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ISOLATED HUMAN G-PROTEIN COLiPLED RECEPTORS, NUCLEIC ACID
MOLECDLES ENCODING HUMAN GPCR PROTEINS, AND USES THEREOF
RELATED APPLICATIONS
S The present application claims priority to U.S. Application No. 09,G9d,821,
tiled October
2~, 2000 (CIj000$99) and U.S. Serial No. 091781,559, filed February 13, 2001
(CL000899-CIP)
FIELD OF THE INVENTION
The present invention is in the field of G-Protein coupled receptors (GPCRs)
that are
involved in cell signaling, particularly neurotransmitter signaling,
recombinant DNA molecules,
and protein production. The present invention specifically provides novel GPCR
peptides and
proteins and nucleic acid molecules encoding such peptide and protein
molecules, all of which
are useful in the development of human therapeutics and diagnostic
compositions and methods.
BACKGROUND OF THE INVENTION
G-protein coupled receptors
G-pratein caupled receptors (GPCRs) constitute a major class oFproteins
responsible For
transducing a signal within a cell. GPCRs have three structural domains: an
amino terminal
extracellular domain, a transmembrane domain containing seven transmembrane
segments, three
extracellular loops, and three intracellular loops, and a carboxy ternlinal
intracellular domain. Upon
binding of a ligand to an extracellular portion of a GPCR, a signal is
transduced within the cell that
results in a change in a biological or physiological property of the cell.
GPCRs, along with G-
proteins and eFfectors (intracellular enzymes and channels modulated by G-
proteins), are the
components of a modular signaling system that connects the state of
intracellular second
messengers to extracellulal- inputs.
GPCR genes and gene-products are potential causative agents of disease
(Spiegel et rrl., J
C'lil~, Il~ai,sl, 93;1 I 19-1 125 ( 1993); McItusick c~l crl., J. Iblr?cl
Gc~)ml, 3(J:1-2G (1993)). Specific
defects in the rhodopsin gene and the V2 vasopressin receptor gene have been
shown to cause
various forms of retinitis pigmentosum (Nathans ct crl" ft)l)nl. Rcu. Gel~~tl.
?6:103-~I?~l( 1992)), and
nephl'ogeIllC dlabeteS Ill~lpldLl~ ~)'IOItIIll~ill t'I Cll., Ihll)l. r1~(ll.
CiC'r?C'h ?:I~UI-1~0~11993))_ These
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receptors are of critical importance to both the central nervous system and
peripheral physiological
processes. Evolutionary analyses suggest that the ancestor of these proteins
originally developed in
concert with complex body plans and nervous systems.
'The GPCR protein superfamily can be divided into Five families: I~Aamily I,
receptors
typified by rhodopsin and the (32-purinergic receptor and currently
represented by over 2DD unique
members (Dohlman e! al.,~lr~nzi. Rev. Biochc~rn. «1;653-68$ (1991)); Family
II, the parathyroid
hotmonelcalcitoninlsecretin receptor family (Juppner et al., Science 25-l:1
D24-1 D26 ( 1991 ); Lin e!
al., Scier2ce 25:1:1D22-ID24 (1991)); Family III, the metabotropic glutamate
receptor family
(Nakanishi, Science 258 597:6D3 (1992)); Family IV, the cAMP receptor family,
important in the
1D chemotaxis and development of D. di,scoideZrrrZ (Klein e! al., Science 2-
11:1467-1172 (1988)); and
Family V, the fungal mating pheromone receptors such as STE2 (Kurjan, ~ln~zt.
Rev. Biochenz.
61:1097-1129 ( 1992)).
There are also a small number of other proteins that present seven putative
hydrophobic
segments and appear to be unrelated to GPCRs; they have not been shown to
couple to G-proteins.
I 5 Dnosophila expresses a photoreceptor-specific protein, bride of sevenless
(boss), a seven-
transmembrane-segment protein that has been extensively studied and does not
show evidence of
being a GPCR (I-Iart ~t al., Pnoc. Ncrtl. ~lcad Sci. LISA 90:SD47-SD51 (
1993)). The geneyi~~led (fz)
in Drosophila is also thought to be a protein with seven transmembrme
segments. bike boss, fz has
not been shown to couple to G-proteins (Vinson e1 crl., NalZn°e 338:263-
264 (1989)).
2D G proteins represent a family of heterotrimeric proteins composed of a, (3
and y subunits,
that bind guanine nucleotides. These proteins are usually linked to cell
surface receptors, e.g.,
receptors containing seven transmembrane segments. Following ligand binding to
the GPCR, a
conformational change is transmitted to the G protein, which causes the a-
subunit to exchange a
bound GDP molecule for a GTP molecule and to dissociate from the (3y-subunits.
The GTP-bound
25 form of the a-subunit typically functions as an eFfector-modulating moiety,
leading to the
production of second messengers, such as cAMP (e.g., by activation of adenyl
cyclase),
diacylglycerol or inositol phosphates. Greater than 2D different types of a-
subunits are known in
humans. These subunits associate with a smaller pool of (3 and y suhunits.
Examples of
mammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins are described
ehtensively in
3D Lodish of al., ~t=Itrlr~catlcw C'cll Bioky~~y. (~cientiluc American gooks
lnc., New York, N'.Y., 1995), the
contents of which are incorporated herein by reference. GPCRs, G proteins and
G protein-linked
effector and second messenger s~°stems hove been reviewed in Thi.W-
Pmolc~ir~ 1 irTked R~co~p!cu~ Fcrc~!
l3o~k, Watson cn al., eds.. Academic Press ( 1994).
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Aminer~c GPCRs
One Family oFthe GPCRS, Family II. contains receptors For acetylcholine,
catccholanline, and indoleamine ligands (hereaFter reFerred to as biogenic
amines). The bio~~~nic
amine receptors (amincrgic GPCRs) represent a large group oFGPCRs that share a
common
evolutionary ancestor and which are present in both vertebrate
(deuterostonle), and invertebrate
(protostome) lineages. This Family oFGPCRs includes, but is not limited to the
5-HT-like, the
dopamine-like, the acetylcholine-like, the adrenaline-like and the melatonin-
like GPCRs.
Dopamine receptors
The understanding oFthe dopaminergic system relevance in brain Function and
disease
1 D developed several decades ago from three diverse observations following
drug treatments. These
were the observations that dopamine replacement therapy improved Parkinson's
disease symptoms,
depletion of dopamine and other catecholamines by reserpine caused depression
and antipsychotic
drugs blocked dopamine receptors. The Ending that the dopamine receptor
binding affinities of
typical antipsychotic drugs correlate with their clinical potency led to the
dopamine overactivity
hypothesis of schizophrenia (Snyder, S.H., ,~ni J PSyGhiaby 133, 197-2D2
(1976); Seeman, P. and
Lee, T., Science I ~3c~, 1217-9 (1975)). Today, dopamine receptors aI°e
crucial targets in the
pharmacological therapy of schizophrenia, Parkinson's disease, Tourette's
syndrome, tardive
dyskinesia and Huntington's disease. The dopaminergic system includes the
nigrostriatal,
mesocorticolimbic and tuberoinfi.lndibular pathways. The nigrostriatal pathway
is part of the striatal
motor system and its degeneration leads to Parkinson's disease; the
mesocorticolimbic pathway
plays a key role in reinforcement and in emotional expression and is the
desired site of action of
antipsychotic drugs; the tuberoinfundibular pathways regulates prolactin
secretion from the
pituitary.
Dopamine receptors are members of the G protein coupled receptor superfamily,
a large
group proteins that share a seven helical membrane-spanning structure and
transduce signals
through coupling to heterotrimeric guanine nucleotide-binding regulatory
proteins (G proteins).
Dopamine receptors are classified into subfamilies: Dl-like (Dl and DS) and
D'~-like (D2, D3 and
D4) based on their different ligand binding profiles, signal transduction
properties, sequence
homologies and genomic organizations (Civelli, O~, F~unzow, .1R, and Grandy,
D.K., ,~Irznu Reu
I'hcrrrncrcol Toxfcml33, 281-307 (1993)). The Dl-like receptors, D1 and D5,
stimulate cAMP
synthesis through coupling with Gs-like proteins and their Belles do not
contain intron s within their
pl'otelll Codlng CeglOnS. OIl the otheI' hatld, the D~-llkf= reCeptol'S, D?.
D3 alld Dzl, lllhlblt Cf~IVIP
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synthesis through their interaction with Gi-like proteins and share a similar
genomic organization
which includes introns within their protein coding regions.
Sel'otolllll I'eCeptorS
Serotonin (5-I-Iydroxytryptamine; 5-HT) was first isolated from blood serum,
where if was
shown to promote vasoconstriction (Rapport, M.M., Green, A.A. and Page, LI-I.,
J Biol C'hern 17<,
1213-1251 (148). Interest on a possible relationship between 5-HT and
psychiatric disease was
spurred by the observations that hallucinogens such as LSD and psilocybin
inhibit the actions of 5-
HT on smooth muscle preparations {Gaddum, J.H. and Hameed, K.A., Br J
Pharmucol 9, 240-248
(1954)). This observation lead to the hypothesis that brain 5-HT activity
might be altered in
psychiatric disorders (Wooley, D.W. and Shaw, E., Proc Nail Accld Sci LIS~I -
10, 228-231 (1951);
Gaddum, J.H, and Picarelli, Z.P., Bf~ J Phay~nzacol 12, 323-328 (1957)). This
hypothesis was
strengthened by the introduction of tricyclic antidepressants and monoasnine
oxidase inhibitors For
the treatment of major depression and the observation that those drugs
affected noradrenaline and 5-
HT metabolism. Today, drugs acting on the serotoninergic system have been
proved to be effective
I 5 in the pharmacotherapy of psychiatric diseases such as depression,
schizophrenia, obsessive-
compulsive disorder, panic disorder, generalized anxiety disorder and social
phobia as well as
migraine, vomiting induced by cancer chemotherapy and gastric motility
disorders.
Serotonin receptors represent a very large and diverse family of
neurotransmitter receptors.
To date thirteen 5-HT receptor proteins coupled to G proteins plus one ligand-
gated ion channel
receptor (5-HT3) have been described in mammals. This receptor diversity is
thought to reflect
serotonin's ancient origin as a neurotransmitter and a hormone as well as the
many different roles of
5-HT in mammals. The 5-HT receptors have been classified into seven
subfamilies or groups
according to their different ligand-binding affinity profiles, molecular
structure and intracellular
transduction mechanisms (Hoyer, D. et al., Phcrrrrurcol. Rev. -16, 157-203
(1991)).
Adrener~ic GPCRs
The adrenergic receptors comprise one o~the largest and most extensively
characterized
families within the G-protein coupled receptor "superFamily". This superfamily
includes not only
adrenergic receptors, but also muscarinic, cholinergic, dopaminergic,
serotonergic, and
histaminergic receptors. Numerous peptide receptors include glucagon,
somatostatin, and
vasopressin receptors, as well as sensory receptors for vision (rhodopsin),
taste, and olfaction,
also belong to this growing Family. Despite the diversity of signalling
molecules, G-protein
coupled receptors all possess a similar overall primary structure,
characterized by 7 putative
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membrane-spanning .alpha, helices (Probst et al., 199?). In the most basic
sense, the adrenergic
receptors are the physiological sites of action of the catecholamines,
epinephrine and
norepinephrine. Adrencrgic receptors were initially classil7ed as either
.alpha. or .beta. by
Ahlquist, who demonstrated that the order of potency For a series of agonists
to evolve a
physiological response was distinctly different at the 2 receptor subtypes
(Ahlquist, 1918).
functionally, .alpha, adrenergie receptors were shown to control
vasoconstriction, pupil dilation
and uterine inhibition, while .beta. adrenergic receptors were implicated in
vasorelaxation,
myocardial stimulation and bronchodilation (Regan et al., 1990). Eventually,
pharmacalogists
realized that these responses resulted from activation of several distinct
adrenergic receptor
subtypes. .beta. adrenergic receptors in the heart were defined as
.beta.
l, while those in the
lung and vasculature were termed .beta.
2 (Lands et al., 1967).
.alpha. Adrenergic receptors, meanwhile, were first classified based on their
anatomical
location, as either pre or post-synaptic (.alpha.
2 and .alpha.
l,
respectively) (Langer et
al., 1970. This classification scheme was confounded, however, by the presence
of .alpha.
2receptors in distinctly non-synaptic locations, such as platelets (Berthelsen
and Pettinger, 1977).
With the development of radioligand binding techniques, .alpha. adrenergic
receptors could be
distinguished pharmacologically based on their affinities for the antagonists
prazosin or
yohimbine {Stark, 1981 ). Definitive evidence for adrenergic receptor
subtypes, however, awaited
purification and molecular cloning of adrenergic receptor subtypes. In 19$6,
the genes For the
hamster .beta.
2 (Dickson et al., 19$6) and turkey .beta.
l adrenergic
receptors {Yarden
et al., 19$6) were cloned and sequenced. I-Iydropathy analysis revealed that
these proteins
contain 7 hydrophobic domains similar to rhodopsin, the receptor for light.
Since that time the
adrenergic receptor family has expanded to include 3 subtypes of .beta.
receptors (Emorine et al,,
1989), 3 subtypes of .alpha.
l receptors (Schwinn et al., 1990), and 3
distinct types of
,beta.
2 receptors (Lomasney et al., 1990).
The cloning, sequencing and expression of alpha receptor subtypes from animal
tissues
has led to the subclassitication of the alpha 1 receptors into alpha 1d
(formerly known as alpha
1 a or 1 al l d), alpha 1 b and alpha 1 a (Formerly known as alpha 1 c)
subtypes. Each alpha 1
receptor subtype exhibits its own pharmacologic and tissue speciFtcities. The
designation "alpha
la" is the appellation recently approved by the IUPI-IARN~omenclature
Committee Far the
previously designated "alpha 1 c" cloned subtype as outlined in the 1995
Receptor and Ion
Channel l~lomenclature Supplement ( Watson and Girdlestone, 1995). The
designation alpha 1 a is
used throughout this application to reFer to this subtype. At the same time,
the receptor Formerly
t
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designated alpha 1 a was renamed alpha 1 d. The new nomenclature is used
throughout this
application. Stable cell lines expressing these alpha 1 receptor subtypes are
referred to herein;
however, these cell lives were deposited with the American Tfype Culture
Collection (ATCC)
under the old nomenclature. For a review of the classification ol~alpha 1
adrenoceptor subtypes,
see, Martin C. Michel, et al., Naunyn-Schmiedeberg's Arch. Pharmacol. (1995)
352;1-10.
The differences in the alpha adrenergic receptor subtypes have relevance in
pathophysiologic conditions. Benign prostatic hyperplasia, also known as
benign prostatic
hypertrophy or BPH, is an illness typically affecting men over f fty years of
age, increasing in
severity with increasing age. The symptoms of the condition include, but are
not limited to,
increased difficulty in urination and sexual dysfunction. These symptoms are
induced by
enlargement, or hyperplasia, of the prostate gland. As the prostate increases
in size, it impinges
on free-flow of fluids through the male urethra. Concommitantly, the increased
noradrenergic
innervation of the enlarged prostate leads to an increased adrenergic tone of
the bladder neck and
urethra, further restricting the flow of urine through the urethra.
The .alpha.
2 receptors appear to have diverged rather early from either
.beta. or
.alpha.
l receptors. The .alpha.
2 receptors have been broken down
into 3 molecularly
distinct subtypes termed .alpha.
2 C2, .alpha.
2 C4, and .alpha.
2 C10 based on their
chromosomal location. These subtypes appear to correspond to the
pharmacologically defined
.alpha.
2B, .alpha.
2C, and .alpha.
2A subtypes, respectively
(Bylund et al., 1992).
While all the receptors of the adrenergic type are recognized by epinephrine,
they are
pharmacologically distinct and are encoded by separate genes. These receptors
are generally
coupled to different second messenger pathways that are linked through G-
proteins. Among the
adrenergic receptors, .beta.
l and .beta.
2 receptors activate the
adenylate cyclase,
.alpha.
2 receptors inhibit adenylate cyclase and .alpha.
l receptors
activate
phospholipase C pathways, stimulating breakdown of polyphosphoinositides
(Chung, F. Z. et al.,
J. Biol. Chem., 263:1052 (1988)). .alpha.
l and .alpha.
2 adrenergic
receptors differ in
their cell activity for drugs.
Issued US patent that disclose the utility of members of this family of
proteins include,
but are not limited to, 6,063,785 Phthalimido arylpiperazines useful in the
treatment of benign
prostatic hyperplasia; 6,060,492 Selective .beta.3 adrenergic agonists;
6,057,350 Alpha 1 a
adrenergic receptor antagonists; 6,06,192
Phenylethanolaminotetralincarboxamide derivatives;
6,016,183 Method of synergistic treatment for benign prostatic hyperplasia;
6,013,253 Fused
piperldine substituted arylsulfonamides as .beta. 3-a~.~onists; G.0~3.22~1
Compositions and
6
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methods for treatment of neurological disorders and neurodegenerative
diseases; 6,037,354
Alpha la adrenergic receptor antagonists; 6,034,106 Oxadiazole
benzenesulfonamides as
selective .beta.
3 Agonist for the treatment of Diabetes and Obesity; 6,01
I ,048 Thiarole
benzenesulfonamicles as .beta.3 agonists for treatment of diabetes and
obesity; 6,008,361
5,994,506 Adrenergic receptor; 5,994,294 Nitrosated and nitrosylated .alpha.-
adrenergic receptor
antagonist compounds, compositions and their uses; 5,990,128 .alpha.
l C
specific
compounds to treat benign prostatic hyperplasia; 5,977,151 Selective .beta.3
adrenergic agonist;
5,977,1 15 Alpha I a adrenergic receptor antagonists; 5,939,443 Selective
.beta,3 adrenergic
agonists; 5,932,538 Nitrosated and nitrosylated ,alpha.-adrenergic receptor
antagonist
compounds, compositions and their uses; 5,922,722 Alpha 1 a adrenergic
receptor antagonists 26
5,908,830 and 5,861,309 DNA endoding human alpha 1 adrenergic receptors.
Purinergic GPCRs
Purinoceptor P2Y 1
P2 purinoceptors have been broadly classified as P2X receptors which are ATP-
gated
channels; P2Y receptors, a family of G protein-coupled receptors, and P2Z
receptors, which
mediate nonselective pores in mast cells. Numerous subtypes have been
identified for each of the
P2 receptor classes. P2Y receptors are characterized by their selective
responsiveness towards ATP
and its analogs. Some respond also to UTP. Based on the recommendation for
nomenclature of P2
purinoceptors, the P2Y purinoceptors were numbered in the order of cloning.
P2Y1, P2Y2 and
P2Y3 have been cloned from a variety of species. P2Y 1 responds to both ADP
and ATP. Analysis
of P2Y receptor subtype expression in human bone and 2 osteoblastic cell lines
by RT-PCR showed
that all known human P2Y receptor subtypes were expressed: P2Y1, P2Y2, P2Y4,
P2Y6, and P2Y7
(Maier et al. 1997). In contrast, analysis of brain-derived cell lines
suggested that a selective
expression of P2Y receptor subtypes occurs in brain tissue,
Leon et al. generated P2Y I -null mice to define the physiologic role of the
P2Y 1 receptor (J.
Clin. Invest. 104: 1731-1737(1999)). These mice were viable with no apparent
abnormalities
affecting their development, survival, reproduction, or morphology of
platelets, and the platelet
count in these animals was identical to that of wildtype mice. However,
platelets Frotn P2Y1-
deficient mice were unable to aggregate in response to usual concentrations of
ADP and displayed
impaired aggregation to other agonists, while high concentrations oFADP
induced platelet
aggregation withou t shape change. In addition, ADP-induced inhibition of
adenylyl cyelase still
7
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occurred, demonstrating the existence ofan ADf receptor distinct from P2Y1.
PZY1-null mice had
no spontaneous bleeding tendency but were resistant to thromboemholism induced
by intravenous
inic:ction ofADI' or collagen and adi°enaline. f-Ience, the P?Yl
recoptor plays an essential role in
thrambotic states and represents a potential target for antithrombo tic drugs.
Somers et al. mapped
the P2RY 1 gen a between Ranking markers D3S 1279 and D3S 1280 at a position
173 to 174 cM
from the most telomeric markers on the short arm of chromosome 3. (Genomics
~l~f: 127-130
1997)).
Purinoc~tor P2Y2
The chloride ion secretory pathway that is defective in cystic fibrosis (CF)
can be bypassed
by an alternative pathway for chloride ion transport that is activated by
extracellular nucleotides.
Accordingly, the P2 receptor that mediates this effect is a therapeutic target
For improving chloride
secretion in CF patients. Parr et al. reported the sequence and functional
expression of a cDNA
cloned from human airway epithelial cells that encodes a protein with
properties of a P2Y
nucleotide receptor. (Proc. Nat. Acad. Sci. 91: 3275-3279 (1994)) The human
P2RY2 gene was
mapped to chromosome 11 q 13.5-q 1 ~.1.
Purinoceptor P2RY~I
The P2RY~1 receptor appears to be activated specifically by UTP and LJDP, but
not by ATP
and ADP. Activation ofthis uridine nucleotide receptor resulted in increased
inositol phosphate
fomlation and calcium mobilization. The LJNR gene is located on chromosome
Xql3.
Purinoceptor P2 Y6
Somers et al. mapped the P2RY6 gene to 11 q 13.5, between palymorphic markers
D 11 S 131 ~I and D 115916, and P2RY2 maps within less than 4 cM of P2RY6.
