AlaValThr ThrAspAla
330 335 340
accaggccggatgcc gccagtcag gggctg ctccgaccc tccgactcc 1112
ThrArgProAspAla AlaSerGln GlyLeu LeuArgPro SerAspSer
345 350 355
cactctctgtcttcc ttcacacag tgtccc caggattcc gccctc 1157
HisSerLeuSerSer PheThrGln CysPro GlnAspSer AlaLeu
360 365 370
tgaacacaca tgccattgcg ctgtccgtgc ccgactccca acgcctctcg ttctgggagg 1217
cttacagggt gtacacacaa gaaggtgggc tgggcacttg gacctttggg tggcaattcc 1277

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
- JO -
agcttagcaa cgcagaagag tacaaagtgt ggaagccagg gcccagggaa ggcagtgctg 1337
ctggaaatgg cttctttaaa ctgtgagcac gcagagcacc ccttctccag cggtgggaag 1397
tgatgcagag agcccacccg tgcagagggc agaagaggac gaaatgcctt tgggtgggca 1457
gggcattaaa ctgctaaaag ctggttagat ggaacagaaa atgggcattc tggatctaaa 1517
ccgccacagg ggcctgagag ctgaagagca ccaggtttgg tggacaaagc tactgagatg 1577
cctgttcatc tgctgacttc tgtctaggct catggatgcc accccctttc atttcggcct 1637
aggcttcccc tgctcaccac tgaggcctaa tacaagagtt cctatggaca gaactacatt 1697
ctttctcgca tagtgacttg tgacaattta gacttggcat ccagcatggg atagttgggg 1757
caaggcaaaa ctaacttaga gtttccccct caacaacatc caagtccaaa ccctttttag 1817
gttatccttt cttccatcac atCCCCtttt ccaggcctcc tccattttag gtccttaata 1877
ttCtttCttt ttCtCtCtCt CtCgtttCtC tCttCtCtCt CCtCtCCtCt CCtCtCtCtt 1937
ctcctcttct ctctctctcc ctctctctcc tttgtccaga gtaaggataa aattctttct 1997
actaaagcac tggttctcaa actttttggt ctcagacccc actcttagaa attgaggatc 2057
tcaaagagct ttgcttatat tttgttcttt tgatacttac catactagaa attaaagcga 2117
atacattttt aaaataaata cacatgcaca cattacatta gccatgggag caataatgtc 2177
accacacaca cttcatgaag cctctggaaa actctacagt atacttgtga gagaatgaga 2237
gtgaaaggga caaataacat ctgtgtagca gtattatgaa aatagcttga ccttgtggac 2297
ttcctcagag ggttggtccc tggatcacac tttgagaacc atacttgtcc tgaagtattg 2357
gagttcatgt ctaacttctt cccagggcat tatgtacagt gctttttatt actgtgggga 2417
gagggcagtg ctaaataaat taatcactac tgataaaaaa aaaaaaaaaa aaaaaaaaaa 2477
aaa 2480
<210> 20
<211> 372
<212> PRT
<213> Homo Sapiens
<400> 20
Met Leu Ala Asn Ser Ser Ser Thr Asn Ser Ser Val Leu Pro Cys Pro
1 5 10 15
Asp Tyr Arg Pro Thr His Arg Leu His Leu Val Val Tyr Ser Leu Val
20 25 30
Leu Ala Ala Gly Leu Pro Leu Asn Ala Leu Ala Leu Trp Val Phe Leu
35 40 45
Arg Ala Leu Arg Val His Ser Val Val Ser Val Tyr Met Cys Asn Leu
50 55 60
Ala Ala Ser Asp Leu Leu Phe Thr Leu Ser Leu Pro Val Arg Leu Ser
65 70 75 80

