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NOVEL PROTEINS AND NUCLEIC ACIDS ENCODING SAME
BACKGROUND OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides. More
particularly,
the invention relates to nucleic acids encoding novel G-protein coupled
receptor (GPCR)
polypeptides, as well as vectors, host cells, antibodies, and recombinant
methods for
producing these nucleic acids and polypeptides.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of nucleic acid sequences
encoding
novel polypeptides. The novel nucleic acids and polypeptides are referred to
herein as
GPCRX, or GPCRI, GPCR2, GPCR3, GPCR4, GPCRS, GPCR6, GPCR7, GPCRB, GPCR9,
and GPCR10 nucleic acids and polypeptides. These nucleic acids and
polypeptides, as well as
derivatives, homologs, analogs and fragments thereof, will hereina8er be
collectively
designated as "GPCRX" nucleic acid or polypeptide sequences.
In one aspect, the invention provides an isolated GPCRX nucleic acid molecule
encoding a GPCRX polypeptide that includes a nucleic acid sequence that has
identity to the
nucleic acids disclosed in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22,
24, 28, 30, 32, 34,
36, and 38. In some embodiments, the GPCRX nucleic acid molecule will
hybridize under
stringent conditions to a nucleic acid sequence complementary to a nucleic
acid molecule that
includes a protein-coding sequence of a GPCRX nucleic acid sequence. The
invention also
includes an isolated nucleic acid that encodes a GPCRX polypeptide, or a
fragment, homolog,
analog or derivative thereof. For example, the nucleic acid can encode a
polypeptide at least
80% identical to a polypeptide comprising the amino acid sequences of SEQ ID
NOS:2, 4, 6,
8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and 83. The nucleic acid
can be, for example,
a genomic DNA fragment or a cDNA molecule that includes the nucleic acid
sequence of any
of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36,
and 38.
Also included in the invention is an oligonucleotide, e.g., an oligonucleotide
which
includes at least 6 contiguous nucleotides of a GPCRX nucleic acid (e.g., SEQ
ID NOS:1, 3, 5,
7, 9, 11, 13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38) or a complement
of said
oligonucleotide.
Also included in the invention are substantially purified GPCRX polypeptides
(SEQ ID
NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and 83). In
certain embodiments,
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the GPCRX polypeptides include an amino acid sequence that is substantially
identical to the
amino acid sequence of a human GPCRX polypeptide.
The invention also features antibodies that immunoselectively bind to GPCRX
polypeptides, or fragments, homologs, analogs or derivatives thereof.
In another aspect, the invention includes pharmaceutical compositions that
include
therapeutically- or prophylactically-effective amounts of a therapeutic and a
pharmaceutically-
acceptable carrier. The therapeutic can be, e.g., a GPCRX nucleic acid, a
GPCRX
polypeptide, or an antibody specific for a GPCRX polypeptide. In a further
aspect, the
invention includes, in one or more containers, a therapeutically- or
prophylactically-effective
amount of this pharmaceutical composition.
In a further aspect, the invention includes a method of producing a
polypeptide by
culturing a cell that includes a GPCRX nucleic acid, under conditions allowing
for expression
of the GPCRX polypeptide encoded by the DNA. If desired, the GPCRX polypeptide
can then
be recovered.
In another aspect, the invention includes a method of detecting the presence
of a
GPCRX polypeptide in a sample. In the method, a sample is contacted with a
compound that
selectively binds to the polypeptide under conditions allowing for formation
of a complex
between the polypeptide and the compound. The complex is detected, if present,
thereby
identifying the GPCRX polypeptide within the sample.
The invention also includes methods to identify specific cell or tissue types
based on
their expression of a GPCRX.
Also included in the invention is a method of detecting the presence of a
GPCRX
nucleic acid molecule in a sample by contacting the sample with a GPCRX
nucleic acid probe
or primer, and detecting whether the nucleic acid probe or primer bound to a
GPCRX nucleic
acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the
activity of a
GPCRX polypeptide by contacting a cell sample that includes the GPCRX
polypeptide with a
compound that binds to the GPCRX polypeptide in an amount sufficient to
modulate the
activity of said polypeptide. The compound can be, e.g., a small molecule,
such as a nucleic
acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other
organic (carbon
containing) or inorganic molecule, as further described herein.
Also within the scope of the invention is the use of a therapeutic in the
manufacture of
a medicament for treating or preventing disorders or syndromes including,
e.g., diabetes,
metabolic disturbances associated with obesity, the metabolic syndrome X,
anorexia, wasting
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disorders associated with chronic diseases, metabolic disorders, diabetes,
obesity, infectious
disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative
disorders,
Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic
disorders,
or other disorders related to cell signal processing and metabolic pathway
modulation. The
therapeutic can be, e.g., a GPCRX nucleic acid, a GPCRX polypeptide, or a
GPCRX-specific
antibody, or biologically-active derivatives or fragments thereof.