(Genomics ~~: 127-
130 (1997)) This was fhe first chromosomal clustering of this gene family to
be described.
Adenine and uridine nucleotides, in addition to their well established role in
intracellular
energy metabolism, phosphorylation, and nucleic acid synthesis, also are
important extracellular
signaling molecules. P2Y metabotropic receptors are GPCRs that mediate the
effects of
extracellular nucleotides fo regulate a wide variety of physiological
processes. At least ten
subfamilies of P2Y receptors have been identified, Flhese receptor subfamilies
differ greatly in their
sequences and in their nucleotide agonist selectivities and eFticacies.
It has been demonstrated that the P2Y1 receptors are strongly expressed in the
brain, but the
P2Y2, P?Y~1 and P?Y6 receptors are also present. -Ihc localisation oCone or
more of these subtypes
8
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on neurons, on glia cells. on brain vasculature or on vEntricle ependimal
cells was found by in situ
mRNA hybridisation and studies on thosE cells in culture. 'I"he P2Y 1
receptors are prt7minent on
17eu1"oIlS. ~hhe Coupling ol'Cel'ta117 P2Y reCeptD1' SLIbtypeS t0 N-type Ca?~'
Chanl7ElS C>I'to partlCLlla1'
k.+ channels was also demonstrated.
It has also been demonstrated that several P2Y receptors mediate potent growth
stimulatory
effects on sn7ooth n7uselE cells by stimulating intracellular pathways
including Gq-proteins, protein
kinase C and tyrosine phosphorylation, leading to increased immediate early
gene expression, cell
number, DNA and protein synthesis. It has been further demonstrated that P2Y
regulation plays a
mitogenic rale in response to the development of artherosclerosis.
It has Further been demonstrated that P2Y receptors play a critical role in
cystic fibrosis.
The volume and composition of the liquid that lines the airway surface is
modulated by active
transport of ions across the airway epithelium. This in turn is regulated both
by autonomic agonists
acting on basolateral receptors and by agonists acting on luminal receptors.
Specifically,
extracellular nucleotides present in the airway surface liquid act on luminal
P2Y receptors to control
both Cl- secretion and Na+ absorption. Since nucleotides are released in a
regulated manner from
airway epithelial cells, it is likely that their control over airway ion
transport forms part of an
autocrine regulatory system localised to the luminal surface ofairway
epithelia. In addition to this
physiological role, P2Y receptor agonists have the potential to be of crucial
benefit in the treatment
of CF, a disorder of epithelial ion transport. The airways of people with CF
have defective C1-
secretion and abnormally high rates of Na+ absorption. Since P2Y receptor
agonists can regulate
both these ion transport pathways they have the potential to pharmacologically
bypass the ion
transport defects in CF.
For a detailed description of a putative neurotransmitter GPCR involved in
neurotransmitter
signaling, see Zeng el al., Bioche~rz. Biophya". Res. ConZnzatn. 242 (3), 575-
578 (1998).
GPCRs, particularly GPCRs involved in neurotransmitter signaling, are a major
target far
drug action and development. Accordingly, it is valuable to the field of
pharmaceutical
development to identify and characterize previously unknown GPCRs. The present
invention
advances the state of the art by providing a previously unidentified human
GPCR.
SUM1V><ARY O>F THE INVENTION
The present invention is based in part on the identiFcation of nucleic acid
sequences that
encode an7ino acid sequences of human GPC"R peptides and proteins that are
involved in cell
c~
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signaling. particularly neurotransmitter signaling, allelic variants thereof
and other mammalian
orthologs thereof. These unique peptide sequences, and nucleic acid sequences
that encode these
peptides. Can be Llsed a5 Illod(:1S Iol'the develOplnent Ul'hL1171a17
t17e1'apeLltIC targets, aid 111 the
identification oh therapeutic proteins, and serve as targets For the
development of human
therapeutic agEnts.
The proteins of the present inventions are GPCRs that participate in signaling
pathways,
particularly neurotransmitter signaling pathways, in cells that express these
proteins. Experimental
data as provided in Figure 1 indicates expression in humans in the stomach,
placenta, kidney,
skeletal muscle, liver, bone marrow, and thymus. As used herein, a "signaling
pathway" refers to
I 0 the modulation (e.g., stimulation or inhibition) of a cellular
functionlactivity upon the binding of a
ligand to the GPCR protein. Examples of sLlch functions include mobilization
of intracellular
molecules that participate in a signal transduction pathway, e.g.,
phosphatidylinositol 4,5-
bisphosphate (PIPz), inositol 1,~I,S-triphosphate (IP3) and adenylate cyclase;
polarization of the
plasma membrane; production or secretion of molecules; alteration in the
structure oFa cellular
component; cell proliferation, e.g., synthesis of DNA; cell migration; cell
differentiation; and cell
survival
The response mediated by the receptor protein depends on the type of cell it
is expressed on.
Some information regarding the types of cells that express other members of
the subfamily of
GPCRs of the present invention is already known in the art (see references
cited in Background and
information regarding closest homologous protein provided in Figure 2;
Experimental data as
provided in Figure 1 indicates expression in humans in the stomach, placenta,
kidney, skeletal
muscle, liver, bone marrow, and thymus). For example, in some cells, binding
of a ligand to the
receptor protein may stimulate an activity such as release of compounds,
gating of a channel,
cellular adhesion, migration, differentiation, etc., through
phosphatidylinositol or cyclic AMP
metabolism and turnover while in other cells, the binding of the ligand will
produce a diFferent
result, Regardless of the cellular activityhesponse modulated by the
particular GPCR of the present
invention, a skilled artisan will clearly know that the receptor protein is a
GPCR and interacts with
G proteins to praduce one or more secondary signals, in a variety of
intracellular signal trap sduction
pathways, e.g., through phosphatidylinositol or cyclic AMP metabolism and
turnover, in a cell thus
participating in a biological process in the cells or tissues that express the
GPCR. Experimental data
as provided in Figure 1 indicates that GPCR proteins of the present invEntion
are Expressed in
humans in the stomach, placenta, Icidn Ey. sl:elctal muscle, liver. bone
marrow, and thymus.
SpeciFically, a virtual northern blot shows Expression in the stomach. In
addition, PCR-bawd tissue
CA 02425897 2003-04-23
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screening panels indicate expression in placenta. kidney, skeletal muscle,
liver, bone marrow. and
thymus tissue,
As used herein, "phosphatidylinositol turnover and metabolism" refers to the
molecules
involved in the turnover and metabolism oFphosphatidylinositol X1,5-
bisphosphate (PIPZ) as well as
to the activities oFthese molecules. PIPS is a phospholipid Found in the
cytosolic leaflet of the
plasma membrane. Binding of ligand to the receptor activates, in some cells,
the plasma-membrane
enzyme phospholipase C that in turn can hydrolyze PIPS to produce 1,2-
diacylglycerol (DAG} and
inositol 1,4,5-triphosphate (IP;). Once formed IPA can diffuse to the
endoplasmic reticulum surface
where it can bind an IP3 receptor, e.g., a calcium channel protein containing
an IP3 binding site. IP3
binding can induce opening of the channel, allowing calcium ions to be
released into the cytoplasm.
IPA can also be phosphorylated by a specific kinase to Form inositol 1,3,1,5-
tetraphosphate (IP,~}, a
molecule that can cause calcium entry into the cytoplasm from the
extracellular medium. IP,; and
IPA can subsequently be hydrolyzed very rapidly to the inactive products
inositol 1,~-biphosphate
(IPA} and inositol 1,3,4-triphosphate, respectively. These inactive products
can be recycled by the
cell to synthesize PIPS. The other second messenger produced by the hydrolysis
of PIPZ, namely
1,2-diacylglycerol (DAG), remains in the cell membrane where it can serve to
activate the enzyme
protein kinase C. Protein kinase C is usually found soluble in the cytoplasm
of the cell, but upon an
increase in the intracellular calcium concentration, this enzyme can move to
the plasma membrane
where it can be activated by DAG. The activation of protein kinase C in
different cells results in
various cellular responses such as the phosphorylation of glycogen synthase,
or the phosphorylation
of various transcription factors, e.g., NF-kB. The language
"phosphatidylinositol activity", as used
herein, refers to an activity of PIPZ or one of its metabolites.
Another signaling pathway in which the receptor may participate is the cAMP
turnover
pathway. As used herein, "cyclic AMP turnover and metabolism" refers to the
molecules
involved in the turnover and metabolism oFcyclic AMP (CAMP) as well as to the
activities of
these molecules. Cyclic AMP is a second messenger produced in response to
ligand-induced
stimulation of certain G protein coupled receptors. In the cAMP signaling
pathway, binding of a
ligand to a GPCR can lead to the activation of the enzyme adenyl cyclase,
which catalyzes the
synthesis of cAMP. The newly synthesized CAMP can in turn activate a cAMP-
dependent
protein kinase. This activated kinase can phosphorylate a voltage-gated
potassium channel
protein, or an associated protein, and lead to the inability ofthe potassium
channel to open
during an action potential. The inability oFthe potassium channel to open
results in a decrease in
the outward Flow oFpotassium, which normally repolarizes the membrane oFa
neuron. leading to
CA 02425897 2003-04-23
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prolonged membrane depolarization,
f3y targeting an agent to modulate a GPCR, the signaling activity and
biological process
mediated by the receptor can be agonised or antagonised iv specific cells and
tissues.
Experimental data as provided in F figure 1 indicates expression in humans in
the stomach,
placenta, kidney, skeletal muscle, liver, bone marrow, and thymus. Such
agonistn and
antagonism serves as a basis for modulating a biological activity in a
therapeutic context
(mammalian therapy) or toxic context (anti-cell therapy, e.g. anti-cancer
agent).
DESCRIPTION OF THE FIGURE SHEETS
FIGURE 1 provides the nucleotide sequence of a cDNA molecule that encodes the
GPCR
oFthe present invention. (SEQ ID NO: I) In addition, structure and functional
information is
provided, such as ATG start, stop and tissue distribution, where available,
that allows one to
readily determine specific uses of inventions based on this molecular
sequence. Experimental
data as provided in Figure 1 indicates expression in humans in the stomach,
placenta, kidney,
I S skeletal muscle, liver, bone marrow, and thymus.
FIGURE 2 provides the predicted amino acid sequence of the GPCR of the present
invention. (SEQ ID N0:2) In addition structure and Functional information such
as protein
family, function, and modification sites is provided where available, allowing
one to readily
determine specific uses of inventions based on this molecular sequence.
FIGURE 3 provides genamie sequences that span the gene encoding the GPCR
protein of
the present invention. (SEQ ID N0:3) In addition, structure and functional
information, such as
intron/exon structure, promoter location, etc., is provided where available,
allowing one to
readily determine specific uses of inventions based on this molecular
sequence. As illustrated in
Figure 3, G 171 I T is a known SNP variant.
DETAILED DESCRIPTION OF THE INVENTION
General Description
The present invention is based on the sequencing of the human genome. During
the
sequencing and assembly of the human genome, analysis of the sequence
information revealed
previously unidentified fragments of the human genome that encode peptides
that share
structural and/or sequence homolof~y to protein/peptitieldomains identified
and characterised
1?
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within the art as being a GPCR protein or part of a GPCR protein, that are
involved in cell
signaling, particularly neurotransmitter signaling. Utilising these sequences,
additional genomic
sequences were assembled and transcript and/or cDNA sequences were isolated
and
characterized. Based on this analysis, the present invention provides amino
acid sequences of
human GPCR peptides and proteins that are involved in cell signaling,
particularly
neurotransmitter signaling, nucleic acid sequences in the farm of transcript
sequences, cDNA
sequences andlor genomic sequences that encode these GPCR peptides and
proteins. nucleic acid
variation (allelic information), tissue distribution of expression, and
information about the closest
art known proteiupeptide/domain that has structural or sequence homology to
the GPCR of the
present invention.
In addition to being previously unknown, the peptides that are provided in the
present
invention are selected based on their ability to be used for the development
of commercially
important products and services. Specifically, the present peptides are
selected based on
homology andlor structural relatedness to known GPCR proteins involved in cell
signaling,
particularly neurotransmitter signaling, and the expression pattern observed.
Experimental data
as provided in Figure 1 indicates expression in humans in the stomach,
placenta, kidney, skeletal
muscle, liver, bone marrow, and thymus. The art has clearly established the
commercial
importance of members of this family of proteins and proteins that have
expression patterns
similar to that of the present gene. Some of the more specific features of the
peptides of the
present invention, and the uses thereof, are described herein, particularly in
the Background of
the Invention and in the annotation provided in the Figures, and/or are knawn
within the art for
each of the known GPCR proteins involved in cell signaling, particularly
neurotransmitter
signaling.
Specific Embodiments
Peptide Molecules
The present invention provides nucleic acid sequences that encode protein
molecules that
have been identified as being members of the GPCR family of proteins and are
involved in cell
signaling. particularly neurotransmitter si~,~naling (protein sequences are
provided in Figure ?,
cDNA sequences are provided in Figure 1 and genomic sequences are provided in
Figure 3).
The peptide sequences provided in higure ?. as well as the obvious variants
described herein.
particularly allelic variants as ideniiCaed herein and u,sin~~ the information
in Figure 3, will be
I,
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reFerred herein as the GPCR peptides of the present invention, GPCR peptides,
or
peptides/proteins of the present invention,
The present invention provides isolated peptide and protein molecules that
consist o(~,
consist essentially of, or comprise the amino acid seduences of the GPCR
peptides disclosed in
figure 2, (encoded by the nucleic acid molecule shown in figure I, cDNA
sequence. or Figure 3,
genomic sequence), as well as all obvious variants of these peptides that are
within the art to
make and use. Some of these variants are described in detail below.
As used herein, a peptide is said to be "isolated" or "purified" when it is
substantially free
of cellular material or free of chemical precursors or other chemicals. The
peptides of the present
invention can be purified to homogeneity or other degrees of purity. The level
of purification will
be based on the intended use. The critical feature is that the preparation
allows for the desired
function of the peptide, even if in the presence of considerable amounts of
other components (the
features of an isolated nucleic acid molecule is discussed below).
In some uses, "substantially free of cellular material" includes preparations
of the peptide
I 5 having less than about 30°~'0 (by dry weight} other proteins (i.e.,
contaminating protein), less than
about 20°~'0 other proteins, less than about 10°r'o other
proteins, or less than about 5°r'° other proteins.
When the peptide is recombinantly produced, it can also be substantially free
of culture medium,
i.e" culture medium represents less than about 20°~'° of the
volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes
preparations of the peptide in which it is separated From chemical precursors
or other chemicals that
are involved in its synthesis. In one embodiment, the language "substantially
free of chemical
precursors or other chemicals" includes preparations of the GPCR peptide
having less than about
30°r'° (by dry weight) chemical precursors or other chemicals,
less than about 20°r'° chemical
precursors or other chemicals, less than about 10°l° chemical
precursors or other chemicals, or less
than about 5°r°° chemical precursors or other chemicals.
The isolated GPCR peptide can be puril7ed From cells that naturally express
it, purified from
cells that have been altered to express it (recombinant}, or synthesized using
known protein
synthesis methods. Experimental data as provided in Figure 1 indicates
expression in humans in the
stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus.
For example, a nucleic
acid molecule encoding the GPCR peptide is cloned into an expression vector.
the expression vector
introduced into a host cell and the protein expressed in the host cell. 'hhe
protein can then be
isolated from the cells by an appropriate purification scheme using standard
protein purification
tochuiques_ Many of these techniques are described in detail below.
1~
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Accordingly, the present invention provides protein s that Consist of the
amino acid
SeqlleIlCeS pl'oVlCled I11 I"lgLlI'e 2 (S~';Q ID NO;2), loI' eXalnple,
pl'Ote111S ellCOded by the CDNA
llLlClelC aCICI SCqLlenGeS SllQWl1 In flglll'e 1 (S~'rQ ID NO; 1 ~ and tile
genoLIllC SeqLlenGeS pl'OVIdCLI In
Figure 3 (ShQ ID N0:3). 'IAhe amino acid Sequence oFsuch a protein is provided
in figure 2. A
protein consists ofan amino acid sequence when the amino acid sequence is the
final amino acid
sequence of the protein.
The present invention Further provides proteins that consist essentially of
the amino acid
sequences provided in Figure 2 (SEQ ID N0:2), for example, proteins encoded by
the cDNA
nucleic acid sequences shown in Figure I (SEQ ID NO:1 ) and the genomic
sequences provided in
Figure 3 (SEQ ID N0:3). A protein consists essentially of an amino acid
sequence when such an
amino acid sequence is present with only a few additional amino acid residues,
for example from
about I to about 100 or so additional residues, typically From 1 to about 20
additional residues in the
final protein.
The present invention filrther provides proteins that comprise the amino acid
sequences
I S provided in Figure 2 (SEQ ID N0:2), for example, proteins encoded by the
cDNA nucleic acid
sequences shown in Figure 1 (SEQ ID NO: l ) and the genomic sequences provided
in Figure 3
(SEQ ID N0:3). A protein comprises an amino acid sequence when the amino acid
sequence is at
least part ofthe final amino acid sequence of the protein. In such a fashion,
the protein can be only
the peptide or have additional amino acid molecules, such as amino acid
residues (Contiguous
encoded sequence) that are naturally associated with it or heterologous amino
acid residues/peptide
sequences. Such a protein can have a few additional amino acid residues or can
comprise several
hundred or more additional amino acids. The preferred classes of proteins that
are comprised of the
GPCR peptides oFthe present invention are the naturally occurring mature
proteins. A brief
description of how various types of these proteins can be madelisolated is
provided below.
The GPCR peptides of the present invention can be attached to heterologous
sequences to
Form chimeric or fusion proteins. Such Ghimeric and Fusion proteins comprise a
GPCR peptide
operatively linked fo a heterologous protein having an amino acid sequence not
substantially
homologous to the GPCR peptide. "Operatively linked'' indicates that the GPCR
peptide and the
heterologous protein arc Fused in-Ii'anle. The heterologous protein can be
fused to the N'-terminus
or C-terminus of the GPCR peptide.
In some uses, the fusion protein does not aFFect thL: activity of the GPCR
peptide pc~r.ve. For
example, the Fusion protein can include, but is not limited to, en~,ymatic
fusion proteins, for example
beta-galactosidase I~usions, yeast two-hybrid GAL Fusions, poly-I-Its fusions,
MYC-tagged, Hl-
l~
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
tagged and Ig I'usions. Such fusion proteins, particularly poly-His fusions,
can facilitate the
purification ofrecombinasit GPCR peptide, In certain host cells (e.g.,
mammalian host cells),
expression andior secretion of a protein can be increased by using a
heterologous signal sequence.
A chimeric or fusion protein can be produced by standard recombinant DN'A
technidues,
F"or example, DNA fragments coding for the different protein sequences are
ligated together in-
frame in accordance with conventional techniques. In another embodiment, the
fusion gene can be
synthesized by conventional techniques including automated DNA synthesizers.
Alternatively, PCR
amplification of gene fragments can be carried out using anchor primers which
give rise to
complementary overhangs between two consecutive gene fragments which can
subsequently be
I 0 annealed and re-amplified to generate a ehimerie gene sequence (see
Ausubel et al., Current
Protocols irr tllolecatlar Biology, 1992). Moreover, many expression vectors
are commercially
available that already encode a fusion moiety (e.g., a CST protein). A GPCR
peptide-encoding
nucleic acid can be cloned into such an expression vector such that the fusion
moiety is linked in-
frame to the GPCR peptide.
As mentioned above, the present invention also provides and enables obvious
variants of the
amino acid sequence of the proteins of the present invention, such as
naturally occurring mature
forms of the peptide, alleliclsequenee variants of the peptides, non-naturally
occurring
recombinantly derived variants ofthe peptides, and orthologs and paralogs
ofthe peptides. Such
variants can readily be generated using art-known techniques in the fields of
recombinant nucleic
acid technology and protein biochemistry. It is understood, however, that
variants exclude any
amino acid sequences disclosed prior to the invention.
Such variants can readily be identifiedlmade using molecular techniques and
the sequence
information disclosed herein. Further, such variants can readily be
distinguished from other
peptides based on sequence and/or structural homology to the GPCR peptides of
the present
?S invention. The degree ofhomologylidentity present will be based primarily
on whether the peptide
is a functional variant or non-functional variant, the amount of divergence
present in the paralog
family and the evolutionary distance between the ortholags.
To determine the percent identity oftwo amino acid sequences or two nucleic
acid
sequences, the sequences are aligned for optimal comparison purposes (e.g.,
gaps can be
introduced in one or both of a first and a second amigo acid or nucleic acid
sequence for optimal
alignment and non-homologous sequences can brr disregarded for comparison
purposes). In a
preferred embodiment, the length of~a ref~:rence sequence aligned for
comparison purposes is at
least 30%, ~0°~'0, Sp°r'°, GO°~o, 70%, 80°
o, or c)()°,~o or more: of the length of the reference sequence.