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-31 -
Tyr Tyr Ala Leu His His Trp Pro Phe Pro Asp Leu Leu Cys Gln Thr
85 90 95
Thr Gly Ala Ile Phe Gln Met Asn Met Tyr Gly Ser Cys Ile Phe Leu
100 105 170
Met Leu Ile Asn Val Asp Arg Tyr Ala Ala Ile Val His Pro Leu Arg
115 120 125
Leu Arg His Leu Arg Arg Pro Arg Val Ala Arg Leu Leu Cys Leu Gly
130 135 140
Val Trp Ala Leu Ile Leu Val Phe Ala Val Pro Ala Ala Arg Val His
145 150 155 160
Arg Pro Ser Arg Cys Arg Tyr Arg Asp Leu Glu Val Arg Leu Cys Phe
165 170 175
Glu Ser Phe Ser Asp Glu Leu Trp Lys Gly Arg Leu Leu Pro Leu Val
180 185 190
Leu Leu Ala Glu Ala Leu Gly Phe Leu Leu Pro Leu Ala Ala Val Val
195 200 205
Tyr Ser Ser Gly Arg Val Phe Trp Thr Leu Ala Arg Pro Asp Ala Thr
210 215 220
Gln Ser Gln Arg Arg Arg Lys Thr Val Arg Leu Leu Leu Ala Asn Leu
225 230 235 240
Val Ile Phe Leu Leu Cys Phe Val Pro Tyr Asn Ser Thr Leu Ala Val
245 250 255
Tyr Gly Leu Leu Arg Ser Lys Leu Val Ala Ala Ser Val Pro Ala Arg
260 265 270
Asp Arg Val Arg Gly Val Leu Met Val Met Val Leu Leu Ala Gly Ala
275 280 285
Asn Cys Val Leu Asp Pro Leu Val Tyr Tyr Phe Ser Ala Glu Gly Phe
290 295 300
Arg Asn Thr Leu Arg Gly Leu Gly Thr Pro His Arg Ala Arg Thr Ser
305 310 315 320
Ala Thr Asn Gly Thr Arg Ala Ala Leu Ala Gln Ser Glu Arg Ser Ala
325 330 335
Val Thr Thr Asp Ala Thr Arg Pro Asp Ala Ala Ser Gln Gly Leu Leu
340 345 350
Arg Pro Ser Asp Ser His Ser Leu Ser Ser Phe Thr Gln Cys Pro Gln
355 360 365
Asp Ser Ala Leu
370
<210> 21
<211> 19
<212> DNA
<213> Artificial Sequence
<220>

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-32-
<223> Description of Artificial Sequence: Primer LW1282
<400> 21
taatacctgc actgcccac 19
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW 1283
<400> 22
tctttccttc tcttctcact cc 22
<210> 23
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW 1373
<400> 23
gcataagctt atgctaacac tgaataaaac ag 32
<210> 24
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1374
<400> 24
gcatctcgag tcacatgctg taggatttgg 30
<210> 25
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Peptide
<400> 25
Ala Pro Arg Thr Pro Gly Gly Arg Arg
1 5
<210> 26
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1248

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-, -,
<400> 26
gcatgaattc caatatactt ccccatacct ac 32
<210> 27
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1249
<400> 27
gcatggatcc ggaaaagaag gagaagaaag 30
<210> 28
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1278
<400> 28
accgctgcct ttttagtc 18
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1279
<400> 29
ccttctttct gggtacataa gtc 23
<210> 30
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1405
<400> 30
aagcataaca tggatgaaac aggaaatctg 30
<210> 31
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1406
<400> 31
aagcataact atactttaca tatttcttc 29

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
<210> 32
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1280
<400> 32
tctgcacaca gctcttccat gg 22
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1281
<400> 33
tcccttgtcc agttggttga gg 22
<210> 34
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1385
<400> 34
gcataagctt ccatggaact tcataacctg 30
<210> 35
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1386
<400> 35
gcatctcgag ttacccccac agcgctgcag 30
<210> 36
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1329
<400> 36
gcatctcgag tcagcctaag gttatgttg 29
<210> 37
<211> 29
<212> DNA

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-35-
<213> Artificial Sequence
<220>
<223> Description of. Artificial Sequence: Primer LW1377
<400> 37
gcataagctt atgaacacca cagtgatgc 29
<210> 38
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1387
<400> 38
gagaaatatt tttctaaaaa aacctgtttt tgcaaaaacg g 41
<210> 39
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1388
<400> 39
ccgtttttac aaaaacaggt ttttttagaa aaatatttct c 41
<210> 40
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1314
<400> 40
gcatgaattc ccaccttcat catctacctc 30
<210> 41
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1315
<400> 41
gcatggatcc gaagaccaaa aagacccag 29
<210> 42
<211> 30
<212> DNA
<213> Artificial Sequence