For example, the compositions of the present invention will have efficacy for
treatment
of patients suffering from: developmental diseases, MHCII and III diseases
(immune
diseases), taste and scent detectability Disorders, Burkitt's lymphoma,
corticoneurogenic
disease, signal transduction pathway disorders, Retinal diseases including
those involving
photoreception, Cell growth rate disorders; cell shape disorders, feeding
disorders; control of
feeding; potential obesity due to over-eating; potential disorders due to
starvation (lack of
appetite), noninsulin-dependent diabetes mellitus (NIDDM1), bacterial, fungal,
protozoal and
viral infections (particularly infections caused by HIV-1 or HIV-2), pain,
cancer (including but
not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus
cancer),
anorexia, bulimia, asthma, Parkinson's disease, acute heart failure,
hypotension, hypertension,
urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; Albright
Hereditary
Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma,
allergies, benign
prostatic hypertrophy, and psychotic and neurological disorders, including
anxiety,
schizophrenia, manic depression, delirium, dementia, severe mental
retardation.
Dentatorubro-pallidoluysian atrophy (DRPLA) Hypophosphatemic rickets,
autosomal
dominant (2) Acrocallosal syndrome and dyskinesias, such as Huntington's
disease or Gilles
de la Tourette syndrome andlor other pathologies and disorders of the like.
The polypeptides can be used as immunogens to produce antibodies specific for
the
invention, and as vaccines. They can also be used to screen for potential
agonist and
antagonist compounds. For example, a cDNA encoding GPCRX may be useful in gene
therapy, and GPCRX may be useful when administered to a subject in need
thereof. By way
of nonlimiting example, the compositions of the present invention will have
efficacy for
treatment of patients suffering from bacterial, fungal, protozoal and viral
infections
(particularly infections caused by HIV-1 or HIV-2), pain, cancer (including
but not limited to
Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia,
bulimia,
asthma, Parkinson's disease, acute heart failure, hypotension, hypertension,
urinary retention,
osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright
Hereditary
Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma,
allergies, benign
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prostatic hypertrophy, and psychotic and neurological disorders, including
anxiety,
schizophrenia, manic depression, delirium, dementia, severe mental retardation
and
dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome
and/or other
pathologies and disorders.
The invention further includes a method for screening for a modulator of
disorders or
syndromes including, e.g., diabetes, metabolic disturbances associated with
obesity, the
metabolic syndrome X, anorexia, wasting disorders associated with chronic
diseases,
metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-
associated
cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease,
Parkinson's Disorder,
immune disorders, and hematopoietic disorders or other disorders related to
cell signal
processing and metabolic pathway modulation. The method includes contacting a
test
compound with a GPCRX polypeptide and determining if the test compound binds
to said
GPCRX polypeptide. Binding of the test compound to the GPCRX polypeptide
indicates the
test compound is a modulator of activity, or of latency or predisposition to
the aforementioned
disorders or syndromes.
Also within the scope of the invention is a method for screening for a
modulator of
activity, or of latency or predisposition to an disorders or syndromes
including, e.g., diabetes,
metabolic disturbances associated with obesity, the metabolic syndrome X,
anorexia, wasting
disorders associated with chronic diseases, metabolic disorders, diabetes,
obesity, infectious
disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative
disorders,
Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic
disorders or
other disorders related to cell signal processing and metabolic pathway
modulation by
administering a test compound to a test animal at increased risk for the
aforementioned
disorders or syndromes. The test animal expresses a recombinant polypeptide
encoded by a
GPCRX nucleic acid. Expression or activity of GPCRX polypeptide is then
measured in the
test animal, as is expression or activity of the protein in a control animal
which recombinantly-
expresses GPCRX polypeptide and is not at increased risk for the disorder or
syndrome. Next,
the expression of GPCRX polypeptide in both the test animal and the control
animal is
compared. A change in the activity of GPCRX polypeptide in the test animal
relative to the
control animal indicates the test compound is a modulator of latency of the
disorder or
syndrome.
In yet another aspect, the invention includes a method for determining the
presence of
or predisposition to a disease associated with altered levels of a GPCRX
polypeptide, a
GPCRX nucleic acid, or both, in a subject (e.g., a human subject). The method
includes
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measuring the amount of the GPCRX polypeptide in a test sample from the
subject and
comparing the amount of the polypeptide in the test sample to the amount of
the GPCRX
polypeptide present in a control sample. An alteration in the level of the
GPCRX polypeptide
in the test sample as compared to the control sample indicates the presence of
or predisposition
S to a disease in the subject. Preferably, the predisposition includes, e.g.,
diabetes, metabolic
disturbances associated with obesity, the metabolic syndrome X, anorexia,
wasting disorders
associated with chronic diseases, metabolic disorders, diabetes, obesity,
infectious disease,
anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders,
Alzheimer's
Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders.