1G
CA 02425897 2003-04-23
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~lAhe amino acid residues or nucleotides at corresponding amino acid positions
or nucleotide
poSltIOIIS al'C then COIllpat'ed. Whell a posltlOn 117 the llCSt SeqUeIlCe lS
OCCLlpled by the Sallle
an7111C) aCld 1'CSldlle Or 1lLICleOtlde aS the GQI'reSpondlllg pOS1t1011 In
the $eC011d SeqllCIlCe, tllell the
InOleCUIeS al'e IdentICal at that pOSltl011 laS LlSed lleCeln alnlllo aCICl Or
nLlClelC aCld "Idelltlty" 1S
equivalent to amino acid or Nucleic acid "homology"). The percent identity
between the two
sequences is a function of the number of identical positions shared by the
sequences, taking into
account the number of gaps, and the length of each gap, which need to be
introduced for optimal
alignment of the two sequences.
The comparison of sequences and determination of percent identity and
similarity
between two sequences can be accomplished using a mathematical algorithm.
(C~rr~pattational
Molecatlcrr Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988;
Bioc'orraprttir2g.'
Infor'malic,~~ and Genoryre Pr'oject,s, Smith, D.W., ed., Academic Press, New
York, 1993; C'omprtter'
Analysis of SedZter?ce Dala, Par'/ l, Griffin, A.M., and Griffin, H.G., eds.,
Humana Press, New
Jersey, 1994 S~grtence Analysis in Molecztlar- Biology, van Eleinje, G.,
Academic Press, 1987; and
Sec7Ztence Ar2alysis Pr'iryzer~, Gribskov, M. and Devereux, J., eds., M
Stockton Press, New York,
1991 ). In a preferred embodiment, the percent identity between two amino acid
sequences is
determined using the Needleman and Wunsch (J, tt~lol, f3iol. (~18):4~1~1-X53
(1970)) algorithm
which has been incorporated into the GAP program in the GCG software package
(available at
http:l/www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and
a gap weight
of 16, 14, 12, 10, 8, 6, or 4 and a length weight of l, 2, 3, ~l, 5, or 6. In
yet another preferred
embodiment, the percent identity between two nucleotide sequences is
determined using the
GAP program in the GCG software package (Devereux, J., et crl.,
Natclc~ic~4cicls Ro,r. l2(1):387
(19$x)) (available at http:l/www.gcg.com), using a NWSgapdna.CMP matrix and a
gap weight of
40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, ~, 5, or 6. In another
embodiment, the
percent identity between two amino acid or nucleotide sequences is determined
using the
algorithm of >f. Meyers and W. Miller (CABIOS, X1:1 1-17 ( 1989)) which has
been incorporated
into the ALIGN pragram Cversion 2.0), using a PAM I 20 weight residue table, a
gap length
penalty of 12 and a gap penalty of ~.
'fhe nucleic acid and protein sequences ofthe present invention can further be
used as a
"query sequence" to perform a search against sequence databases to, for
example, identify other
Family members or related sequences. Such searches can be performed using the
NBLAST and
XBLAS~f programs (version 2.0) of Altschul, et al. (.l, ~tlol. 73io1. 215:403-
10 ( 1990)). BLAS'f
nucleotide searches cal be performed with the NBLAS°IT program, score =
1 Op, wordlength = 12
17
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WO 02/34913 PCT/USO1/31454
t0 Obtalll (lllCleOtlde SeqllellCeS 110111010~O11S tC? the lluCIC:IC acid
17101eCL11eS OI'the lIlVeIltlOn.
BLAS~C pri>tein searches can be performed with the ~BI~AST program, score =
50. wordlength =
3 t0 Obtalll am111O aClC1 SeqllellCeS 1101nOlOgOLIS t0 the prOtelnS 01' the
lnvellt1011. ~1'l) Ob ti1111 gapped
alignmelets for comparison purposes, <iappcd BLA~S~h can be utilized rls
described in Altschul et
al. (lVzac~lc~ic~ ~lc~icl.~ Rc.~,S~. 25( 17):3389-3102 ( 1997)). When
utilizing BLAS'C and gapped BLAST
programs, the default parameters of the respective programs (e.g., XBLASlI'
and NBLAS'C) can
be used.
Full-length pre-processed forms, as well as mature processed forms, of
proteins that
comprise one of the peptides of the present invention can readily be
identified as having complete
sequence identity to one of the GPCR peptides of the present invention as well
as being encoded by
the same genetic locus as the GPCR peptide provided herein. As indicated by
tile data presented in
Figure 3, the map position was determined to be all chromosome 6 by radiation
hybrid mapping.
Allelic variants of a GPCR peptide can readily be identified as being a human
protein
having a high degree (significant) of sequence homologylidentity to at least a
portion of the GPCR
peptide as well as being encoded by the same genetic locus as the GPCR peptide
provided herein.
Genetic locus can readily be determined based on the genomic information
provided in Figure 3,
such as the genomie sequence mapped to the reference human. As indicated by
the data presented
in Figure 3, the map position was determined to be on chromosome 6 by
radiation hybrid mapping.
As used herein, two proteins (or a region of the proteins) have significant
homology when the
amino acid sequences are typically at least about 70-80°l°, 80-
90%, and more typically at least
about 90-95°r'° or more homologous. A significantly homologous
amino acid sequence,
according to the present invention, will be encoded by a nucleic acid sequence
that will hybridize
to a GPCR peptide encoding nucleic acid molecule under stringent conditions as
more fully
described below.
Figure 3 provides information on a SNP variant, 6171 IT, which has been found
in a
gene encoding the GPCR protein of the present invention. The change in the
amino acid
sequence caused by this SNP is indicated in Figure 3 and can readily be
determined using the
universal genetic code and the protein sequence provided in Figure ? as a
reference.
Paralogs ofa GPCR peptide can readily be identified as leaving some degree of
significant
sequence homolo~y/identify fo at least a portion of~ihe GPCR peptide. as being
encoded by a gene
fram humans, and as having similar activity or function. Two proteins will
typically be eon sidered
paralogs when the amino acid sequences are typically at least about 60% or
greater, and more
typically at least about 70°I° or greater homology through a
given region or domain. Such
18
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
paralogs will be encoded by a nucleic acid sequence that will hybridize to a
GfCR peptide
encoding nucleic acid molecule under moderate to stringent conditions as more
(lolly described
below.
Urtholags of a GfCR peptide can readily be identit7ed as having some degree of
significant
sequence homologylidentity to at least a portion of the GPCR peptide as well
as being encoded by a
gene from another organism. Preferred orthologs will be isolated from mammals,
preferably
primates, for the development of human therapeutic targets and agents. Such
orthologs will be
encoded by a nucleic acid sequence that will hybridize to a GPCR peptide
encoding nucleic acid
molecule under moderate to stringent conditions, as more fully described
below, depending on
the degree of relatedness of the two organisms yielding the proteins.
hlon-naturally occurring variants of the GPCR peptides of the present
invention can readily
be generated using recombinant techniques. Such variants include, but are not
limited to deletions,
additions and substitutions in the amino acid sequence of the GPCR peptide.
For example, on a
class of substitutions are conserved amino acid substitution. Such
substitutions are chose that
substitute a given amino acid in a GPCR peptide by another amino acid of like
characteristics.
Typically seen as conservative substitutions are the replacements, one for
another, among the
aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl
residues Ser and °fhr;
exchange of the acidic residues Asp and Glu; substitution between the amide
residues Asn and Gln;
exchange of the basic residues I,ys and Arg; and replacements among the
aromatic residues Phe and
Tyr. Guidance concerning which amino acid changes are likely to be
phenotypically silent are
found in Bowie et crl., Science 217:1306-1310 (1990).
Variant GPCR peptides can be fully functional or can lack function in one or
more
activities, e.g. ability to bind ligand, ability to bind G-protein, ability to
mediate signaling, etc.
Fully functional variants typically contain only conservative variation or
variation in non-critical
residues or in non-critical regions. Figure 2 provides the result of protein
analysis that identifies
critical domains/regions. Functional variants can also contain substitution of
similar amino acids
that result in no change or an insignificant change in function.
Alternatively, such substitutions may
positively or negatively affect function to some degree.
Non-functional variants typically contain one or more nan-conservative amino
acid
substitutions, deletions. insertions. inversions, or truncation or a
substitution. insertion, inversion, or
deletion in a critical residue or critical region.
Amino acids that are essential for function can be identit7ed by methods known
in the art,
such as site-directed mutagenesis or alanine:-scanning mutageneSis
(Cunninghatn ei crl , ,Sc~ic~nc~c~
1 ~)
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
2-I-1:108 i-1 (1$5 ~ 1989)), particularly using the results provided in Figure
2. 'IAhe latter procedure
introduces single alanine mutations at every residue in the molecule. The
resulting mutant
molecules are then tested for biological activity such as ligand/eF(octor
molecule binding or in
assays such as an in vitro prolil~rative activity. ~it~s that are critical For
ligand-receptor binding can
also be determined by structural analysis such as crystallization, nuclear
magnetic resonance or
photoahlinity labeling (Smith e/ al.. J. tt~lol_ Biul. 2?-1:899-904 (1992); de
Vos e/ crl. ~Sciejzoe
2»:306-312 (1992)).
The present invention further provides fragments of the GPCR peptides, in
addition to
proteins and peptides that comprise and consist of such fragments,
particularly those comprising the
residues identified in Figm-e 2. The fragments to which the invention
pertains, however, are not to
be construed as encompassing fragments that may be disclosed publicly prior to
the present
invention.
As used herein, a fragment comprises at least 8, 10, 12, I ~l, 16, or more
contiguous amino
acid residues from a GPCR peptide. Such fragments can be chosen based on the
ability to retain
I S one or more of the biological activities of the GPCR peptide or could be
chosen for the ability to
perform a function, e.g. ability to bind ligand or effector molecule or act as
an immunogen.
Particularly important fragments are biologically active fragments, peptides
which are, for example,
about 8 or more amino acids in length. Such Fragments will typically comprise
a domain or motif of
the GPCR peptide, e.g., active site, a G-protein binding site, a transmembrane
domain or a ligand-
binding domain. Further, possible fragments include, but are not limited to,
domain or motif
containing fragments, soluble peptide fragments, and fragments containing
immunogenic structures.
Predicted domains and functional sites are readily identifiable by computer
programs well-known
and readily available to those of skill in the art (e.g., PROSITE analysis).
The results of one such
analysis are provided in Figure 2.
Polypeptides often contain amino acids other than the 20 amino acids commonly
referred to
as the 20 naturally occurring amino acids. Further, many amino acids,
including the terminal amino
acids, may be modified by natural processes, such as processing and other post-
translational
modifications, or by chemical modification technidues well known in the art.
Common
modifications that occur naturally in GPCR peptides are described in basic
texts, detailed
ip monographs. and the research literature, and they are well known to those
ofskill in the ari(some o~
these features are identified in Figure 2).
Known modifications include, but are not limited to, acetylation, acylation,
ADN-
ribosylatfon, amidation, covalent attachment of Ilavin, covalent attachment of
a heme moiety,
'' 0
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
covalent attachment ol~a nucleotide ar nucleotide derivative, covalent
attachment of a lipid or lipid
derivative, covalent attachment ofphosphotidylinositol. cross-linking,
cyclization, disulfide bond
lbrmation, demethylation, formation ofcovaleni crosslinks, formation
ofcystinc, formation of
pyroglutamate, fo~7mylation, gamma carboxylation, glycosylation, Gf I anchor
formation,
hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic
processing,
phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-
RNA mediated
addition of amino acids to proteins such as arginylation, and ubiquitination.
Such modifications are well-knomm to those of skill in the art and have been
described in
great detail in the scientific literature. Several particularly common
modifications, glycosylation,
lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues,
hydroxylation and
ADP-ribosylation, For instance, are described in most basic texts, such as Py-
oteins - SlrztclZtre and
Molecztlar Pr~operlie,s, 2nd >~d., T.E. Creighton, W. H. Freeman and Company,
New York (1993).
Many detailed reviews are available on this subject, such as by Wold, F.,
Po.stIrarZ.slalional Covalent
~~odificcrlion o~Py~oleins, B.C. Johnson, Ed., Academic Press, New York 1-12
(1983); Seifter el al.
(Melh. Enynlol. 182: 626-616 ( 1990)} and Rattan et al. (~l nn. N. ~'. ~I cad,
Sci. 663:8-62 ( 1992)).
Accordingly, the GPCR peptides of the present invention also encompass
derivatives or
analogs in which a substituted amino acid residue is nat one encoded by the
genetic code, in which
a substituent group is included, in which the mature GPCR peptide is fused
with another compound,
such as a compound to increase the half=life of the GPCR peptide (for example,
polyethylene
glycol), or in which the additional amino acids are fused to the mature GPCR
peptide, such as a
leader or secretory sequence or a sequence for purification of the mature GPCR
peptide or a pro-
protein sequence.
ProteinlPeptide Uses
The proteins of the present invention can be used in substantial and specific
assays
related to the functional information provided in the Figures and Back Ground
Section; to raise
antibodies or to elicit another immune response: as a reagent (including the
labeled reagent} in
assays designed to quantitatively determine levels of the protein (or its
binding partner or
receptor) in biological fluids; and as markers for tissues in which the
corresponding protein is
preferentially expressed (either constitutively or at a particular stage of
tissue differentiation or
development or in a disease state). Where the preat~:in binds or potentially
binds to another
protein (such as, for example, in a receptor-ligand interaction j, the protein
can be used to identify
~' I
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
the binding partner so as to develop a system to identify inhibitors of the
binding interaction.
Any or all of these research utilities are capable of being developed into
reagent grade or kit
lbrmat for commercialisation as commercial products.
Methods for performing the uses listed above are well known to those skilled
in the art.
References disclosing such methods include "Molecular Cloning: A Laboratory
Manual", 2d ed..
Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 1989,
and "Methods in En~ymology: Guide to Molecular Cloning Techniques'', Academic
PreSS,
Bergen S. L. and A. R. I~immel eds., 19$7.
The potential uses of the peptides of the present invention are based
primarily on the
source of the protein as well as the classjaction of the protein. For example,
GPCRs isolated
from humans and their human/mammalian orthologs serve as targets for
identifying agents For
use in mammalian therapeutic applications, e.g. a human drug, particularly in
modulating a
biological or pathological response in a cell or tissue that expresses the
GPCR. Experimental
data as provided in Figure 1 indicates that GPCR proteins of the present
invention are expressed
1 S in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone
marrow, and thymus.
Specifically, a virtual northern blot shows expression in the stomach. In
addition, PCR-based
tissue screening panels indicate expression in placenta, kidney, skeletal
muscle, liver, bone
marrow, and thymus tissue. Approximately 70°r~° ofall
pharmaceutical agents modulate the
activity of a GPCR. A combination of the invertebrate and mammalian ortholog
can be used in
selective screening methods to find agents specific for invertebrates. The
structural and
functional information provided in the Background and Figures provide specific
and substantial
uses for the molecules of the present invention, particularly in combination
with the expression
information provided in Figure 1. Experimental data as provided in Figure 1
indicates expression
in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone
marrow, and thymus.
Such uses can readily be determined using the information provided herein,
that known in the art
and routine experimentation.
The proteins of the present invention (including variants and fragments that
may have been
disclosed prior to the present invention) are useful far biological assays
related to GPCRs that are
involved in cell signaling, particularly n eurotransmitter signaling. Such
assays involve any of the
known GPCR functions or activities or properties useful for diagnosis and
treatment of GPCR-
related conditions that are specituc For the subfamily of GPCRs that the one
ofthe present invention
belongs to, particularly in cells and tissues that express this receptor.
Experimental data as provided
iv figure 1 indicates that GPCR proteins ofthe present invention are expressed
in human s in the
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
stomach, placenta, kidney, skeletal muscle, liver, hone marrow, and thymus.
Specifically, a virtual
northern blot shows expression in the stomach. In addition, PCR-based tissue
screening panels
indicate expression ill placenta, kidney, skeletal muscle, liver, bone marrow,
and thymus tissue
~hhe pl'Ote111S Rf' the prGSf'.nt IIlVellt1011 al'e al SO LISefUI 111 tll'L!g
SCI'Et;llln~ aSSayS, IIl Cell-baSC;d
or cell-free systems. Cell-based systems can he native, i.e., cells that
normally express the receptor
protein, as a biopsy or expanded in cell culture, Experimental data as
provided in Figure 1 indicates
expression in humans in the stomach, placenta, kidney, skeletal muscle, liver,
bone marrow, and
thymus. In an alternate embodiment, cell-based assays involve recombinant host
cells expressing
the receptor protein.
The polypeptides can be used to identify compounds that modulate receptor
activity of the
protein in its natural state, or an altered form that causes a specific
disease or pathology associated
with the receptor. Both the GPCRs of the present invention and appropriate
variants and fragments
can be used in high-throughput screens to assay candidate compounds for the
ability to bind to the
receptor. These compounds can be ftlrther screened against a functional
receptor to deternline the
effect of the compound on the receptor activity. hurther, these compounds can
be tested in animal
or invertebrate systems to determine activityleffectiveness. Compounds can be
identified that
activate (agonist) or inactivate (antagonist) the receptor to a desired
degree.
Further, the proteins of the present invention can be used to screen a
compound for the
ability to stimulate or inhibit interaction between the receptor protein and a
molecule that normally
interacts with the receptor protein, e.g. a ligand or a component ofthe signal
pathway that the
receptor protein normally interacts (for example, a G-protein or other
interactor involved in cAMP
or phosphatidylinositol turnover andlor adenylate cyclase, or phospholipase C
activation). Such
assays typically include the steps of combining the receptor protein with a
candidate compound
under conditions that allow the receptor protein, or Fragment, to interact
with the target molecule,
and to detect the formation of a complex between the protein and the target or
to detect the
biochemical consequence of the interaction with the receptor protein and the
target, such as any of
the associated effects of signal transduction such as G-protein
phosphorylation, cAMP or
phosphatidylinositol turnover, and adenylate cyclase or phospholipase C
activation.
Candidate compounds include, for example, 1 ) peptides such as soluble
peptides, including
Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g.,
Lam et ul., tV'crlm~e
3~-l:8?-$~l ( 1991 ); I-IoLlghten ct ul" r'~crlm~~a 35-1:81-$6 ( 1 ~q 1 )) and
combinatorial chemistry-derived
molecular libraries made ol'I~- andlor L- conligurafion amino acids: ?)
phosphopeptides Ie.g.,
members of random and partially degenerate, direcfed phosphopeptide:
libraries, see, e.g" Son gyang
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
m ttl,, C'c~ll 7?:767-778 ( 1993 )); 3 ) antibodies (e.g.. polyclonal.
monoclonal, humanised, anti-
idit>typic. chimerie, and single chain antibodies as well as Fah, Iv'~ab')?,
Fab expression library
fragments, and epitope-binding fragments of antibodies); and ~I) small organic
and inorganic
molecules (e.g., molecules obtained (rote combinatorial and natural product
libraries),
One candidate compound is a soluble fragment of the receptor that competes for
ligand
binding. Other candidate compounds include mutaait receptors or appropriate
fragments containing
mutations that affect receptor Function and thus compete for ligand.
Accordingly, a Fragment that
competes for ligand, for example with a higher aF~nity, or a fragment that
binds ligand but does not
allow release, is encompassed by the invention.
The invention further includes other end point assays to identify compounds
that modulate
(stimulate or inhibit) receptor activity. The assays typically involve an
assay of events in the signal
transduction pathway that indicate receptor activity. Thus, a cellular process
such as proliferation,
the expression of genes that are up- or down-regulated in response to the
receptor protein dependent
signal cascade, can be assayed. In one embodiment, the regulatory region of
such genes can be
operably linked to a marker that is easily detectable, such as luciferase.
Any of the biological or biochemical functions mediated by the receptor can be
used as an
endpoint assay. These include all of the biochemical or biochemicallbiological
events described
herein, in the references cited herein, incorporated by reference for these
endpoint assay targets, and
other Functions known to those of ordinary skill in the art or that can be
readily identified using the
information provided in the Figures, particularly Figure 2. Specifically, a
biological Function of a
cell or tissues that expresses the receptor can be assayed. Experimental data
as provided in Figure I
indicates that GPCR proteins of the present invention are expressed in humans
in the stomach,
placenta, kidney, skeletal muscle, liver, bone marrow, and thymus,
Specifically, a virtual northern
blot shows expression in the stomach. In addition, PCR-based tissue screening
panels indicate
expression in placenta, kidney, skeletal muscle, liver, bone marrow, and
thymus tissue.
Binding and/or activating compounds can also be screened by using chimeric
receptor
proteins in which the amino terminal extracellular domain, as parts thereof,
the entire
transmembrane domain or subregions, such as any of the seven transmembrane
segments or any of
the intracellular or extracellular loops and the carboxy terminal
intracellular domain, or parts
thereof, can be replaced by heterologou s domains or subregions. For example,
a G-protein-binding
region can be used that interacts with a diFferent G-protein then that which
is recognized by the
native receptor. Accordingly, a different set of signal transduction
components is available as an
end-point assay For activation. Alternatively, the entire transmembrane
portion or subregions Csuch
~ ~-I
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
as transtnembrane segments or intracellular or extracellular loops) can be
replaced with the entire
transmembrane portion or subregicans specific to a host cell that is different
from the bast cell ti~om
which th a amino terminal exiracellular domain and/or the G-protein-binding
regian are derived.
IAhis allows for assays to be performed in alher than the specific bast cell
from which the receptor is
derived. Alternatively, the amino terminal extracellular domain {andlor other
ligand-binding
regions) could be replaced by a domain (and/or other binding region) binding a
different ligand,
thus, providing an assay for test compounds that interact with the
heterologous amino terminal
extracellular domain (or region) but still cause signal transduction. Finally,
activation can be
detected by a reporter gene containing an easily detectable coding region
operably linked to a
transcriptional regulatory sequence that is part of the native signal
transduction pathway.