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-3G-
<220>
<223> Description of Artificial Sequence: Primer Lw1326
<400> 42
gcatgaattc atgatggtgg atcccaatgg 30
<210> 43
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1327
<400> 43
gcatctcgag cctagggctc tgaagcg 27
<210> 44
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1415
<400> 44
ccatgtatat atttctttgc atgctttcag gcattgacat cc 42
<210> 45
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1416
<400> 45
ggatgtcaat gcctgaaagc atgcaaagaa atatatacat gg 42
<210> 46
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1308
<400> 46
gcatgaattc actcacttct catctccttc 30
<210> 47
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1309
<400> 47

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-37-
gcatggatcc aatctccttt gtcttcactc 30
<210> 48
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1324
<400> 48
gatcggatcc atggaaagcg agaacag 27
<210> 49
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1325
<400> 49
gatcctcgag tcaggctatg tgcttattaa acacc 35
<210> 50
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1306
<400> 50
gcatgaattc ttctacttca tcatcctcc 29
<210> 51
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1307
<400> 51
gcatggatcc aaaggccatc acaacaag 28
<210> 52
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer GV599
<400> 52
ggcagaagaa ggctattggt cttagacgag 30

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
_ ;g
<210> 53
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer GV600
<400> 53
ctgaaacagc gcctcagctc cc 22
<210> 54
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1482
<400> 54
agctatggcg aactatagcc atgcagc 27
<210> 55
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW148
<400> 55
agtcctcata taacacagta aggttcc 27
<210> 56
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1310
<400> 56
gcatgaattc gcagaagaag gctattgg 28
<210> 57
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1311
<400> 57
gcatggatcc gcagtaaaga agggttgtg 29
<210> 58
<211> 19

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-39-
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1442
<400> 58
gccattctgt ccacagaag 19
<210> 59
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1443
<400> 59
tcagttgctg ttatggcac 19
<210> 60
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1440
<400> 60
aagcggatgt ttagacctct tgtg
24
<210> 61
<211> 23
<212> DNA
<213 > Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1441
<400> 61
aacagtcatg aataggaatt gag 23
<210> 62
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1472
<400> 62
gcatgaattc tgccatgtca atcatttctc tc 32
<210> 63
<211> 31

CA 02386509 2002-04-16
WO 01/31014 PCT/US00/29601
-40-
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1473
<400> 63
gcatggatcc gttctgcatt ttccaggtct c 31
<210> 64
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1411
<400> 64
gcatgaattc tgccaaacat catcctgac 29
<210> 65
<211> 29
<212> DNA
<213> Artifi<:ial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1412
<400> 65
gcatggatcc tacacagcca caacaaccc 29
<210> 66
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1448
<400> 66
aagcggtacc atgttagcca acagctcctc 30
<210> 67
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1449
<400> 67
aagctctaga tcagagggcg gaatcctgg 29
<210> 68
<211> 43
<212> DNA

CA 02386509 2002-04-16
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-41 -
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 217A
<400> 68
taggtcggta gtcaggacac gggagaacag aactgttggt tga 43
<210> 69
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 217B
<400> 69
gcccctgtgg cggtttagat ccagaatgcc cattttctgt tccatctaac ca 52
<210> 70
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1480
<400> 70
ggttctacct ggacttatgg 20
<210> 71
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer LW1481
<400> 71
taatgaatga gtaagtgccc 20
<210> 72
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer CON103a
<400> 72
tttattaata ttggaaggga caaactggag agcacagaac at 42
<210> 73
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer CON103b
<400> 73

CA 02386509 2002-04-16
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-42-
aaagccacca tggaagccat gccaaagatg atgctgggca agaa 44
<210> 74
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 1332
<400> 74
tcctactgtc atgaaccc 18
<210> 75
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer 1333
<400> 75
cagaagaagt tgtccagc 18