Also, the
expression levels of the new polypeptides of the invention can be used in a
method to screen
for various cancers as well as to determine the stage of cancers.
In a further aspect, the invention includes a method of treating or preventing
a
pathological condition associated with a disorder in a mammal by administering
to the subject
a GPCRX polypeptide, a GPCRX nucleic acid, or a GPCRX-specific antibody to a
subject
(e.g., a human subject), in an amount sufficient to alleviate or prevent the
pathological
condition. In preferred embodiments, the disorder, includes, e.g., diabetes,
metabolic
disturbances associated with obesity, the metabolic syndrome X, anorexia,
wasting disorders
associated with chronic diseases, metabolic disorders, diabetes, obesity,
infectious disease,
anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders,
Alzheimer's
Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders.
In yet another aspect, the invention can be used in a method to identity the
cellular
receptors and downstream effectors of the invention by any one of a number of
techniques
commonly employed in the art. These include but are not limited to the two-
hybrid system,
affinity purification, co-precipitation with antibodies or other specific-
interacting molecules.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although methods and materials similar or equivalent to those
described herein can
be used in the practice or testing of the present invention, suitable methods
and materials are
described below. All publications, patent applications, patents, and other
references
mentioned herein are incorporated by reference in their entirety. In the case
of conflict, the
present specification, including definitions, will control. In addition, the
materials, methods,
and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the
following
detailed description and claims.
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DETAILED DESCRIPTION OF THE INVENTION
The invention is based, in part, upon the discovery of novel nucleic acid
sequences that
encode novel polypeptides. The novel nucleic acids and their encoded
polypeptides are
referred to individually as GPCR1, GPCR2, GPCR3, GPCR4, GPCRS, GPCR6, GPCR7,
GPCRB, GPCR9, and GPCR10. The nucleic acids, and their encoded polypeptides,
are
collectively designated herein as "GPCRX".
The novel GPCRX nucleic acids of the invention include the nucleic acids whose
sequences are provided in Tables 1A, 1D, 1G, 2A, 3A, 4A, 4C, 4G, 5A, SC, 5G,
6A, 7A, 8A,
9A, 10A, lOC and IOF, inclusive ("Tables 1A - 10F"), or a fragment,
derivative, analog or
homolog thereof. The novel GPCRX proteins of the invention include the protein
fragments
whose sequences are provided in Tables 1B, 1E, 1H, 2B, 3B, 4B, 4H, 5B, 5D, 5H,
6B, 7B, 8B,
9B, 10B, 10D, and 10G, inclusive ("Tables 1B - 10G"). The individual GPCRX
nucleic acids
and proteins are described below. Within the scope of this invention is a
method of using
these nucleic acids and peptides in the treatment or prevention of a disorder
related to cell
signaling or metabolic pathway modulation.
G-Protein Coupled Receptor proteins (GPCRs) have been identified as a large
family
of G protein-coupled receptors in a number of species. These receptors share a
seven
transmembrane domain structure with many neurotransmitter and hormone
receptors, and are
likely to underlie the recognition and G-protein-mediated transduction of
various signals.
Human GPCR generally do not contain introns and belong to four different gene
subfamilies,
displaying great sequence variability. These genes are dominantly expressed in
olfactory
epithelium. See, e.g., Ben-Arie et al., Hum. Mol. Genet. 1994 3:229-235; and,
Online
Mendelian Inheritance in Man (OMIM) entry # 164342
(http://www.ncbi.nlm.nih.gov/entrez/
dispomim.cgi?).
The olfactory receptor (OR) gene family constitutes one of the largest GPCR
multigene families and is distributed among many chromosomal sites in the
human genome.
See Rouquier et al., Hum. Mol. Genet. 7(9):1337-45 (1998); Malnic et al., Cell
96:713-23
(1999). Olfactory receptors constitute the largest family among G protein-
coupled receptors,
with up to 1000 members expected. See Vanderhaeghen et al., Genomics 39(3):239-
46
(1997); Xie et al., Mamm. Genome 11(12):1070-78 (2000); Issel-Tarver et al.,
Proc. Natl.
Acad. Sci. USA 93(20):10897-902 (1996). The recognition of odorants by
olfactory receptors
is the first stage in odor discrimination. See Krautwurst et al., Cell
95(7):917-26 (1998); Buck
et al., Cell 65(1):175-87 (1991). Many ORs share some characteristic sequence
motifs and
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have a central variable region corresponding to a putative ligand binding
site. See Issel-Tarver
et al., Proc. Natl. Acad. Sci. USA 93:10897-902 (1996).