The proteins of the present invention are also useful in competition binding
assays in
methods designed to discover compounds that interact with the receptor. Thus,
a compound is
exposed to a receptor polypeptide under conditions that allow the compound to
bind or to otherwise
interact with the polypeptide {Hodgson, Bioltechnology, 1992, Sept 10(9);973-
80). Soluble
receptor polypeptide is also added to the mixture. If the test compound
interacts with the soluble
receptor polypeptide, it decreases the amount of complex formed or activity
from the receptor
target. This type of assay is particularly useful in eases in which compounds
are sought that interact
with specific regions of the receptor. Thus, the soluble polypeptide that
competes with the target
receptor region is designed to contain peptide sequences corresponding to the
region of interest.
To perform cell free drug screening assays, it is sometimes desirable to
immobilize either
the receptor protein, or fragment, or its target molecule to facilitate
separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to accommodate
automation of the
assay.
Techniques for immobilizing proteins on matrices can be used in the drug
screening assays.
In one embodiment, a fission protein can be provided which adds a domain that
allows the protein to
be bound to a matrix. For example, glutathione-S-transferase fusion proteins
can be adsorbed onto
glutathione sepharose beads {Sigma Chemical, St. Louis, MO) or glutathione
derivatized microtitre
plates, which are then combined with the cell lysates {e.g.,''S-labeled) and
the candidate
compound, and the mixture incubated under conditions conducive to complex
Formation {e.g., at
physiological conditions for salt and pf-I). Following incubation, the beads
are washed to remove
any unbound label, and the matrix immobilized and radiolabel determined
directly, or in the
supernatant alter the complexes are dissociated. Alternatively, the complexes
can be dissociated
from the matrix, separated by ~D~-PAGC~. and the level of receptor-binding
protein found in the
~i
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
bead fraction quantitated from the gel Using standard electrophoretic
technidues. laor example,
either the polypeptide or its target molecule can be inlmobili~ed utilizing
conjugation of biotin and
streptavidin using techniques well known in the art. Alternatively, antibodies
reactive with the
protein but which do not interfere with binding of the protein to its target
molecule can be
derivati~ed to the wells of the plate, and the protein trapped in the wells by
antibody conjugation.
Preparations of a receptor-binding protein and a candidate compound are
incubated in the receptor
protein-presenting wells and the amount of complex trapped in the well can be
quantitated.
Methods for detecting such complexes, in addition to those described above for
the GS'I°-
immobilized complexes, include immunodeteetion of complexes using antibodies
reactive with the
receptor protein target molecule, or which are reactive with receptor protein
and compete with the
target molecule, as well as enzyme-linked assays which rely on detecting an
enzymatic activity
associated with the target molecule.
Agents that modulate one of the GPCRs of the present invention can be
identified using one
or more of the above assays, alone or in combination. It is generally
preferable to use a cell-based
I 5 or cell free system first and then confirm activity in an animal or other
model system. Such model
systems are well known in the art and can readily be employed in this context.
Modulators of receptor protein activity identified according to these drug
screening assays
can be used to treat a subject with a disorder mediated by the receptor
pathway, by treating cells or
tissues that express the GPCR. Experimental data as provided in Figure 1
indicates expression in
humans in the stomach, placenta, kidney, skeletal muscle, liver, bone marrow,
and thymus. These
methods of treatment include the steps of administering a modulator of the
GPCR's activity in a
pharmaceutical composition to a subject in need of such treatment, the
modulator being identified as
described herein.
In yet another aspect of the invention, the GPCR proteins can be used as "bait
proteins"
in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No.
5,283,317; Zervos et al.
( 1993) Cell 72:223-232; Madura et al. (1993) J. Biol. C'hc~m4 268:12016-
12051; Bartel et al.
( 1993 ) l3iolec~hniqite,r I 1:920-92~; Iwabuchi et al. ( 1993) ()f~c~o~crTt'
8:1693-1696; and Brent
W094/10300), to identify other proteins, which bind to or interacf with the
GPCR and are
involved in GPCR activity. Such GPCR-binding proteins are also likely to be
involved in the
propagation of signals by the GPCR proteins or GfCR targets as, for example,
downstream
elements of a GPCR-mediated signaling pathway. Alternatively. such GPCR-
binding proteins
are likely to be GPCR inhibitors.
FI'he tWO-h ybl'Id SySt~:ln 1S based 011 the Illotllllal' nature ofInOSt
tl'aIlSC1'1pt1011 IaCtol'S,
76
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
which consist of separable DNA-binding and activation domains. Briefly. the
assay utilizes two
diFFerent DNA constructs. In one construct. the gene that codes For a GPCR
protein is Fused to a
gene encoding the DNA binding domain oFa known transcription factor (e.g.. GAG-
4). In the
other construct, a DNA sequence, from a library of DNA sequences. that encodes
an unidentified
protein ("prey" or "sample") is fused to a gene that codes For the activation
domain of the known
transcription Factor. If the "bait" and the "prey" proteins are able to
interact, ij~ vivo, forming a
GPCR-dependent complex, the DNA-binding and activation domains of the
transcription Factor
are braught into close proximity. This proximity allows transcription of a
reporter gene (e.g.,
LacZ) which is operably linked to a transcriptional regulatory site responsive
to the transcription
factor. l;xpression of the reporter gene can be detected and cell colonies
containing the
functional transcription factor can be isolated and used to obtain the cloned
gene which encodes
the protein which interacts with the GPCR protein.
This invention Further pertains to novel agents identified by the above-
described
screening assays. Accordingly, it is within the scope of this invention to
further use an agent
identified as described herein in an appropriate animal model. For example, an
agent identified
as described herein (e.g., a GPCR modulating agent, an antisense GPCR nucleic
acid molecule, a
GPCR-specific antibody, or a GPCR-binding partner) can be used in an animal or
other model to
determine the efficacy, toxicity, or side effects of treatment with such an
agent. Alternatively, an
agent identified as described herein can be used in an animal or other model
to determine the
mechanism of action of such an agent. Furthermore, this invention pertains to
uses of novel
agents identified by the above-described screening assays for treatments as
described herein.
The GPCR proteins of the present invention are also useful to provide a target
for
diagnosing a disease or predisposition to disease mediated by the peptide.
Accordingly, the
invention provides methods For detecting the presence, or levels of the
protein (or encoding
mRl~f'A) in a cell, tissue, or organism. Experimental data as provided in
Figure 1 indicates
expression in humans in the stomach, placenta, kidney, skeletal muscle, Liver,
bone marrow, and
thymus. 'hhe method involves contacting a biological sample with a compound
capable of
interacting with the receptor protein such that the interaction can be
detected. Such an assay can be
provided in a single detection format or a mufti-detection format such as an
antibody chip array.
One agent for detecting a protein in a sample is an antibody capable of
selectively binding to
protein. A biological sample includes tissues, cells and biological Iluids
isolated from a subject, as
well as tissues, cells and fluids present within a subject.
~hhe peptides oFihe present invention also provide targets Cor diagnosing,
active protein
~7
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
activity, disease, or predisposition to disease. in a patient having a variant
peptide, particularly
activities and conditions that are known For other members ohthe Family of
proteins to which the
present one bilongs_ ~fhus, the peptide can be isolated From a biological
sample and assayed far the
presence of a genetic mutation that results in aberrant peptide. Al~his
includes amino acid
substitution, deletion, insertion, rearrangement, (as the result of aberrant
splicing events), and
inapprapriate post-translational modification. Analytic methods include
altered electrophm°etic
mobility, altered tryptic peptide digest, altered receptor activity in cell-
based or cell-free assay,
alteration in ligand or antibody-binding pattern, altered isoelectric point,
direct amino acid
sequencing, and any other of the known assay techniques useful for detecting
mutations in a protein.
Such an assay can be provided in a single detection format or a multi-
detection format such as an
antibody chip array.
In vitro techniques for detection of peptide include enzyme linked
immunosorbent assays
{ELISAs), Western blots. immunoprecipitations and immunofluorescence using a
detection reagent,
such as an antibody or protein binding agent. Alternatively, the peptide can
be detected in vioo in a
subject by introducing into the subject a labeled anti-peptide antibody or
other types of detection
agent. For example, the antibody can be labeled with a radioactive marker
whose presence and
location in a subject can be detected by standard imaging techniques.
Particularly useful are
methods that detect the allelic variant of a peptide expressed in a subject
and methods which detect
Ii-agments of a peptide in a sample.
The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics
deal with
clinically significant hereditary variations in the response to dnlgs due to
altered drug disposition
and abnarmal action in affected persons. See, e.g., Eichelbaum, M. (Clip. Exp.
Phanmacol. Phvsiol.
23(10-11):983-985 (1996)), and Linden M.W. (Clira. Chern. ~13(2):25~-266
(1997)). The clinical
outcomes of these variations result in severe toxicity of therapeutic drugs in
certain individuals or
therapeutic failure of drugs in certain individuals as a result of individual
variation in metabolism.
Thus, the genotype of the individual can determine the way a therapeutic
compound acts on the
body or the way the body metabolizes the compound. Further, the activity of
drug metabolizing
enzymes effects both the intensity and duration ofdrug action, Thus, the
phamiacogenomics of the
individual permit the selection of effective compounds and effective dosages
of such compounds Ior
prophylactic or therapeutic treatment based on the individual's genotype. The
discovery oI'genetic
polymorphisms in some drug metabolizing enzymes has explained why some
patients do not obtain
the expected drug elTects, show an exaggerated drug effect, or experience
serious toxicity From
standard dru~~ dosages. folymorphisms can be expressed in the phenotype ol~th~
extensive
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
metaboli~er and the phenotype oFihe poor metaholizer. Accordingly, genetic
polymorphism may
lead to allelic protein variants oFihe receptor protein in which one or more
of the receptor tlmctions
in one population is diFFerent From those in another population. The peptides
thus allow a target to
ascertain a genetic predisposition that can alT ect treatment modality, 'Thus,
in a ligand-based
treatment, polymorphism may give rise to amino terminal extracellular domains
and/or other ligand-
binding regions that are more or less active in ligand binding, and receptor
activation. Accordingly,
ligand dosage would necessarily be modified to maximize the therapeutic effect
within a given
population containing a polymorphism. As an alternative to genotyping,
specific polymorphic
peptides could be identified.
The peptides are also useful for treating a disorder characterized by an
absence of,
inappropriate, or unwanted expression of the protein. Experimental data as
provided in Figure 1
indicates expression in humans in the stomach, placenta, kidney, skeletal
muscle, liver, bone
marrow, and thymus. Accordingly, methods for treatment include the use of the
GPCR protein or
fragments.
Antibodies
The invention also provides antibodies that selectively bind to one of the
peptides of the
present invention, a protein comprising such a peptide, as well as variants
and fragments thereof,
As used herein, an antibody selectively binds a target peptide when it binds
the target peptide and
?0 does not significantly bind to unrelated proteins. An antibody is still
considered to selectively bind
a peptide even if it also binds to other proteins that are not substantially
homologous with the target
peptide so long as such proteins share homology with a fragment or domain of
the peptide target of
the antibody. In this ease, it would be understood that antibody binding to
the peptide is still
selective despite some degree oFcross-reactivity.
As used herein, an antibody is defined in terms consistent with that
recognized within the
art: they are multi-subunit proteins produced by a mammalian organism in
response to an antigen
challenge. The antibodies of the present invention include polyclonal
antibodies and monoclonal
antibodies, as well as fragments ofsuch antibodies, including, but not limited
to, I~ab or IM(ab')~, and
Fv fragments.
Many methods arc known for generating and/or identiFyint~ antibodies to a
given target
peptide. Several such methods are described by° I-Iarlow, Antibodies,
Cold Spring Harbor Press.
( 1989)_
'' 9
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
In general. to generate antibodies, an isolated peptide is used as an
inltllunogen and is
administered to a mammalian organism, such as a rat, rabbit or mouse. The
fLlll-length protein, an
antigenic peptide fragment or a fusion protein can he used. fal-ticularly
important fragments are
tlloSe COVeI'lllg tullCtlollal dC)Illalns, SLICK as the CIoIl1a111S ldentlhed
111 I~1gL11'e ?, alld dOmaln 01'
sequence homology or divergence amongst the family, such as those that can
readily be identified
using protein alignment methods and as presented in the Figures.
Antibodies are preferably prepared from regions or discrete fragments of the
GPC1Z
proteins. Antibodies can be prepared from any region of the peptide as
described herein.
However, preferred regions will include those involved in function/activity
and/or
receptorlbinding partner interaction. Figure 2 can be used to identify
particularly important
regions while sequence alignment can be used to identify conserved and unique
sequence
Fragments.
An antigenic fragment will typically comprise at least 8 contiguous amino acid
residues.
The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more
amino acid residues.
Such fragments can be selected on a physical property, such as fragments con-
espond to regions that
are located on the surface of the protein, e.g., hydrophilic regions or can be
selected based an
sequence uniqueness (see Figure 2).
Detection on an antibody of the present invention can be facilitated by
coupling (i.e.,
physically linking} the antibody to a detectable substance. Examples of
detectable substances
include various enzymes, prosthetic groups, fluorescent materials, luminescent
materials,
bioluminescent materials, and radioactive materials. Examples of suitable
enzymes include
horseradish peroxidase, alkaline phosphatase, (3-galactosidase, or
acetylcholinesterase; examples of
suitable prosthetic group complexes include streptavidinlbiotin and
avidilllbiotin; examples of
suitable fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent materials
include luciferase,
luciferin, and aequorin, and examples of suitable radioactive material include
I''~I, III, ~'S or ~H.
Antibody L.Fses
~() Ahhe antibodies can be used to isolate one oFthe proteins of the present
invention by standard
techniLlues, such as affinity chromatography or immunoprecipitation. The
antibodies can facilitate
the pLlrIflGat1011 of the llatLlI'al prote111 fl'om CClls allCl rCCO
I11b111aTltly p1'odLICed protein eXpl'eSSed In
i()
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
hOSt Cells. 111 CldC1lt10n, SLICK alltlbodlES are LlSef'Lll t0 dEteCt the
preSC'.IlCe Of otle oI the pl"OtelllS Of the
pI'eSEllt lnVen tlon lIl Cells of tISSLIe$ t0 deten11111e the pattern
ol'expl'eSS10I1 ol~tll(: protElll alTlong
val'louS tlSSIIC:S In all ol'gr11115n1 and ovCa' the Coul'Se Of'1101-111a1
C1L'Velopmellt. I>xpel'11T1elltal data as
provided in Figure 1 indicates that UPC'IZ proteins of the present invention
are expressed in humans
in the stomach, placenta, kidney, skeletal mLlscle, livEr, bon a marrow, and
thymus. Specifically, a
virtual narth em blot shows expression in the stomach. In addition, PCIZ-based
tissue screening
panels indicate expression in placenta, kidney, skeletal muscle, liver, bone
marrow, and thymus
tissue. FuI-ther, such antibodies can be used to detect protein in suet, ijZ
uitro, or in a cell lysate or
supernatant in order to evaluate the abundance and pattern of expression.
Also, such antibodies can
be used to assess abnormal tissue distribution or abnormal expression during
development or
progression of a biological condition. Antibody detection of circulating
fragments of the fidl length
protein can be used to identify turnover.
Further, the antibodies can be used to assess expression in disease states
such as in active
stages of the disease or in an individual with a predisposition toward disease
related to the protein's
function. When a disorder is caused by an inappropriate tissue distribution,
developmental
expression, level of expression of the protein, or expressed/processed form,
the antibody can be
prepared against the normal protein. Experimental data as provided in Figure 1
indicates expression
in humans in the stomach, placenta, kidney, skeletal muscle, liver, bone
marrow, and thymus. If a
disorder is characterized by a specific mutation in the protein, antibodies
specific for this mutant
protein can be used to assay for the presence ofthe specific mutant protein.
The antibodies can also be used to assess normal and aberrant subcellular
localization of
cells in the various tlSSUeS in an organism. Experimental data as provided in
Figure 1 indicates
expression in humans in the stomach, placenta, kidney, skeletal muscle, liver,
bone marrow, and
thymus. The diagnostic uses can be applied, not only in genetic testing, but
also in monitoring a
treatment modality. Accordingly, where treatment is ultimately aimed at
correcting expression level
or the presence of aben-ant sequence and aberrant tissue distribution or
developmental expression,
antibodies directed against the protein or relevant fragments can be used to
monitor therapeutic
efficacy.
Additionally, antibodies are useful in pharmacogenomic analysis. Thus,
antibodies prepared
a0 against polylnorphic proteins can be used to identify individuals that
require modified treatment
modalities. The antibodies are also useful as diagnostic tools as an
immunological marker fi)r
aberrant protein analyzed by electrophoretic mobility, isoele:ctric point,
Cryptic peptide digest. and
other physfcal assays known to those ill the art.
;l
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Fl~he antibodies are also useFul far tissue typing. IJxperimental data as
provided in Figure 1
indicates expression in humans in the stomach, placenta, kidney, skeletal
muscle, liver, bone
marrow, and thymus. 'hhus, where a specific protein has been correlated with
expression in a
specific tissue, antibodies that are specific For this protein can be used to
ir(entity a tissue type.
'fhe antibodies are also useful for inhibiting protein function, For example,
blocking the
binding of the GPCR peptide to a binding partner such as a ligand. 'these uses
can also be applied
in a therapeutic context in which treatment involves inhibiting the protein's
Function. An antibody
can be used, for example, to block binding, thus modulating (agonizing or
antagonizing) the
peptides activity. Antibodies can be prepared against specific fragments
containing sites required
for function or against intact protein that is associated with a cell or cell
membrane. See Figure 2 for
structural information relating to the proteins of the present invention.
The invention also encompasses kits for using antibodies to detect the
presence of a protein
in a biological sample. The kit can comprise antibodies such as a labeled or
labelable antibody and
a compound or agent for detecting protein in a biological sample; means for
determining the amount
of protein in the sample; means for comparing the amount of protein in the
sample with a standard;
and instructions for use. Such a kit can be supplied to detect a single
protein or epitope or can be
configured to detect one of a multitude of epitopes, such as in an antibody
detection array. Arrays
are described in detail below for nucleic acid arrays and similar methods have
been developed for
antibody arrays.
ucleic Acid Molecules
The present invention fm-ther provides isolated nucleic acid molecules that
encode a GPCR
peptide or protein of the present invention (cDNA, transcript and genomic
sequence). Such nucleic
acid molecules will consist of, consist essentially of, or comprise a
nucleotide sequence that encodes
one of the GfCR peptides of the present invention, an allelic variant thereoF
or an ortholog or
paralog thereof.
As used herein, an "isolated" nucleic acid molecule is one that is separated
from other
nucleic acid present in the natural source of the nucleic acid. Preferably, an
"isolated" nucleic acid
is free of sequences which naturally flank the nucleic acid (i.e., sequences
located at the 5' and 3'
ends of the nucleic acid) in the genomic D~IA of the organism from which the
nucleic acid is
derived. I-lowever, there can be some flanking nucleotide seque.races, for
example up to about SKB,
~1K13, iKl3, ?KI3, or 1 KI3 or less, particularly contiguous peptide encoding
sequences and peptide
CA 02425897 2003-04-23
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encoding sequences within the same gene but separated by introns in the
~ellomic sequence. The
important point is that the nucleic acid is isolated from remote and
unimportant tlanl:ing sequences
SLICK theft It 0111 be SLIb~eCtCd t0 the SpeCIflC I11an1pLilat1011s deSCl'lbed
llerelll SLICK a5 CeCOITlblllallt
expression, preparation of probes and primers, and other uses specific to the
nucleic acid sequences.
S Moreover, an "isolated" nucleic acid molecule, such as a transcript/cDNA
molecule, can be
substantially free of ocher cellular material, or culture medium when produced
by recombinant
techniques, or chemical precursors or other chemicals when chemically
synthesized. However, the
nucleic acid molecule can be fused to other coding or regulatory sequences and
still be considered
isolated.
For e~cample, recombinant DNA molecules contained in a vector are considered
isolated.
Further examples of isolated DNA molecules include recombinant DNA molecules
maintained in
heterologous host cells or purified (partially or substantially) DNA molecules
in solution. Isolated
RNA molecules include irr vivo or in vitro RNA transcripts of the isolated DNA
molecules of the
present invention. Isolated nucleic acid molecules according to the present
invention further include
such molecules produced synthetically.
Accordingly, the present invention provides nucleic acid molecules that
consist of the
nucleotide sequence shown in Figure 1 or 3 (SEQ ID NO:I, cDNA sequence and SEQ
ID N0:3,
genomic sequence), or any nucleic acid molecule that encodes the protein
provided in Figure 2,
SEQ ID N0:2. A nucleic acid molecule consists of a nucleotide sequence when
the nucleotide
sequence is the complete nucleotide sequence oFthe nucleic acid molecule.
The present invention further provides nucleic acid molecules that consist
essentially of the
nucleotide sequence shown in Figure I or 3 (SEQ ID NO:I, cDNA sequence and SEQ
ID N0:3,
genomic sequence), or any nucleic acid molecule that encodes the protein
provided in Figure 2,
SEQ LD N0:2. A nucleic acid molecule consists essentially of a nucleotide
sequence when such a
nucleotide sequence is present with only a few additional nucleic acid
residues in the final nucleic
acid molecule.
The present invention further provides nucleic acid molecules that comprise
the nucleotide
sequences shown in Figure 1 or 3 (SEQ ID NO:I, cDNA sequence and SEQ ID N0:3,
genonlic
sequence), or any nucleic acid molecule that encodes the protein provided in
Figure 2, SCQ ID
NO:2. A nucleic acid molecule comprises a nucleotide sequence when the
nucleotide sequence is at
leasf part of the final nucleotide seduence of the nucleic acid Molecule. In
such a fashion, the
nucleic acid molecule can be only the nucleotide sequence or have additional
nucleic acid residues,
such as nucleic acid resiclucs that arc naturally associated with it or
lveterolof~ous nucleotide
"
CA 02425897 2003-04-23
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sequences. Such a nucleic acid molecule can have a few additional nucleotides
or can comprises
several hundred or more additional nucleotides. A brief description of how
various types of these
nucleic acid molecules can be readily madelisolated is provided below.
In Figures 1 and 3, both coding and non-coding sequences are provided, Because
of the
source ofthe present invention, human genomic sequences (Figure a) and cDNA
sequences
(Figure I ), the nucleic acid molecules in the Figures will contain genomic
intronic sequences, 5'
and 3' non-coding sequences, gene regulatory regions and non-coding intergenic
sequences. In
general such sequence features are either noted in Figures 1 and 3 or can
readily be iden tiFed
using computational tools known in the art. As discussed below, some of the
non-coding
regions, particularly gene regulatory elements such as promoters, are useful
for a variety of
purposes, e.g. control of heterologous gene expression, target for identifying
gene activity
modulating compounds, and are particularly claimed as fragments of the genomic
sequence
provided herein.
The isolated nucleic acid molecules can encode the mature protein plus
additional amino or
I 5 carboxyl-terminal amino acids, or amino acids interior to the mature
peptide (when the mature form
has more than one peptide chain, for instance). Such sequences may play a role
in processing of a
protein from precursor to a mature form, facilitate protein trafficking,
prolong or shorten protein
half life or facilitate manipulation of a protein for assay or production,
among other things. As
generally is the case in situ, the additional amino acids may be processed
away from the mature
protein by cellular enzymes.
As mentioned above, the isolated nucleic acid molecules include, but are not
limited to, the
sequence encoding the GPCR peptide alone, the sequence encoding the mature
peptide and
additional coding sequences, such as a leader or secretory sequence (e.g., a
pre-pro or pro-protein
sequence), the sequence encoding the mature peptide, with or without the
additional coding
?5 sequences, plus additional non-coding sequences, far example introns and
non-coding 5' and 3'
sequences such as transcribed but non-translated sequences that play a role in
transcription, mRNA
processing (including splicing and polyadenylation signals), ribosome binding
and stability of
mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence
encoding, for
Example, a peptide that facilitates purification _
Isolated nucleic acid molecules can be in the Form of RNA, such as mRNA, or in
the form
DNA, including cDNA and genomic DNA obtained by cloning or produced by
chemical synthetic
techniqu es or by a combination thereof The nucleic acid, especially DNA, can
be double-stranded
or single-stranded. Single-stranded nucleic acid can be the codin~~ strand
(sense strand) or the non-
=I
CA 02425897 2003-04-23
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coding strand (anti-sense strand).
The invention fuI-ther provides nucleic acid molecules that encode fragments
of the peptides
of the present invention as well as nucleic acid molecules that encodE obvious
variants of the GI'CR
proteins of the present invention that are described above. Such nucleic acid
molecules may be
naturally occul-ring, such as allelic variants (same locus), paralogs
(different locus), and orthologs
(different oI°ganism), ar may be constructed by recombinant DNA methods
or by chemical
synthesis. Such non-naturally occurring variants may be made by mutagenesis
techniques,
including those applied to nucleic acid molecules, cells, ar organisms.
Accordingly, as discussed
above, the variants can contain nucleotide substitutions, deletions,
inversions and insertions.
Variation can occur in either or both the coding and non-coding regions. The
variations can
produce both conservative and non-conservative amino acid substitutions.
The present invention further provides non-coding fragments of the nucleic
acid molecules
provided in Figures 1 and 3. Preferred non-coding fragments include, but are
not limited to,
promoter sequences, enhancer sequences, gene modulating sequences and gene
termination
I S sequences. Such fragments are useful in controlling heterologous gene
expression and in
developing screens to identify gene-modulating agents. A promoter can readily
be identified as
being 5' to the ATG stal-t site in the genomic sequence provided in Figure 3,
A fragment comprises a contiguous nucleotide sequence greater than 12 or more
nucleotides. Further, a fragment could at least 30, X10, 50, 100, 2~0 or 500
nucleotides in length.
The length of the fragment will be based on its intended use. For example, the
fragment can encode
epitope bearing regions of the peptide, or can be usefid as DNA probes and
primers. Such
fragments can be isolated using the known nucleotide sequence to synthesize an
oligonucleotide
probe. A labeled probe can then be used to screen a cDNA library, genomie DNA
library, or
mRNA to isolate nucleic acid corresponding to the coding region. Further,
primers can be used in
PCR reactions to clone speciFic regions of gene.
A probelprimer typically comprises substantially a purified oligonucleotide or
oligonucleotide pair. The oligonucleotide typically comprises a region of
nucleotide sequence that
hybridizes under stringent conditions to at least about 12, 20, ?5, X10, s0 or
more consecutive
I7LlcleOtldeS.
Orthologs, homologs, and allelic variants can be identified using methods well
known in the
art. As described in the Peptide Section, these variants comprise a nucleoside
sequence encoding a
peptide that is typically 60-70°~0, 70-80°%0, 80-
90°~°0, avd more typically at least about 90-95a~"o or
more homolo4~oLls to the nucleotide seduencc shown in the Figure sheets or a
IragmEnt of this
;s
CA 02425897 2003-04-23
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sequence. Such nucleic acid molecules can readily be identified as being able
to hybridi2e under
moderate to stringent conditions, to the nucleotide sequence shown in the
Figure sheets or a
f~raglnent ol~the sequence. Allelic variants can readily be. determined by
genetic locus of"the
encoding gene. As indicated by the data presented in Figure 3, the map
position was detel-Inined to
be on clwomosorne 6 by radiation hybrid mapping.
Figure 3 provides information on a SNP variant, G 171 1 T, which has been
found in a gene
encoding the GPCR protein of the present invention. The change in the amino
acid sequence caused
by this SNP is indicated in Figure 3 and can readily be determined using the
universal genetic code
and the protein sequence provided in Figure 2 as a reference.
As used herein, the term "hybridizes under stringent conditions" is intended
to describe
conditions for hybridization and washing under which nucleotide sequences
encoding a peptide at
least 6p-70% homologous to each other typically remain hybridized to each
other. The conditions
can be such that sequences at least about 60°~'0, at least about 70%,
or at least about 80% or more
homologous to each other typically remain hybridized to each other. Such
stringent conditions are
known to those skilled in the art and can be found in Czts°renl
Protocols in Molecztlar~ Biology, John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization
conditions are
hybridization in 6~ sodium chloride/sodium citrate (SSC) at about ~fSC,
followed by one or more
washes in 0.2 X SSC, 0.1°~'o SDS at 50-65C. Examples of moderate to low
stringency hybridization
conditions are well known in the art.
Nucleic Acid Molecule Uses
The nucleic acid molecules of the present invention are useful for probes,
primers, chemical
intermediates, and in biological assays. The nucleic acid molecules are useful
as a hybridization
probe for messenger RNA, transcripUcDNA and genomic DNA to isolate full-length
cDNA and
genomic clones encoding the peptide described in Figure 2 and to isolate cDNA
and genomic
clones that correspond to variants (alleles, orthologs, etc.) producing the
same or related peptides
shown in Figure 2. As illustrated in Figure' 3, 6171 1T is a known SNP
variant.
'I'he probe can correspond to any sequence along the entire length of"the
nucleic acid
mOleCUIeS pl'OVlded 111 the Flgttres. ACCOI"dlllgly, It COtild be deI'ived
frOITl ~~ IlOnCOdIIl~ CegIOIIS, the
COdlng reg1O11, alld 3~ nOncOd111g reglOnS. I'IOW(:Vel'" aS dlSGLlSSed,
fCagIT7eI7tS are nOt t0 be COIIStI'tled
as encompassing Fragments disclosed prior to the present invention.
'I"he nucleic acid molecules arc also useful as primers For PCR to amplify any
given region
,6
CA 02425897 2003-04-23
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of a nucleic acid molecule and are useful to synthesize antisense molecules of
desired length and
sequence.
Al~he nucleic acid molecules are also useful for constructing recombinant
vectors. Such
vectors include expression vectors that express a portion of, or all ol~, the
peptide sequences.
Vectors also include insertion vectors, used to integrate into another nucleic
acid molecule
sequence, such as into the cellular genome, to alter in ,S~ilzt Expression of
a gene andlor gene product.
For example, an endogenous coding sequence can be replaced via homologous
recombination with
all or part of the coding region containing one or more specifically
introduced mutations.
The nucleic acid molecules are also useful for expressing antigenic portions
of the proteins.
The nucleic acid molecules are also useful as probes for determining the
chromosomal
positions of the nucleic acid molecules by means of in sitzt hybridization
methods. As indicated by
the data presented in Figure 3, the map position was determined to be on
chromosome 6 by
radiation hybrid mapping.
The nucleic acid molecules are also useful in making vectors containing the
gene regulatory
I 5 regions of the nucleic acid molecules of the present invention.
The nucleic acid molecules are also useful for designing ribozymes
corresponding to all, or
a part, of the mRNA produced from the nucleic acid molecules described herein.
The nucleic acid molecules are also useful for making vectors that express
part, or all, of the
peptides.
The nucleic acid molecules are also useful for constructing host cells
expressing a part, or
all, of the nucleic acid molecules and peptides.
The nucleic acid molecules are also useful for constructing transgenic animals
expressing
all, or a part, of the nucleic acid molecules and pepfides.
The nucleic acid molecules are also useful as hybridization probes for
determining the
presence, level, form and distributian ofnucleic acid expression. Experimental
data as provided in
Figure 1 indicates that GPCR proteins of the present invention are expressed
in humans in the
stomach, placenta, kidney, skeletal muscle, liver, bone marrow, and thymus.
Specifically, a virtual
northern blot shows expression in the stomach. In addition, PCR-based tissue
screening panels
indicate expression in placenta, kidney, skeletal muscle. liver, bone marrow,
and thymus tissue.
Accordingly, the probes can be used to detect the presence of, or to determine
levels o1; a specific
nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid
whose level is
determined can be DNA or RNA. Accordingly, probes corresponding to the
peptides described
herein can be used to assess expression andlor gene copy number iv a given
cell, tissue, or
~7
CA 02425897 2003-04-23
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organism. 'hh ese uses are relevant For diagnosis of disorders involving an
increase or decrease in
GPCR protein expression relative to normal results.
In uiiro techniques for detection of mRNA include Northern hybridizations and
fn ,~~itn
hybridizations. Ire ollrr~ techniques for detecting DNA include Southern
hybridizations and in .~'ilu
hybridization.
Probes can be used as a part oFa diagnostic test kit for identifying cells or
tissues that
express a GPCR protein, such as by measuring a level of a receptor-encoding
nucleic acid in a
sample of cells from a subject e.g., mRNA or genomie DNA, or determining if a
receptor gene has
been mutated. Experimental data as provided in Figure I indicates that GPCR
proteins of the
present invention are expressed in humans in the stomach, placenta, kidney,
skeletal muscle, liver,
bane marrow, and thymus. Specifically, a virtual northern blot shows
expression in the stomach. In
addition, PCR-based tissue screening panels indicate expression in placenta,
kidney, skeletal
muscle, liver, bone marrow, and thymus tissue.
Nucleic acid expression assays are useful for drug screening to identify
compounds that
modulate GPCR nucleic acid expression.
The invention thus provides a method for identifying a compound that can be
used to treat a
disorder associated with nucleic acid expression of the GPCR gene,
particularly biological and
pathological processes that are mediated by the GPCR in cells and tissues that
express it.
Experimental data as provided in Figure 1 indicates expression in humans in
the stomach, placenta,
kidney, skeletal muscle, liver, bone marrow, and thymus. The method typically
includes assaying
the ability of the compound to modulate the expression of the GPCR nucleic
acid and thus
identifying a compound that can be used to treat a disorder characterized by
undesired GPCR
nucleic acid expression. The assays can be performed in cell-based and cell-
free systems. Cell-
based assays include cells naturally expressing the GPCR nucleic acid or
recombinant cells
genetically engineered to express specific nucleic acid sequences.
The assay for GPCR nucleic acid expression can involve direct assay of nucleic
acid levels,
such as mRNA levels, or on collateral compounds involved in the signal
pathway. Further, the
expression of genes that are up- or down-regulated in response to the GPCR
protein signal pathway
can also be assayed. In this embodiment the regulatory regions of these genes
can be operably
linked to a reporter gene such as luciferase.
Thus, madulators of GPCR gene etpression can be identified in a method wherein
a cell is
contacted with a candidate compound and the expression of mRNA determined.
'The level of
expression of GPCR mRNA in the presence of the candidate compound is compared
to the level of
s
CA 02425897 2003-04-23
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Expression oFGPCR mRNA in the absence ofthe candidate compound, 'fhe candidate
compound
can then he identified as a modulator of nucleic acid expression based on this
comparison and he
used, for example to treat a disorder characterioed by aber i°ant
nucleic acid Expression. When
expression of mRNA is statistically significantly greater in the presence
oFthe candidate compound
than in its absence, the candidate compoLmd is identified as a stimulator of
nucleic acid expression.
When nucleic acid expression is statistically significantly less in the
presence of the candidate
compound than in its absence, the candidate compound is identified as an
inhibitor of nucleic acid
expression.
The invention further provides methods of treatment, with the nucleic acid as
a target, using
a compound identified through drug screening as a gene modulator to modulate
GPCR nucleic acid
expression, particularly to modulate activities within a cell or tissue that
expresses the proteins.
Experimental data as provided in Figure 1 indicates that GPCR proteins of the
present invention are
expressed in humans in the stomach, placenta, kidney, skeletal muscle, liver,
bone marrow, and
thymus. Specifically, a virtual northern blot shows expression in the stomach.
In addition, PCR-
based tissue screening panels indicate expression in placenta, kidney,
skeletal muscle, liver, bone
marrow, and thymus tissue. Modulation includes both up-regulation (i.e.
activation or agonization)
or down-regulation (suppression or antagonization) or nucleic acid expression.
Alternatively, a modulator for GPCR nucleic acid expression can be a small
molecule or
drug identified using the screening assays described herein as long as the
drug or small molecule
inhibits the GPCR nucleic acid expression in the cells and tissues that
express the protein.
Experimental data as provided in Figure 1 indicates expression in humans in
the stomach, placenta,
kidney, skeletal muscle, liver, bone marrow, and thymus.
The nucleic acid molecules are also useful for monitoring the effectiveness of
modulating
compounds on the expression or activity of the GPCR gene in clinical trials or
in a treatment
regimen. 'thus, the gene expression pattern can serve as a barometer for the
continuing
effectiveness of treatment with the compound, particularly with compounds to
which a patient can
develop resistance. The gene expression pattern can also serve as a marker
indicative of a
physiological response of the aFfected cells to the compound. Accordingly,
such monitoring would
allow either increased administration of the compound or the administration of
alternative
compounds to which the patient has not become resistant. Similarly, iFthe
level ol~nucleic acid
expression Falls below a clesirable level, administration of the compound
could be commensurately
decreased.
'Fhe nucleic acid molecules are also useCl~l in diaf~nostic assays Ior
qualitative: changes in
CA 02425897 2003-04-23
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GPCR nucleic acid, and particularly in qualitative changes that lead to
pathology. The nucleic acid
molecules can be used to detect mutations in GPCR genes and gen a expression
products such as
mRNA. The nucleic acid molecules can be used as hybridization probes to detect
naturally-
occurring genetic mutation s in the GPCR gone and thereby to determine whether
a subject with the
mutation is at risk for a disorder caused by the mutation. Mutations include
deletion, addition, or
substitution of one or more nucleotides in the gone, chromosomal rean-
angement, such as inversion
or transposition, modification of genomic DNA, such as aberrant methylation
patterns or changes in
gone copy number, such as amplification. Detection of a mutated form of the
GPGR gone
associated with a dysfunction provides a diagnostic tool for an active disease
or susceptibility to
disease when the disease results from overexpression, underexpression, or
altered expression of a
GPCR protein.
Individuals carrying mutations in the GPCR gene can be detected at the nucleic
acid level by
a variety of techniques. Figure 3 provides information on a SNP variant, G
1711 T, which has been
found in a gene encoding the GPCR protein of the present invention. The change
in the amino acid
sequence caused by this SNP is indicated in Figure 3 and can readily be
determined using the
universal genetic code and the protein sequence provided in Figure 2 as a
reference. As indicated
by the data presented in Figure 3, the map position was determined to be on
chromosome 6 by
radiation hybrid mapping. Genomic DNA can be analyzed directly or can be
amplified by using
PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses,
detection of the
mutation involves the use of a probe/primer in a polymerase chain reaction
(PCR) (see, e.g. U.S.
Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,
alternatively, in a
ligation chain reaction (LCR) (see, e.g., Landegran el nl., Scierzce 2-
11:10'77-1080 (1988); and
Nakazawa et al., PIVAS 91:360-364 (1994)), the latter of which can be
particularly useful far
detecting point mutations in the gene (see Abravaya et girl., Nzxcleic.~cic~s
Res~ 23:675-682 ( I 995)).
This method can include the steps of collecting a sample of cells from a
patient, isolating nucleic
acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting
the nucleic acid sample
with one or mare primers which speciFcally hybridize to a gene under
conditions such that
hybridization and amplification of the gene (if present) occurs, and detecting
the presence or
absence of an amplification product, or detecting the size of the
amplification product and
comparing the length to a control sample, Deletions and insertions can be
detected by a change in
sine ofthe amplified product compared to the narnlal genotype. faint mutations
can be identifed
toy hybridizing amplified DNA to normal RNA or antisense DNA sequences.
Alternatively, mutations in a GPC'R gene can be directly iclentificd, for
example, by
CA 02425897 2003-04-23
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alterations in restriction enzyrtne digestion patterns determined by gel
electrophoresis.
Further, sequence-specihic ribozymes ~U.S. Patent No. 5,198,531 ) can be used
to score for
tho presence of specific mutations by development or loss of a ribozyme
cleavage site. Perfectly
matched sequences can be distinguished from mismatched sequences by nuclease
cleavage
digestion assays or by differences in melting temperature.
Sequence changes at specific locations can also be assessed by nuclease
protection assays
such as RNase and S I protection or the chemical cleavage method. Furthermore,
sequence
differences between a mutant GPCR gene and a wild-type gene can be determined
by direct DNA
sequencing. A variety of automated sequencing procedures can be utilized when
performing the
diagnostic assays (Naeve, C.W., (1995) BiotechzZigzres 79:418), including
sequencing by mass
spectrometry (see, e.g., PCT International Publication No. WO 91/16101; Cohen
et al., .~dv.
C"hroznatogr. 36:127-162 ( 1996); and Griffin et al.,,~lppl. BioclZem.
Biol~chzzol. 38:17-159 (1993)).
Other methods for detecting mutations in the gene include methods in which
protection
from cleavage agents is used to detect mismatched bases in RNAIRNA or RNAIDNA
duplexes
I S (Myers et al., Sciezzce 23a:12~12 (1985)); Cotton et czl., PNAS 8~:~1397
(1988); Saleeba et al., Meth.
Erzymol. 217:286-295 ( 1992)), electrophoretic mobility of mutant and wild
type nucleic acid is
compared (Orita et al., Plf~l~S' c~6:2766 (1989); Cotton et al., MZttal.
Rc,S~. 285:125-1 ~I~l ( 1993); and
Hayashi e1 al., Genel. anal, Tech. ~lppl. 9:73-79 (1992)), and movement of
mutant or wild-type
fragments in polyacrylamide gels containing a gradient of denaturant is
assayed using denaturing
gradient gel electrophoresis (Myers el al., Natazre 313:95 (1985)). Examples
of other techniques
for detecting point mutations include selective oligonucleotide hybridization,
selective
amplif canon, and selective primer extension.
The nucleic acid molecules are also useful for testing an individual for a
genotype that while
not necessarily causing the disease, nevertheless affects the treatment
modality. Thus, the nucleic
acid molecules can be used to study the relationship between an individual's
genotype and the
individual's response to a compound used for treatment {pharmacogenomic
relationship).
Accordingly, the nucleic acid molecules described herein can be used to assess
the mutation content
of the GPCR gene in an individual in order to select an appropriate compound
or dosage regimen
for treatment. As illustrated in Figure 3, 0,171 1T is a known SNP variant.
Thu s nucleic acid molecules displaying genetic variations that affect
treatment provide a
diagnostic target that can be used to tailor treatment in an individual.
Accordingly, the production
of recombinant cells and animals containing these polymorphisms allow
effective clinical design of
treatment compounds and dosage re«imens.
~l
CA 02425897 2003-04-23
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The nucleic acid molecules are thus useful as an tiscnse constructs to control
GP CR gene
expression in cells, tissues, and organisms. A DNA antisen se nucleic acid
molecule is designed to
be complementary to a region oFthe gene involved in transcription, preventing
transcription and
hence production of GPCR protein. An antisense RNA or DNA nucleic acid
molecule would
hybridize to the mRNA and thus block translation of mRNA into GPCR protein.
Alternatively, a class of a~~tisense molecules can be used to inactivate mRNA
in order to
decrease expression of GPCR nucleic acid. Accordingly, these molecules can
treat a disorder
characterized by abnormal or undesired GPCR nucleic acid expression. This
technique involves
cleavage by means of ribozymes containing nucleotide sequences complementary
to one or more
regions in the mRNA that attenuate the ability of the mRNA to be translated.
Possible regions
include coding regions and particularly coding regions corresponding to the
catalytic and other
functional activities of the GPCR protein, such as ligand binding.
The nucleic acid molecules also provide vectors For gene therapy in patients
containing cells
that are aberrant in GPCR gene expression. Thus, recombinant cells, which
include the patient's
cells that have been engineered ex vivo and returned to the patient, are
introduced into an individual
where the cells produce the desired GPCR protein to treat the individual.
The invention also encompasses kits ~or detecting the presence of a GPCR
nucleic acid in a
biological sample. Experimental data as provided in Figure 1 indicates that
GPCR proteins of the
present invention are expressed in humans in the stomach, placenta, kidney,
skeletal muscle, liver,
bone marrow, and thymus. Specifically, a virtual northern blot shows
expression in the stomach, In
addition, PCR-based tissue screening panels indicate expression in placenta,
kidney, skeletal
muscle, liver, bone marrow, and thymus tissue. For example, the kit can
comprise reagents such as
a labeled or labelable nucleic acid or agent capable of detecting GPCR nucleic
acid in a biological
sampled means for determining the amount of GPCR nucleic acid in the sample;
and means For
comparing the amount oFGPCR nucleic acid in the sample with a standard. The
compound or
agent can be packaged in a suitable container. The kit can Further comprise
instructions For using
the kit to detect GPCR protein mRNA or DNA.
Nucleic Acid Arrays
l~he present invention Further provides nucleic acid detection kits, such as
arrays or
microarrays of nucleic acid molecules that are based on the sequence
inFormation provided in
higures 1 and 3 (SEQ ID NUS:I and i j~
CA 02425897 2003-04-23
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As used herein "Arrays' or "Microarrays" refers to an array oFdistinet
polynucleotides or
oligonucleotides synthesized on a substrate, such as paper. nylon or other
type oFmembrane,
Intel', Glllp, glaSS Slide, Or any Othel' SLlltable SOlICi SLIpp01't. Ill 017e
e171bOd1111ent, the 1711C1'Oal'1'ay IS
prepared an~i used according to the methods described in U'S Patent 5,837,832,
Chee et al., fC"F
application W09511 1905 (Chee et al.), Lockhart, D. .l. et al. ( 1996; Nat.
Biotech. l~: I675-1680)
and Schena, M. et al. (1996; Nroc. Natl. Acad. Sci. 93: 1061-10619), all of
which are
incorporated herein in their entirety by reference. In other embodiments, such
arrays are
produced by the methods described by Brown et. al., US Patent N'o. 5,807,522.
The microarray or detection kit is preferably composed of a large number of
unique,
single-stranded nucleic acid sequences, usually either synthetic antisense
oligonucleotides or
fragments of cDNAs, fixed to a solid support. The oligonucleotides are
preferably about 6-60
nucleotides in length, more preferably 15-30 nucleotides in length, and most
preFerably about 20-
25 nucleotides in length. Far a certain type of microarray or detection kit,
it may be preferable to
use oligonucleotides that are only 7-20 nucleotides in length. The microarray
or detection kit
may contain oligonucleotides that cover the known 5', or 3', sequence,
sequential
oligonucleotides which cover the full length sequence; or unique
oligonucleotides selected from
particular areas along the length of the sequence. Polynucleotides used in the
microarray or
detection kit may be oligonucleotides that are specific to a gene or genes of
interest.
In order to produce oligonueleotides to a known sequence for a microarray or
detection
kit, the genes) of interest (or an ORF identified from the contigs of the
present invention) is
typically exan7ined using a computer algorithm which starts at the 5' or at
the 3' end of the
nucleotide sequence. Typical algorithms will then identify aligomers of
defined length that are
unique to the gene, have a GC content within a range suitable for
hybridization, and lack
predicted secondary structure that may interfere with hybridisation. In
certain situations it may
be appropriate to use pairs oFoligonucleotides on a microarray or detection
kit, The "pairs" will
be identical, except for one nucleotide that preferably is located in the
center of the sequence,
The second oligonucleotide in the pair (mismatched by one) serves as a
control. The number of
oligonucleotide pairs may range from two to one million. The oligomers are
synthesized at
designated areas on a substrate using a light-directed chemical process. The
substrate may be
paper, nylon or other type oFmembrane, Filter, chip. glass slide or any other
suitable solid
support.
In another aspect. an oligonucleotide may be synthesized on the surface ol~
the substrate
by using a chemical coupling procedure and an inkaet application apparatus, as
described in PC'h
3
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
application W095/251 1 16 (Baldescllweiler et al.) which is incorporated
herein in its entirety by
reference. In another aspect, a "gridded" array analogous to a dot (or slot)
blot may be used to
arrange and linl. cDNA fragments or oligonucleotides to the surlace ofa
substrate using a
vacuum system, thermal, UV, mechanical or chemical bonding procedurta. An
array, such as
those described above, may be produced by hand or by using available devices
slot blot or dot
blot apparatus), materials (any suitable solid support), and machines
(including robotic
instruments), and may contain 8, 24, 96, 381, 1536, 6141 or more
oligonucleotides, or any other
number between two and one million which lends itself to the eCFicient use of
commercially
available instrumentation.
In order to conduct sample analysis using a mieroarray or detection kit, the
RNA or DNA
from a biological sample is made into hybridization probes. The mRNA is
isolated, and eDNA is
produced and used as a template to make antisense RNA (aRNA). The aRNA is
amplified in the
presence of fluorescent nucleotides, and labeled probes are incubated with the
microarray or
detection kit so that the probe sequences hybridize to complementary
oligonucleotides of the
mieroarray or detection kit. Incubation conditions are adjusted so that
hybridization occurs with
precise complementary matches or with various degrees of less complementarity.
After removal
of nonhybridized probes, a scanner is used to determine the levels and
patterns of fluorescence.
The scanned images are examined to determine degree of complementarity and the
relative
abundance of each oligonucleotide sequence on the microarray or detection kit.
The biological
samples may be obtained from any bodily fluids (such as blood, urine, saliva,
phlegm, gastric
juices, etc.), cultured cells, biopsies, or other tissue preparations. A
detection system may be
used to measure the absence, presence, and amount of hybridization for all of
the distinct
sequences simultaneously. This data may be used for large scale correlation
studies on the
sequences, expression patterns, mutations, variants, or polymorphisms among
samples.
Using such arrays, the present invention provides methods to identify the
expression of
the GPCR proteins/peptides of the present invention. In detail, such methods
comprise
incubating a test sample with one or more nucleic acid molecules and assaying
for binding of the
nucleic acid molecule with components within the test sample. Such assays will
typically
involve arrays comprising many genes, at least one ofwhich is a gene of the
present invention
and or alleles of the GPCR gene of the present invention. H figure 3 provides
information on a
SNP variant, G 171 l~I~, which has been found in a gent' encoding the GPCR
protein of the present
invention. 'I°he change in the amino acid sequence caused by this SNP
is indicated in Figure 3
anti can readily be determined using the universal genetic code and the
protein sequence
~l~l
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
provided in higure ? ~ts a reference.
conditions Cor incubating a nucleic acid molecule with a test sample vary.
Incubation
conditions depend on the Format employed in the assay, the detection methods
employed, and the
type and nature ol'the nucleic acid molecule used in the assay, One skilled in
the ~trt will
recognize that any one of the commonly available hybridization, amplification
ar array assay
formats can readily be adapted to employ the novel fragments of the 1-Iuman
genome disclosed
herein. Examples of such assays can be found in shard, T> ~ln Introdz~r~lion
to
Radioitnmztzzoas,scrv rrr~d Related Techniqztes, Elsevier Science Publishers,
Amsterdam, The
Netherlands ( 1986); Bullock, G. R. et al., I'echniqztes in
Imtzzunoc~lochemi,slz_la, Academic
Press, Orlando, FL Vol. 1 (1 982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P.,
Pz°aclice czvtd
Theozy of'Enzynae Inamztnoassays~ Lal~onalo~y Techniques itz Biochenaistjy
crud tLlolecztlar
Biology, Elsevier Science Publishers, Amsterdam, The Netherlands ( 1985).
The test samples of the present invention include cells, protein or membrane
extracts of
cells. The test sample used in the above-described method will vary based on
the assay format,
nature of the detection method and the tissues, cells or extracts used as the
sample to be assayed.
Methods for preparing nucleic acid extracts or of cells are well known in the
art and can be
readily be adapted in order to obtain a sample that is compatible with the
system utilized.
In another embodiment of the present invention, kits are provided which
contain the
necessary reagents to carry out the assays of the present invention.
SpeciFcally, the invention provides a compartmentalized kit to receive, in
close
confnement, one or more containers which comprises: (a) a first container
comprising one of the
nucleic acid molecules that can bind to a fragment of the Human genome
disclosed herein; and
(b) one or more other containers comprising one or more of the following: wash
reagents,
reagents capable of detecting presence of a bound nucleic acid.
2S In detail. a compartmentalized kit includes any kit in which reagents are
contained in
separate containers. Such containers include small glass containers, plastic
containers, strips of
plastic, glass or paper, or arraying material such as silica. Such containers
allows one to
efFtciently transfer reagents from one compartment to another compartment such
that the
samples and reagents are not cross-contaminated, and the agents or solutions
of each container
can be added in a quantitative fashion from one compartment to another. Such
containers will
include a container which will accept the test sample, a container which
contains the nucleic acid
probe. containers which contain wash reagents (such as phosphate buffered
saline, ~I'ris-buffers,
etc.j, and containers which contain the reagents used to detect the bound
probe. Une skilled in
=lj
CA 02425897 2003-04-23
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the art will readily recognize that the previously unidentified GPCR genes of
the present
invention can be routinely identitied using the sequence information disclosed
herein can be
readily incorporated into one of the established kit formats which are well
known in the art,
particularly expression arrays.
Vectorslhost cells
The invention also provides vectors containing the nucleic acid molecules
described herein.
The term "vector" refers to a vehicle, preferably a nucleic acid molecule,
which can transport the
nucleic acid molecules. When the vector is a nucleic acid molecule, the
nucleic acid molecules are
covalently linked to the vector nucleic acid. With this aspect of the
invention, the vector includes a
plasmid, single or double stranded phage, a single or double stranded RNA or
DNA viral vector. or
artificial chromosome, such as a BAC, PAC, YAC, OR MAC.
A vector can be maintained in the host cell as an extrachromosomal element
where it
replicates and produces additional copies of the nucleic acid molecules.
Alternatively, the vector
may integrate into the host cell genome and produce additional copies of the
nucleic acid molecules
when the host cell replicates.
The invention provides vectors for the maintenance (cloning vectors) or
vectors for
expression (expression vectors) ofthe nucleic acid molecules. The vectors can
function in
procaryotic or eulcaryotic cells or in both (shuttle vectors).
Expression vectors contain cis-acting regulatory regions that are operably
linked in the
vector to the nucleic acid molecules such that transcription of the nucleic
acid molecules is allowed
in a host cell. The nucleic acid molecules can be introduced into the host
cell with a separate
nucleic acid molecule capable of affecting transcription. Thus, the second
nucleic acid molecule
may provide a trans-acting factor interacting with the cis-regulatory control
region to allow
transcription of the nucleic acid molecules from the vector. Alternatively, a
trans-acting factor may
be supplied by the host cell. Finally, a trans-acting factor can be produced
from the vector itself. It
is understood, however, that in some embodiments, transcription and/or
translation of the nucleic
acid molecules can occur in a cell-free system.
The regulatory sequence to which the nucleic acid molecules described herein
can be
operably linked include promoters for directing mRNA transcription. These
include, but are not
limited to. the left promoter from bacteriophage 7~, ih a lac, TRP, and TAC
promoters from l;. c~~~ll,
the early and late promoters from SV~10, the CMV iwmediate early promoter, the
ndeno virus early
-I(>
CA 02425897 2003-04-23
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and late promoters, and retrovirus long-terminal repeats.
In addition to control regions that promote transcription, expression vectors
may also
include regions that modulate transcription. such as repressor binding sites
and enhancers.
Examples include the SV~IO enhances, the cytomegalovirus immediate early
enhances, polyoma
enhances, adenovirus enhancers, and retrovirus LTR enhanoers.
In addition to containing sites Far transcription initiation and control,
expression vectors can
also contain sequences necessary for transcription termination and, in the
transcribed region a
ribosome binding site for translation. Other regulatory control elements For
expression include
initiation and termination colons as well as polyadenylation signals. The
person of ordinary skill in
the art would be aware of the numerous regulatory sequences that are useful in
expression vectors.
Such regulatory sequences are described, for example, in Sambrook el al.,
Molecatlaz~ Cloz2iz7g: ,~
Labouatoz~ml~lcrzzztal. Zna', el., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY,
(1989).
A variety of expression vectors can be used to express a nucleic acid
molecule. Such
vectors include chromosomal, episomal, and virus-derived vectors, for example
vectors derived
from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast
chromosomal
elements, including yeast artificial chromosomes. from viruses such as
baculoviruses,
papovavin.tses such as SV40, Vaccinia viruses, adenoviruses, poxvimses,
pseudorabies viruses, and
retroviruses. Vectors may also be derived from combinations of these sources
such as those derived
from plasmid and bacteriophage genetic elements, eg. cosmids and phagemids.
Appropriate
cloning and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et
czl., tl~lolecazlar Clonizzg: ~I Laboz~atozy ~Iflcznz~czl. ?zzd. el., Cold
Spring Harbor Laboratory Press, Gold
Spring Harbor, NY, ( I 989).
The regulatory sequence may provide constitutive expression in one or more
host cells (i.e.
tissue specific) or may provide for inducible expression in one or more cell
types such as by
temperature, nutrient additive, or exogenous factor such as a hormone or other
Iigand. A variety of
vectors providing for constitutive and inducible expression in prokaryotic and
eukatyotic hosts are
well known to those of ordinary skill in the art.
The nucleic acid molecules can be inserted into the vector nucleic acid by
well-known
methodology. Generally, the DNA sequence that will ultimately be expressed is
joined to an
expression vector by cleaving the DNA sequence and the expression vector with
one or more
restriction enzymes and then ligating the fragments together. Procedures for
restriction enzyme
di~,estion and ligation are well known to those of~ordinary skill in the art.
~17
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The vector containing the appropriate nucleic acid molecule can be introduced
into an
appropriate host cell Cor propagation or expression using well-known
techniques. Bacterial ells
include, but are not limited to, E. coli, ~S'lrc~hlona~~oc~.v, and
,~'ulmor~~lla llaphimurimn. I=,ukasyo tic cells
include. but are not limited to, yeast, insect cells such as l~rr~.~~ohl7ilcr,
animal culls such as COS and
CI-10 cells, and plant cells.
As described herein, it may be desirable to express the peptide as a fusion
protein.
Accordingly, the invention provides Fusion vectors that allow for the
producfion of the peptides.
Fusion vectors can increase the expression of a recombinant protein, increase
the solubility of the
recombinant protein, and aid in the purification of the protein by acting for
example as a ligand for
affinity purification. A proteolytic cleavage site may be introduced at the
junction ofthe fusion
moiety so that the desired peptide can ultimately be separated from the fusion
moiety. Proteolytic
enzymes include, but are not limited to, factor Xa, tl-trombin, and
enterokinase. Typical fusion
expression vectors include pGEX (Smith et czl., Gerac~ 67:31-40 (1988)), pMAL
(New England
Biolabs, Beverly, MA) and pRITS (Phatmacia, Piscataway, N.1) which fssse
glutathione S-
transferase (GST), maltose E binding protein, or protein A, respectively, to
the target recombinant
protein. Examples of suitable inducible non-fission E. coli expression vectors
include pTrc (Amann
e! al., GeraL 6:301-315 (19$8)) and pET 1 Id (Studier et al., Genre
Expressiorz Technology; MeClZOCIs
if2 E>?zynzology 18.5:60-89 ( 1990)).
Recombinant protein expression can be maximized in a host bacteria by
providing a genetic
background wherein the host cell has an impaired capacity to proteolytically
cleave the recombinant
protein. (Gottesman, S., Gene Expres.~ion Technolog~~= ~l~lelhocls in
EnzJmzolo~~ 185, Academic
Press, San Diego, California (1990) 1 19-128). Alternatively, the sequence of
the nucleic acid
molecule of interest can be altered to provide preferential codon usage for a
specific host cell, for
example E. ioli. (Wada et crl., Nztclei~.~lcicls Rr~s. X0;2111-.2118 (1992)).
The nucleic acid molecules can alsa be expressed by expression vectors that
are operative in
yeast. Examples of vectors far expression in yeast e.g., ~.S'. oer~eui,~~iae
include pYepSecl (Baldari, et
crl., E~'IrIB(),1 6:229-23d (1987)), pMFa (Kurjan e! crl., Cell 3l):933-
93(1982)), pJRY88 (Scht,sltz ei
al., Ger~c 5-l:1 13-123 ( 1987)), and pYES2 (Invitrogen Cot~aoration, San
Diego, CA).
The nucleic acid molecules can also be expressed in insect cells using, for
example,
baculovirus expression vectors. Baculovirus vectors availahle for expression
of proteins in cultured
insect cells (e.g., Sf 9 cells) include the pAc series (Smith r.?l rrl., ~
lal. C'i.~ll Biol. 3;21 ~6-? 1 d5
(1983)) and the pVI. series (Irucklowcalrrl" ln~r~lpv I ~C1:31-39 X19$9)).
In certain embodiments ofthe invention, the nuclefc acid molecules described
herein are
-18
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
expreSSed 111 Illilllllllallall Ce115 LIS111g 111a1171Tlallan expl'e5S1o11
veCtOT'S. I;XanlpleS of'lTIamI7lallal7
expression vectors include pCDM$ Geed. B. JVolan'e 329:84( 1987)) and
plvl'I"?PC (Kaufman t~~l crl..
~~113(),I. x;187-l~)5 (1987)).
ThL: etpression vectors listed herein are provided by way of example only
ofthe well-
known vectors Available to those of ordinary skill in the art that would be
useful to express the
nucleic acid molecules. '1"he person of ordinary skill in the art would be
aware ofother vectors
suitable for maintenance propagation or expression of the nucleic acid
molecules described herein.
These are found for example in San'lbrook, J., Fritsh, E. F., and Maniatis, T.
Moleczzlar Cloning; A
Laboratory thlcrnztcrl. 2nd, eo', Cold Spring harbor Laborafozy, Cold Spring
Harbor Laboratory
Press, Cold Spring Harbor, NY, 1989.
The invention also encompasses vectors in which the nucleic acid sequences
described
herein are cloned into the vector in reverse orientation, but operably linked
to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense transcript can
be produced to all, or
to a portion, of the nucleic acid molecule sequences described herein,
including bath coding and
non-coding regions. Expression of this antisense RNA is subject to each of the
parameters
described above in relation to expression of fhe sense RNA (regulatory
sequences, constitutive or
indueible expression, tissue-specific expression).
The invention also relates to recombinant host cells containing the vectors
described herein.
Host cells therefore include prokaryotic cells, lower eukaryotic cells such as
yeast, other eukaryotic
cells such as insect cells, and higher eukaryotic cells such as mammalian
cells.
The recombinant host cells are prepared by introducing the vector constructs
described
herein into the cells by techniques readily available to the person afordinary
skill in the art. These
include, but are not limited to, calcium phosphate transfection, DEAF-dextran-
mediated
transfection, cationic lipid-mediated transfection, electroporation,
transduction, infection,
lipofection, and other techniques such as those found in Sambrook, ~l al.
(Molectzlcrr Cloning= ~
hczborcrloz~y t~lczzmcrl. Zntl, rcl , Cold Spz'ing Hcrz~boz~ l~crborcrtoz~~,
Cold Spring I Iarbor Laboratory
Press, Gold Spring I-larbor, NY, 1989.
I-lost cells can contain more than one vector. Thus, different nucleotide
sequences can be
introduced on different vectors ofthe same cell. Similarly, the Nucleic acid
molecules can be
introduced either alone or with other nucleic acid molecules that are not
related to the nucleic acid
molecules such as those providing traps-acting factors for expression vectors.
When more than one
vector is introduced into a cell, the vectors can be introduced independently,
co-introduced or joined
to the nucleic acid molecule vector.
:-l~)
CA 02425897 2003-04-23
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In the case of bacteriophage and viral vectors. these can be introduced into
cells as packaged
or encapsulated virus by Standard procedures for infection and transduction.
Viral vectors can be
replication-competent or replication-defective. In the: case in which viral
replication is defective,
repllcatlon Wlll L>CCllr 111 110St Ge115 pI~OVld111g 111I1Ct1()ll5 that
Cplllplelnellt the deleCIS.
Vectors generally include selectable markers that enable the selection of the
subpopulation
of cells that contain the recombinant vector constructs. The marker can be
contained in the same
vector that contains the nucleic acid molecules described herein or may be on
a separate vector.
Markers include tetracycline or ampicillin-resistance genes for prokaryotic
host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host cells.
However, any marker that
provides selection for a phenotypic trait will be effective.
While the mature proteins can be produced in bacteria, yeast, mammalian cells,
and other
cells under the control of the appropriate regulatory sequences, cell- free
transcription and
translation systems can also be used to produce these proteins using RNA
derived from the DNA
constructs described herein.
Where secretion ofthe peptide is desired, which is difficult to achieve with
multi-
transmembrane domain containing proteins such as GPCRs, appropriate secretion
signals are
incorporated into the vector. The signal sequence can be endogenous to the
peptides or
heterologous to these peptides.
Where the peptide is not secreted into the medium, which is typically the case
with GPGRs,
the protein can be isolated from the host cell by standard disruption
procedures, including freeze
thaw, sonication, mechanical disruption, use of lysing agents and the like.
The peptide can then be
recovered and purified by well-known purification methods including ammonium
sulfate
precipitation, acid extraction, anion or cationic exchange chromatography,
phosphocellulose
chromatography, hydrophobic-interaction chromatography, affinity
chromatography,
hydroxylapatite chromatography, lectin chromatography> or high performance
liquid
chromatography.
It is also understood that depending upon the host cell in recombinant
production of the
peptides described herein. the peptides can have various glycosylation
patterns, depending upon the
cell, or maybe non-glycosylated as when produced in bacteria. In addition, the
peptides may
include an initial modified methionine in some cases as a result of a host-
mediated process.
Uses of vectors and host cells
SO
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
fhe recombinant host calls expressing the peptides described herein have a
variety ofrrses_
I~irst, the cells are useful For producing a GPCR protein or peptide that can
be l-urther purit3ed to
produce desired amounts of GP CR protein or (i~agments. "I~hus, host cells
containing expression
vectors are usefr,tl far- peptide pr~oductiot~.
Host cells are also useful for conducting cell-based assays involving the GPCR
protein or
GPCR protein fragments, such as those described above as well as other formats
known in the ar-t.
Thus, a recombinant host cell expressing a native GPCR protein is useful for
assaying compounds
that stimulate or inhibit GPCR protein function.
Host cells are also useful for identifying GPCR protein mutants in which these
functions are
affected. If the mutants naturally occur and give rise to a pathology, host
cells containing the
mutations are useful to assay compounds that have a desired effect an the
mutant GPCR protein (for
example, stimulating or inhibiting function) which may not be indicated by
their effect on the native
GPCR protein.
Genetically engineered host cells can be further used to produce non-human
transgenic
animals. A transgenic animal is preferably a mammal, for example a rodent,
such as a rat or mouse,
in which one or more of the cells of the animal include a transgene. A
transgene is exogenous DNA
which is integrated into the genome of a cell from which a transgenic animal
develops and which
remains in the genome of the mature animal in one or more cell types or
tissues of the transgenic
animal. These animals are useful for studying the function of a GPCR protein
and identifying and
evaluating modulators of GPCR protein activity. Other examples of transgenic
animals include
non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.
A transgenic animal can be produced by introducing nucleic acid into the male
pronuclei of
a fertilized oocyte, e.g., by microinjection, retroviral infection, and
allowing the oocyte to develop
in a pseudopregnant female foster animal. Any of the GPCR protein nucleotide
sequences can be
introduced as a transgene into the genome of a non-human animal, such as a
mouse.
Any of the regulatory or other sequences useful in expression vectors can form
part of the
transgenic sequence. This includes intronic sequences and polyadenylation
signals, if not already
included. A tissue-specific regulatory sequences) can 6e operably linked to
the transgene to direct
expression of the GPCR protein to particular cells.
Methods for generating transgenic animals via embryo manipulation and
microinjection,
particularly animals such as mice, have become conventional in the art and are
described, for
example. in U~.S. Patent N'os. ~1,73G.8G6 and X1,870,009, both by Leder c1
crl., (.I'.S. Patent No.
X1,873,191 by Wagner cl ctl. and in I-Iogan, f3_. ~'I~Itrrliprtlcrtiry~l the
ltlott.5r Fntbruu, (Cold spring
~l
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
Harbor hahoratory Press, Cold Spring I-larbor, N.Y.. 1986). Similar methods
are used for
production of other transgenic animals, A transgenic lounder animal can be
identified based upon
the presence of the transgene in its gen ome and/or expression of transgenic
mRNA in tissues or
cells of the animals. A transgenic Founder animal can then be used to breed
additional animals
carrying the transgenc. Moreover, transgenic animals carrying a transgene can
further be bred to
ocher transgenic animals carrying other transgenes. A transgenic animal also
includes animals in
which the entire animal or tissues in the animal have been produced using the
homologously
recombinant host cells described herein.
In another embodiment, transgenic non-human animals can be produced which
contain
selected systems that allow for regulated expression of the transgene. One
example of such a
system is the orvlloxP recombinase system ofbacteriophage PI. For a
description ofthe cf~elloxP
recombinase system, see, e.g., Lakso et al. PN~1S $9;6232-6236 ( 1992).
Another example of a
recombinase system is the FLP recombinase system of S, cerevisiae (O'Gorman et
al. Science
21:1351-1355 (1991). If a crelloxP recombinase system is used to regulate
Expression of the
transgene, animals containing transgenes encoding both the C're recombinase
and a selected protein
is required. Such animals can be provided through the consttltction of
"double" transgenic animals,
e.g., by mating two transgenic animals, one containing a transgene encoding a
selected protein and
the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced
according to the methods described in Wilmut, I. e1 al. lVatzn~e 385:810-813
(1997) and PCT
International Publication Nos. WO 97/07668 and WO 97107669. In brief, a cell,
e.g., a somatic cell,
from the transgenic animal can be isolated and induced to exit the growth
cycle and enter G~ phase.
The quiescent cell can then be fused, e.g., tlwough the use of electrical
pulses, to an enucleated
oocyte from an animal of the same species from which the quiescent cell is
isolated. The
reconstructed oocyte is then cultured such that it develops to morula or
blastocyst and then
transferred to pseudopregnant female foster animal. The offspring born of this
female foster animal
will be a clone of the animal from which the cell, e.g.. the somatic cell, is
isolated.
Transgenic animals containing recombinant cells that express the peptides
described herein
are useful to conduct the assays described herein in an ire uiuo context.
Accordingly, the various
physiological factors that are present irmivo and that could effect ligand
binding, GPCR protein
activation, and signal transduction, may not be evident from in vulro cell-Ii-
ee or cell-based assays.
Accordingly, it is useful to provide non-human tt°ans;~t~nic animals to
assay irr uiv>U GPCR protein
function, including ligand interaction, the effect oi~spocific mutant GfCR
proteins on GPCR protein
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
Function and ligand interaction. and the effect of chimcric ~iPCR proteins. It
is also possible to
assess the ~I~fect of mill mutations. that is mutations that substantially or
completely eliminate one or
more Cif'C'R protein functions.
All publications and patents mentioned in the above specification are herein
incorporated
by reference. Various modifications and variations of the described method and
system of the
invention will be apparent to those skilled in the art without departing From
the scope and spirit
of the invention. Although the invention has been described in connection with
specil"ic
preferred embodiments, it should be understood that tl~e invention as claimed
should not be
unduly limited to such specific embodiments. Indeed, various modifications of
the above-
described modes for carrying out the invention which are obvious to those
skilled in the field of
molecular biology or related fields are intended to be within the scope of the
Following claims.
t;
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
~~F~~~~;g~Tr~I~ I.~~'~"I"ItP~
I I d~ ~ L-I:I ~'UIZI?C>F~A'Y' FC~N' f t~J't ~
=wl.a~~~ I~;~I"11"G'I;IJ Hf_1MJ1E~7~ C~~I~'RC~"I'~aLN ~"C~t~I'LAFaI~
F~IV~I:p~~"I'll-~.~, Nn.~~~I~I:I~~' AC"1L~ Mt~I~I~;CtJ~ZE~ EpICQI7lrPt~C
E~~.JMAPJ ~;E~~'I?
b''f?C~"1'I:~rLN~F?, ANt? U~~S '~"FI~RFaE3F
<13a> ~'Laaa899F~CT
<llla> '~~ T3I:~ ASSTGNED
<141> 2aa1-la-24
<15a> a~/~g~,8~1
<15~.> 2a0a-1a-24
<1~0> x91781,559
<1~7~> 2001-13-a~
<160> 6
<17a> FastSEQ ~o~ Wzndaws Vers9.on 4.a
<21a> 1
<211> 2082
<212> I7N'A
<~G1~> I-~IkIT~ail
<9aa> 1
taaataacag cgttaatgag cagcaattca tccctgctgg tggctgtgea gctgtgctae 6a
gcgaacgtga atgggtcctg tgtgaaaatc cccttctcgc cgggatccrg ggtgattctg 22a
tacatagtgt ttggctttgg ggctgtgctg gctgtgtttg gaaacctcct ggtgatgatt 18a
tcaatcctcc atttcaagca gctgcactct ccgaccaatt ttctcgttgc etctctggcc 24a
tgcgctgatt tcttggtggg tgtgactgtg atgcccttca gcatggtcag gacggtggag 3aa
agctgctggt attttgggag gagtttttgt actt~ccaca cctgctgtga tgtggcattt 36a
tgttactctt ctctctttca cttgtgcttc atctccatcg acaggtaeat tgcggttact X20
gaccccctgg tctatcctac caagttcacc gtatctgtgt caggaatttg catcagcgf':g 48a
tcctggatcc tgcccctcat gtacagcggt gctgtgttct acacaggtgt ctatgacgat 54a
gggctggagg aattatctga tgccctaaac tgtataggag gttgtcagac cgttgtaaat 6aa
caaaactggg tgttgacaga ttttctatcc ttctttatac ctacctttat tatgataatt 66a
ctgtatggta acatatttct tgtggctaga cgacaggcga aaaagataga aaatactggt °~2a
agcaagacag aatcatccte agagagttac aaagccagag tggccaggag agagagaaaa ~8a
gcagctaaaa ccctgggggt cacagtggta gcatttatga tttcatggtt accatatagc 840
attgattcat taattgatgc ctttatgggc tttataaccc ctgectgtat ttatgagatt 9aa
tgctgttggt gtgcttatta taactcagcc atgaatcctt tgatttatgc tttattttac 960
ccatggttta ggaaagcaat aaaagttatt gtaactggtc aggttttaaa gaacagttca ~.a2Q
gcaaccatga atttgttttc tgaacatata taagcagttg gatagacgaa gttcaggata ~a8a
cc 1x82
<~~~~>
<~11> 345
<~12> ~Ft"I'
<213> F~~~nan
~ eI t~~ G?'~
Met Sex ~k=~ Asn SRr Sir Lxm~ ~~u vat. Ala Vat ~J! ~ ~~t~ ~y~ "I"yr Ana
1 5 1~ n 1
Asri VaJ! Asn fly Se ~ ~ys Va ~. Toys :T'~l~.e Lara ~k~e her F'r..~ G~.~~~ ~4r
Arg
2 C7
V~~ ~ Ll~.t~ LF7tu "F'y~- :I: ll~ Va 1. ~"fux~ c~ l ~,,~ Fte ~~'1y ~~l~.a
~.j;~.1 L~e~,s AF-~ ~~a 1i. I~)~~
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
40 45
~.~y A:.:n t~eu L,eu VaJ Met. Ll.e S«r L1~ l'~~s ~fi~s Phe L~ys Gin 1."e~ H.a-
s
50 rj"~ ~rJ
Ser ~~ro "T'~v~- Asrr T-'Efl~--~ L"e.e~ V".i~_ Ana 5er P~eta Al~.a wys Ala Asy
L"hca ~~e:c.~
JL~ ~~5 80
Vd~. ~Ly V~.~L Tl-rr V~i~ M~~t- ~~rca I~he ;.~;ez~~ Met Va:~ Arg Thr Vxl~. GLu
S~r-
~ (1
Cys Trp Tyr Phe G1y Arty Ser ~~'~ie Cys "1'hr Phe His 'T'hr Sys ~ys Asp
1g0 1! 0'~ 110
Val A.La Phe Cys "L'yr Ser Ser Lieu Phe Hi.s ~eu Cys Phe I1e Ser :Ck.e
115 12Q 125
Asp Arg Tyr Tle Ala Va1 'I"hr Asp Pro Lieu Val Tyr Pro 'Fhr Lys Phe
130 135 14Q
Thr Va1 Spr Val Ser Gly I1e Cys Tle Ser val Ser Trp 11e Leu Pro
145 150 155 160
Leu Met Tyr Ser Gly Ala Val Phe Tyr Thr fly Val Tyr Asp Asp fly
165 1.70 1~5
T~eu ~J! a flu Leu Ser Asp Ala Leu Asn ~'ys T1e fly ~1y ~'ys ~ln Thr
18Q 18S 1917
Val Va1 Asn ~ln Asn Trp Val Leu Thr Asp Phe Leu Ser Phe Phe Ile
195 200 205
Pro Thr Phe Tle Met Tle lle Leu Tyr fly Asn ale Phe Leu Val Ala
210' 215 220
Arg Arg ~1n Ala Lys Lays Tle g1u Asn Thr fly Ser Lys Thr Slu Ser
225 230 235 24'0
Ser Ser Glu Ser Tyr Lys Ala Arg Val Ala Arg Arg Glu Arg Lys Ala
245 250 255
Ala Lys 'Fhr Leu ~1y Val Thr Val Val Ala Phe Met lle Ser Trp heu
260 265 2~g
Pro Tyr Ser Lle Asp Ser Leu 11e Asp A1a Phe Met Gly Phe Tle Thr
275 280 285
Pro Ala Cys 21e Tyr Glra lle Cys Cys Trp Cys Ala Tyr Tyr Asn Ser
29Q 295 30(~
Ala Met Asn Pro Leu lle Tyr A~.a T~eu Phe Tyr Pro Trp Phe Arg ~ys
3a5 .~~.~ ~1~ 32a
Ala 11e ~ys Val lle Val Thr G1y Gln Va2 heu ~ys Asn Ser Ser Ala
325 330 335
Thr Met Asn ~eu Phe Ser flu His Ile
340 345
<210> 3
<211> 994
<212> ~1~1A
<213> Human
<400> 3
ctt~aggaag acaataatat aataataaca atattttctt cactctgcag tgtctttaca 60
ttcoagggtt gggaacatta ctgaggattc l~Gtt~ccatt ttccagtttc otgtt~atta 1.2Q
tt~ttatttt tttgactgct tttagcatog ggagcaoaaa ggccagteac oaggaattgc X80
aaaoaaatgc gtagtcagag agagagggct cactgcc~cat ttgtc~atgtg gatgcagaca s40
cattg~agat gtgttcc~ag taa~.aatgto ttgagaagag gactggtctt tcca:~oagca 38L7
tctoagaaat gcGggtgtgt otaaaeagoa tgtcgttctt taatg~ttto atgcaatata 3~0
ttttat~caa~~ ctcaagtt~c ~ctcactat.g tattataata atttctgctt gttggtaao~ 420
aaL:g~agatg gaaaattgat tcttaacaga agagaaagag ocaagtattg atg~-tta~ta ~~80
tttaoa~~ct attgtat~-tt tgtaaoaaaa a~oogg9tgg ~-taag~tai:g attgggaa~a 540
agggaatggt t~:aagt~t~at gca~:taagga aaaaoaaato tttgg~,~taa aa~aataatg 6Q~0
ataata"~aa~ ttaatataga gtagagac~~t gttttgtaga ataactt~tr~~ i~agt~aatrsac~ 8~0
tgf~tgaaaat aatcatae L a gtt~a~acug "~~~oactarag ggatt"_~.:at~"
gar.~r~c~.atttt 'DLO
c~ccattr~aaf~ Ocattt~~-~ t ac~etaa~~agg a,~t'~.~at~tt taagr~~gg~:a
at~~~ragg~tk.~~r 7~0~
gataaca~l'-~~~ G~~~a~~ag.~fi'_aa ~~~~~~caag ~~-gt~ ~:~ryt~ntcagwt e~~ataa~
~=ac~ ~at:t->-r-~~~E-afi-"_-
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
a~,r"~~3~z~~k"~t'~t gcaragagaa ar:~tcaaaa":~'~ f~aaaaa~'~aaa atatgaaagq
atatttaaaa ~~g
t:"-aa.~a'aet~~a ttttatcaaa tt aagacfw.~~ _~~~"~acattta t~agti_".~aaa
cast".~tccaa ~~Ct
t'aat-"-t:t~y~t_g caatataatt ttt'4gt~rt_~ant- t t E-~nG-t~ttgt cat'-~aa"~aEk
tgggatttaG- lfon.
aat ~:3,maat:q gaaacttgaa aa'~ti-at at t- r3"~r~ctat aata f_~.~tgal~~at<t
tcctct<qgca 1~.~8r"}
t_"~~a~.s.ai.ttta~-+a tatgtgtttt ttLccc~rt~a"~"~ a'~t"~ac~L=gaaa
~rtt_"~a".~".~adC~a at~=ragtaf'_l_ lb4r~
i-t_t t. Yat:",t~rt~ a at":.aacaaqg a~~e3aa~~"-t t' ~- 1' ~~"°atot_gta
a:~t'~aaC-actc_~a ttal_gagc~~r~ l2gt~
caat~"°a+~r~c ctgctggtgg ctgtgc=ag":f'= gt=qctacgcg
aat°_gt~~a:~tc3 ggtcctgtgt ~
g,~a~tatLeccc ttctcgccgg qatcccqgg6_ gattctgtac atagtgtt tg gctttggggc L3; E~
I~"gtgrwtggct gtgtttggaa acctcci-gqH qai~gatttca atcctccatt tcaagcagct 1~38g
gcact~:t'~ccg accaattttc tcgttgcci:v tctggcctgc gctgatttct tggtgggtgt 144E~
gactgtgatg cccttcagca tggtcaggac ggtggagagc tgctggtatt ttgggaggag 1500
tttttgtact ttccacacct gctgtgatgt gg"uattttgt tactcttctc tctttcactt 156Q
gtgcttcatc tccatcgaca ggtacattgc ggttactgac cccctggtct atcctaccaa 162a
gttcaccgta tctgtgtcag gaatttgcat cagcgtgtcc tggatcctgc ccctcatgta 1680
cagcggtgct gtgttctaca caggtgtcta tgacgatggg ctggaggaat tatctgatgc 5740
cctaaactgt ataggaggtt gtcagaccgt tgtaaatcaa aactgggtgt tgacagattt 7~.8Q0
tctatccttc tttat~ccta cctttattat gataattctg tatggtaaca tatttcttgt 186Q
ggctagacga caggcgaaaa agatagaaaa tactggtagc aagacagaat catcctcaga 1920
gagttacaaa gccagagtgg ccaggagaga gagaaaagca gctaaaaccc tgggggtcac 1988
agtggtagca tttatgattt catggttacc atatagcatt gattcattaa ttgatgcctt 2040
tatgggcttt ataacccctg cctgtattta tgagatttgc tgttggtgtg cttattataa 21Ct0
ctcagccatg aatcctttga tttatgcttt attttaccea tggtttagga aagcaataaa 2160
agttattgta actggtcagg ttttaaagaa cagttcagca accatgaatt tgttttctga 2220
acatatataa gcagttgtat agacgaagtt caggatacct ttaaaattac caagcgaaat 2280
gagtttttaa aaatcaagta agactatgaa tgaatagcaa ataaattgct cttcaaatga 234(>
aaaacaaatc aatgtttttc agtcttgtta agatgtgcac tttcctgtcc cttctgcaaa 24Q0
agtatttact tggctaacaa atgttaaatt cctatttgtt aactgcttta gagctcagca 2460
tatcccactc cctgcagaca ctttttgtct tttaatccat tgactcttcc ctctgctctg 2520
gtatttttcc taaaaatatt tgtttttttt tttttattta ttccctttcc tcttttcttt 258a
acaaagcttt ctactctttc ccagcctgcc aaaaatttca tttgtgaata gcctttatca 2640
aattattggt ttcttttgct ttggttattt taccacagga gtccttttag gtattaattt 27gE~
aatttattca atcttgggag agatctcagg gtgtatgggg caatttgcaa atgaagacat 2760
catcttgacc aggctgttgt aattgteaaa ccagttactg tcattcttgt aattatttcc 2820
tcccccaaag tgggaagcag aagccactgt acttcccaga atgatgttag gatgattatt 288a
tggctgctgt tcttgctatt gcacaaaact gtttaaagag ttggtatgaa tagagccctg 2940
tgttacatta ttcagttcat acacattgaa tattacttgt tcctttaggg aggatatctt 3000
tcaagtgcag tctctagctt tcttttcttt ttttttctat attaaaactt aattacagca 3060
aggaatttgc aagattagaa gccatgtgga atatacatca atgagaagca ttcaggtatt 3120
catccatgtt ttttccattt accaaacatg aaacctgtgc ctatattgta tggaaatcag 318
tgctagatgc cttagacaca ggcataacat cccatttttg tctttaataa gctgtgactc 3?40
tggcaagaag caatgttggt cactgaaaat tttaaaaagg gcgaacacta gctcaaccag 33~g
gtaattaagg ttcaatatca gtagtagtaa ggcatgtggc tattgtgtgt tcttgacatt 3368
atgtaatgag aatggctttt acctctatgt cctttcttcc ccaaatccat aacccccatg ~42p
tagtcatgag aaaaatgtca gacgaaactt tatcgggaat attctgcata atatttgatc 248D
agtatttctc aaaactgtca gtcatcaaaa ataaagtatg agaaactgtc atgatctaca 8S4(?
ggaaactaag aagacatgac aactaaacgt agtatggcat cctaaatgga aagctagaaa 26aQ
gaaaagggac attaggggaa gtgaggaaat ccgaataatg aatggaaatt ttattgctat 366U
attgataaca atattggatc attagttatc acaaatgtac tatatgagtt taagatgtta ~72~J
atgagaaaet tcttgcaagg tatataggaa ttctcattac tatctttgca atttttcagt 3788
aattctacaa ctattctgaa attaaaagtt tattcaaaaa atatagagta cacaattcct 384f~
gcttgataaa gtttctagcc. tgtctatgtg aagacagcaa aqc_acttatc cttacagtca 39L7Q
ttcatttatt cattctgaat atatctttga agactgagtg tgtactagac tcttggttca 396p
gtgtgatcag gaatagaaaa ccaggaactt agaatat'~ttt gtggcaaaac ccaaaatacc 4a2tj
agtaattaag acttggaatg catgggaatt taagctataa aaqgctgtgt ttaaggaaca 4Q8a
~:aggagaaag gagaattcag acctggatgg aaaatqaagq agatgtatta aagaagtggc 41.4rf
attCaagtag ggccttaaat tttaagaagg atttttgta~ ~a:~ggaaagg atgggaagcg 42f~g
attttcaggc atgagcaaag aaactgagaa agtgcaaagt "-~tttdac~r.~aa tatgaaaata 4?6E~
aaacE~a~.~tafg gctggg=gtg gtgggtcacg Fwctgt"pat c"~ as"~"=a° tfi. 'tg
ggaggccaaq 4.~20~
q.gggtggat cacaaggtca aaagatcgag as"~a:~~-"~tq~ -taa"_ae~r3"ai~ gaaa~cecgt
438t'~
cib"~tact~~aa aatacaaaaa i'-t-a~:ctc~s~at gtggi:-3q"'a"°
~.t4"er"_"°t"_~t ag t~c~caca~tt~= 444?
t~~.~g'~ta9~.tct qaggcagq"a:~ ~aat"ctctt'=~~u 'a"_~"~t~~-t:~r~'t~t t"rt;-
~~~"r~t.c~~ actt~gag~=c"~.s 4"~t~rtf
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
~aat~Tatgcca ct<gt-~ic~t~c~ gcrtgggt~aa cxagagl-:~add cta~.t<atcada aaa~-
~~3.:~rad~~ri ~156t~
~.ir'#dc3daagaa dcie4dd~lgaac'3 gs~-aa~aaciclh a~iaa~rlc~t~=te'3
7dr'=r3aCCaelC"C ac~gae~~_~C"ddg 4(~e_~1
~aagt'dgteac gc~ttttaaac cctcctra~-~!~g f-tg~~'at~r-~c,a ar~~~c~tgcac
cte"F~ir~tt~t: 4~a8rt
"=t=tr~fgg~:r"~~c ~~tr c:at:tgt_g qtttggaqac: a~-f-~~t ~yr,cmr~~t r--1-c~C-
wctcaa gt_Et_~-,-~~tc~'_c~ 4'14t1
--~at<~~attf~t t<G-+'a c:+_aagaa acattt:.~:C~e~d d~~fir~ti~~gaf~E t~ir~~3.~G-
iac~°ig tc~r~+~t-"~m~iLr<~ 48Qt7
.aae-ii r3~tc~ca a'g"~adadd~d tttctgatG-r aE~ad~~t'_att.t_L Lr.f~ara~ict-gi'_t
r.trv3t~,a-P r-Et= ~PB~at~
ttck~gttc°tt gctagadcact gl:atattagge"~ ai'att~~aga ~"+'~~:"tgagaag
t~ccl-_~t~+:1~~ 49X1
~+r"aaaaacct gLccaatttt gtttaadtgt gr~att~rtaa~3 cdttrtattaa accatgggta 4980
i:tcttttgat atgaggagct caataacaga g~~tattgtcc aagqaaaata tttdaggaac 5Q40
dccatctcta tcgttgattt ggttcttacc taatgt'_atta ttaatcttgt attggtcttt 5lpg
tggtctttca catacaatac acttcattea atttgtattt ctaadaggta gaggtiggttt 5160
tccdacccat ggtataataa tgagtaagca ctcataacat gttttttttt gtcaattatg 522Q
atgtataaga taaattgtac agtatgtaaa atgggataaa ttatgtgact ttgaaggagg 5280
gactggatgc tgcagactga aaaatctcag aaggttccat ggagaactta gaatataata 5340
r_cagctaagc agtggaggat tggtttaaat agaaaaaaca atagtagaaa dggaatttct 540D
aggcagaata daaatctcat gaaaggtata aatttagtgg tgcatcaaat gcaattaagc 596a
atacaaaaaa ggatagccaa taagatcagt cggcctaaag cacaccattt gggtaagaga 552(3
gtcacaatca atctatccat caatcaacat actattttaa gccctatatc tgcactgtcc 5580
agtatggtag ccatgagcaa catgtggtta ttgaggactt gaaatgtggc tagtctgagc 5648
tgagatdttc tataggtaga atttatatac cagatttcaa agacaaacaa aagaatgtaa 5700
gatgtctctt tttaaaatat tgattacatg ttgaaattat caattttggg atgtattggg 5760
ttaaataaaa atatgttatt tagattadtt tctcttttct ctttttactt tttaaaatgt 5820
ggctactaga aaaatgtaaa attatgtatg tagcttaaat tatatttcta ttggacagca 5888
ctactctaga gaaaacaaaa atgagccata ggacaattct agccctccag gacttaaaaa 5940
tcaagatggg gagagaatgc atgaacataa acaatgaagt aaatatata~ gaactatggt 6000
aaaagttaaa taatatttat ttagcaattg atgtttaaaa acagaggtac aaaactcata 6060
ttaatcttat agtctggtca ttttataaat gagaaaattg ggactcagag agadataaat 6120
taacttgcat gaaattatac agctagtagg ctaggtgcgg ttgctcacgc ctgtaatctc 6180
agcactttgg gaagctgaag tgggaggatc acttgagttc aggagtttga taccagcctg 6240
ggcaacatag tgagaccttg tctgtactaa aaatgadaaa gttagccagg cgtggtggtg 6300
adtgcctgta gtcccagcta ctcaggaggc tcaggtggga ggatcacttg tacacaggag 6360
tttgaggctg cagtgagcag tgatcgcatc tagadacaaa gtgagattct gtctcaaaaa 6420
ataaaaagtt atacagttag caaaaaatct ttgccatact ttgaacccaa attatttgaa 6480
ttctaagctc aatttttttt ctcgacatgg adatgagagt ttaggacaaa caataaggga 6540
tattacaaag aagtacagtt aaataaatgc tatccacaag aagatattta gcatttagag 6600
tttctctcat aagtcagtgg ttcttgataa tttttagdtc atagtcacat tgaaatatgc 6660
tgaaatctat gtattctttc tctagaaaaa tgcacacaat cacatataca caatatttag 6720
tgtactattc tggggg~tcc atadcctttg gggccadtta caggtcatgg atacactgtt 6780
cctaagttgg gcatagacta gacttggctg aagtgaaaac gacctcaact aactctgtgg 6840
cttdacacaa caaaggttta tttcctgctc atgtgaagtc cactatgagt ctgaggagat 6900
dtcagggcaa ttgtcctcaa catagtgcag gttgctttga tttcatggct ccatcttttc 6960
aacaagagac ttctatactt cagcctgaaa gaacacatga acacttaaat ctattagcca 7p20
gaaccaagca gacagtccac ctaatggctt gaaaaacaca ggacgataag cacaatgttt 7080
gctgcacaca aaggcctctt ccaaacactg ttttactgga tgggacatca cattgtgtga 7140
atgatgtatc tctcagggtt tcaatttggc agaaaatgtt ttaaactcat tagtgtttat X200
tttaatcaat attaagtcta agtgagatag ttgctggctt cggctggctg actcaagtat 7260
gttagagtca aggatgctgt acagattttc ctgatgcttg tctaatggtt ccaaagtgac 7320
tacaggagtt ccagccaaat cacacatgtt cctggaaata gggaggagga agacctagga X380
agdggggctc tctgaagccc tacttgacaa cttcagtt+.~c cccaagggag agaagattct 7440
gatgctatgg aggaaattaa cccattggtt gttaggtgag aaataagcat ttctgccaca '500
tgaagtaatc tggaaaatga aatccaacaa gtga~~aataa aactf~cttac caaattctct 7560
caca:~gtatt tgctgtataa atccataagg aagtgaactg agaadaagag aatggaaaaa 7620
ataacaggtt fmtctcaaaga tctctatacc atctttttct taaacttctc cttttgtatt 7680
atttdgtatt aatttttcag ccaatgaaca tgtactatat atgtgttaca tagtaaatgc ~~~0
ectgttgacd atgcagaagt gatgagdatg adtgagdctc aatcc~tttta gtagagtttt X800
caatatttcc caaaaggtag tggttacaag cdgagccaga caqccagtcc ttttatggga X860
cdaagatdtc ttgctdgagd tcacacagtg ggtcagcatc agggacagag tcceeagaat ~9<"-0
ttgcaatagc ctggttgtta ttgtcttgga greaatcdgta ga~~ar~atgta aaggcatatt X980
ttttaatt'tt a=~att*'caag gt~acatgtgr,~ aggatgtgCKa ggttt~f~ttdw ataggtaaa~:
80~f~a
atgtc~c~atc~ gtgqtttg~-t gcacctatca a~~c"~dtE~dr:L tag~ta+-_taa g~:c~aq~~atg
810th
~ac'Statr',3C/',Et=dt ~t~WC~'W=~'Lac3t gr=trytC-~tt'.-jC=C:
!°~'_rac'r"c~fat"--.".'.S= rwGtr3tT~+~~~°da
r=ai~~r'~~w".:'"C:ci~~7 ~~fr~l
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
tgf''c~t ~,:~tgc- tcccttcctiet gtqtcr_~tt'~t attctcatt_g ttcdgc°_t'-
c=ra actgaagaat~ ~?~~'
~,~~a~dt=r-,~t_~ aaaatggc=:a t_at t-=y°_e°r~aa a~caatttat
agar=t"_w El~g ctafitctj"Maf s~,°3r~
tarid~~t-:~"~c~.~ ttgacattct t~car:agdat-t agaaaaaaaa gattt':aa~:i:aa
ttcatat~g!~a ~ ~~lrt~'
~r'1'$'~3el.deldf~ ~l~Ct~gtelP~d t~(.~t'cl~~G.~!=taaa ~txCtaagf:_aa
claei~r7ds:.l,7c'3 riC:as~c3r~r'~ag!~ ~~z~~'TCf
rWl _t~~.ira~~r°~ct r'k~Caact'~f~c'c,1 dr~t~t_dti~drwt'a t,:aaggCtdr
e'1 a.~t t'i~7r_'cw,,t.~,ac1 C:clCjr,dtP~v~l ri rrir~HEl
r"t'".~r'~1_r3r~~acd at~r':ggr~a'lC.~r3t~ agaC".:ac~t:gC~ ~ldtuc7~aatag
c3~d~3~lC~t_r'rdr~ct aat''r.~E3a~~dr~rl ~°_~.jfy
tr an=ai "a ~~ga a6~c.a6=ctgc~t cttcaacaaa ctt~gacaaaa aaa~~aa~~at'-a t_t-
gggaaagE ~3a~t;l
at~ts~~~~t~r=art taataaatgg tgctggaaga actggctagc catat~acaaa aaaatt~gdaa
8t5=t0
ctggr~tfc~r=ct tccttacatc ctatacaaga attaactcaa gac~~gattaa agacttaaat: 8~~~g
gcaa~~rmcad ttaatatttc actagtaggg ctgccctcta ccagat'.~ggct aatggagtca 8-160
cgatgcagag ctatgagatg gagggcaggg gatgctatgg ctaattttaa ccacagcagc 8820
aaacacagaa aaacccacta ggtcgtatag actcctgacc agccccagca tcagatcttg 8880
gtcactgtgg gctatggtga attcataatt caaaacccaa agcctaattt ctgttgtgtt 890'
tggaataaaa ttctaactct gtgattctga tctatgaggc tttgcatagt cttagccccg 9000
cacaactctc tacccactac ctattctgct ctccctttac ttgttatgct ccaaatatat 8060
ttgtccaatc ccagcccctc ggctaccccc cacaggatgc ctcaggagct gtgcactggc 912D
tatcccatct gttgtactag gtcgctcagg agactcgcac agggtttttc atctgttgga 9.80
tcactcagtc ctcagatctt cacggagctg gttccttaca caggtctcag ctttaacatc 9240
gctccctcag aaaaatcaag atctcggcta atgttgttat ccatgtttta ttcttttact 930'0
caccttgttt ttatatcatt tccttcatgg tacatatcag aatgtgttac aatcttattc 9360'
atctgt:ttat tttcttgtgt tttaactgtc tgtctcttaa tgacatgtaa gctgcaggag 940'
gtcagatact tgctgaagta tcactatgag taaatcagac agtttgtact ggtgttttat 9480
ttctttgctc ctatgttgtt gttttacttt ggtcagttga gtaaataaat gagtgaataa 9590
ataaaataga aatagattcc ccagcattgg gaggatacat taaatgtatt cttttttttt 96D0
tttttaaagt tctggggtac atgtgctgga tgtgcagttt tcttacatag gtaaatgtgt 9660
gccatggtgg tttgctgcac ctatcaactc attacctagt tattaagcct ggaaaaatgc 9720
attcttttta taagcttttt tggggagggg cagggtcttg ctgtgaccta ggctggggta 980
cagtggrwatg atcatagctc aatgcagtct caaactcctg ggctcaggtg acactcccat 9840'
gtagctgggg ctacaggcgt gtgcgaccat gcctggctaa tttttaaaaa aaatttattt 9900'
ttgtagaaaa gggatctcat tttgttgccc aggttggtct caaacac 999'7
<z10'> 4
<2~.1> 107.
<~12> I~NA
<~13> Human
<90'Q> 9
gaacagttca gcaaccatga atttgttttc tgaacatata taagcagttg katagacgaa 60
gttcaggata cctttaaaat taccaagcga aatgagtttt t 10'x.
<z10> 5
<2~~> 321
<~~,z> PRT
<2~3> Hurnan
«0'0> 3
Cys "i"?/~ ~ln Val Asn Gly Ser Cys Prc Arg Thr Vat His Thr ~eu G.t.~
10' 15
~1.~ ~ln T~~u Vat. Ile Tyr Leu Thr Cys AJ.a Ala fly Met ~eu Tle I.l.e
zo 2~ 30
Val~ Leta ~~.y Asn Val Phe Va1 Ala Phe Ala Va L Ser "Fyr the .L'ys A.~a
3a ~0 93
~e~' H.~s '~'~ar ~rQ "T'hr Asn the ~.ae~a Leu Lreu her Iaeu J~la Leu A?'a J~s~
'_~0' S5 60
~Iet ~'he L~u ~~! y Leu l.~eu Val Leu ~rca Leu ~~r "I"hr T.~a Arg Ser Val
65 7D' ~S 80
~~t~ See ~'~rs "~'r~a Phe ~'he ~~~!y Ash Phe Leu 0'"~s Jarg Leu fills '~h>~
"T'y~
R5 ~fi A
I~nn.~ asp "2"fns Pry"o~ Fhe C'ys I~e~a "z"hr per ~'tr~" ti'f~t-~ t~.~.s Leu
~'~Js the t.7!.~
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
~L~P 'Lr-~", l~.g
:~er- T~'.e As~.~ Arg f-Ia.:~ ~'ys ALa T:1.F=~ r'y~: Asp Pra Iaeu Lreu "L"yr
!'rte :~~~r
L l r5 1;~'fi7 1~ ~
I~ys f"1're "1"hr. Va 1 Ar"I Va l ALa Lfi:t~ Arch 'i'y'~ L.l,e Laeu A.La Gl~ y
'I'r~~ r-rp y
131 ~ ~a l.tlg
Va D I=rrr AT ~a II E ~r '1"yr "I~'hr Ser E,c-:u E'lm~ lieu "~yr "L""rm Asp
Vci,L Va l rz M ur
125 Lag 15a I~s~r
Thr Arg L,eu S"~r. ~Ln "1'rp I~eu C;~.u flu Met Pra Cys Va1 c:xly See ~y~;
.L6'~ 7_~p 1~a
Gln T~eu ~~eu ~,eu Asn D,ys Phe Trp ~.Ly 'T'rp Leu Asn Phe Pro T~eu F'he
18~ 185 190
Phe Val Pro ~'ys Leu lle Met Ile Ser Leu Tyr Val L,ys l~.e Phe Va.L
~.~5 zoa za5
Val Ala 'Fhr Arg Gln Ala ~ln Gln Tle "~hr Thr ~,eu Ser Lys Ser Geu
211) 215 220
Ala Gly Ala Ala f~ys His ~1u Arg Lys Ala Ala Lys Thr ~eu G~.y 11.e
225 230 235 240
Va1 Val GIy ~1e Tyr lieu Leu Cys 'Frp Leu Pro Phe Thr Tle Asp Thr
245 z50 255
Met Val Asp Ser Iseu Leu His Phe Ile Thr Pra Pro I~eu Val Phe Asp
260 z65 zip
Ile Phe I.Le Trp Phe Ala '~yr Phe Asn Ser Ala Cys Asn Pro Ile 1.1.e
2?5 280 285
Tyr Val Phe Ser Tyr ~1n Trp Phe Arg Lys Ala ~eu Lys Leu 'Fhr I~eu
29(~ z~5 300
Ser ~ln I~ys Val Phe Sex Pra G~.n "Fhr Arg Thr Va1 Asp Leu Tyr ~1n
3a5 3~.0 37.5 321)
Glu
<210> 6
<z11> 296
<21.z> PRT
<z13> Hurnan
<9Q0> 6
~eu Phe ~~!y Asn )~eu Val 21e Met Val Ser lle Ser His Phe ~ys ~ln
1 5 10 15
Leu His Ser Pra Thr Asn Phe Leu T1e Leu Ser Met Ala 1'hr '~'hr Asp
23 25 30
Phe Leu Taeu G.1_y Phe Val Tle Met Pro 'Fyr Ser Ile Met Arg Ser Val
35 4(1 95
Glu Ser Cys "I'rp ~'yr Phe fly Asp fly Phe ~ys Lys Phe His 'I"hr See
50 55 6p
Phe Asp Met Met Leu Arg Leu Thr Ser lle Phe His Leu ~ys Ser Tle
6S 70 75 88
Ala :Lle Asp Arg Phe ryr Ala Va.L ~ys T'yr Fra Leu His "T'yr Thr ~'hr
85 9D ~5
Lys Met "L'hr Asn Sex Thr Tle Lys ~ln ):~eu Iseu Ala Phe Cys Trp Ser
~.tJQ 1Q5 110
Val Pr<a A.La Lieu Phe Ser Phe ~.Ly T.~eu Va.~ beta Ser ~1u Ala Asp Vat.
115 12(~ 1z5
Ser ~ly Met ~a.ll n her Tyr ~~ys :~.~e Leu Val Ala Cys Ptse Asn Phe ~ys
13g 135 leg
Ala ~pu "D'hr Fhe Asrr Lys Phe ~'rp ~.~y "P'hr :L~.e .~eu Phe "L°hr
"~"hr ~'ys
195 15~? 1.55 1.6g
Phe Phe "1"h~ Pr~.~ fly Ser :~l~e Met VaD ~l~.y .ale Tyr fly )!,ors '~~e L'tm
1~5 17fi~ ~.1~',
:LLe Va.l Sr=~ ~.~v:~ c:~Ln His Ala Ar-:I Vat 1')Le 5er 1~Ir.s Vat. Pr~~ ~rlu
Asrr
~ ~~' ~.8!w h uc~
C>
CA 02425897 2003-04-23
WO 02/34913 PCT/USO1/31454
"~'h~ ~.ry5 1~1~~ X11 G+ V~1. ~rys ~~y:.: '' P -; ~.~=j": ~r~Y L~ya ~y5.s Lrys
Ilsp Firs toys
~'r"~J s'~DO! ~~)~
T~~~ 1~~.~ I~ys "?''h r I~F~u ~"r.P y ~' 1~ ~~, ;Y,.a ~ N~Fe ~ I ~~ ~a l FhEr
~reu Al~a C'ys 'k'rF'
_~~J ~l"', ~.W~
1.~~~u I'~o ~ys Fh~ I~."~e~ A7~rfi Vein ~~,an~~ I~r, n::~, ~-rr~ '~'~>r I~a~u
Asia 'I'y~ °;F=r
.' i' ~J rE-.~ C'.? .'..~ ~7 4'
'f qtr Fra I ~.e l~~e~ I ~a ,~Ct~ ~ls~~ D>>av h"r"ar V~ T "~"r~~ ~r~t~ T~rg
'I"yr ~'f~r~ hs,rr
i .J V
~~r Thr Cys Asr~ Fca LreU I.l! ~~ I~ i s C~~ y l~h~ 1''h~ Asri Fra "~'rp F"he
G~.r~
z~Q~ ~'_a 2~0
Lys A1a Fhe Lys "~y.r :~:Le V~.1. ~~r ~.l~y Lys :ale Fhe Se.r Ser l~lis Ser
z75 2801' 285
Glu T'hr Ana Asr> L~eu ~'he ~'rc~ flu
zoo z~~
7