Other examples of seven membrane spanning proteins that are related to GPCRs
are
chemoreceptors. See Thomas et al., Gene 178(1-2):1-5 (1996). Chemoreceptors
have been
identified in taste, olfactory, and male reproductive tissues. See id.;
Walensky et al., J. Biol.
Chem. 273(16):9378-87 (1998); Parmentier et al., Nature 355(6359):453-55
(1992); Asai et
al., Biochem. Biophys. Res. Commun. 221(2):240-47 (1996).
GPCRl
GPCR1 includes a family of three novel G-protein coupled receptor ("GPCR")
proteins
disclosed below. The disclosed proteins have been named GPCRIa, GPCRlb, and
GPCRIc
and are related to olfactory receptors.
GPCRI a
A disclosed GPCRla nucleic acid of 1019 nucleotides is shown in Table 1A. The
disclosed GPCRla open reading frame ("ORF") begins at the ATG initiation codon
at
nucleotides 27-29, shown in bold in Table 1A. The encoded polypeptide is
alternatively
referred to herein as GPCRla or as ba113a10 B. The disclosed GPCRIa ORF
terminates at a
TAG codon at nucleotides 984-986. As shown in Table 1A, putative untranslated
regions 5' to
the start codon and 3' to the stop codon are underlined, and the start and
stop codons are in
bold letters.
Table 1A. GPCRla nucleotide sequence (SEQ ID NO:1).
CCTTCAGTTGACAGAGGAGATACACTATGGTAAGTGCCAATCAGACAGCCTCTGTGACCGAGTTTATTC
TCCTGGGCCTCTCTGCCCACCCAAAGCTGGAGAAAACGTTCTTTGTGCTCATCCTGCTGATGTACCTGG
TGATCCTACTGGGCAATGGGGTCCTCATCCTGATGACTGTGTCCAACTCCCACCTGCACATGCCCATGT
ACTTCTTCCTGGGGAACCTCTCCTTCCTGGACATCTGCTATACAACATCCTCAGTCCCCCTCATCCTTG
ACAGCTTCTTGACCCCCAGGAAAACCATCTCCTTCTCAGCCTGTGCAGTGCAGATGTTCCTCTCCTTTG
CCATGGGAGCCACAGAGTGTGTTCTCCTGAGCATGATGGCGTTTGATCGCTACGTGGCCATCTGCAACC
CCCTTAGGTACCCTGTGGTCATGAGCAAGGCTGCCTACATGCCCCATAAGGCTGCCGGCTCCTGGGTAG
CTGGAAGCACTGCTTCCATGGTGCAGACATCCCTTGCAATGAGGCTGCCCTTCTGTGGAGACAACATCA
TCAACCACTTCACCTGTGAGATTCTGGCTGTCCTGAAGTTGGCCTGTGCTGATATCTCTGTCAATGTGA
TCAGTATGGGAGTGACCAATGTGATCTTCCTGGGGGTCCCGGTTCTGTTCATCTCTTTCTCCTATGTCT
TCATCATTGCCACCATCCTGAGGATCCCCTCAGCTGAGGGGAGGAAAAAGGCCTTCTCCACCTGCTCTG
CCCACCTCACAGTCGTGGTCATCTTCTATGGGACCATCCTCTTCATGTATGGGAAGCCCAAGTCTAAGG
ACCCGCTGGGGGCAGACAAGCAAGACCTTGCAGACAAACTCATTTCCCTTTTCTATGGGGTGGTGACCC
CCATGCTCAACCCCATCATCTACAGCCTGAGGAACAAGGATGTAAAGGCTGCTGTGAGGGACTTGATAT
TTCAGAAATGCTTTGCCTAGTGATGTTTGGGGGAACAGATGTCCTCATAGCTC
A disclosed encoded GPCRIa protein has 319 amino acid residues, referred to as
the
GPCRIa protein. The GPCRla protein was analyzed for signal peptide prediction
and cellular
localization. SignalP results predict that GPCRla is cleaved between position
44 and 55 of
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SEQ ID N0:2, i.e., at the slash in the amino acid sequence GNG-VL. Psort and
Hydropathy
profiles also predict that GPCR1 contains a signal peptide and is likely to be
localized at the
plasma membrane (certainty of 0.6000). The disclosed GPCRI polypeptide
sequence is
presented in Table 1B using the one-letter amino acid code.
Table 1B. Encoded GPCRla protein sequence (SEQ ID N0:2).
MVSANQTASVTEFILLGLSAHPKLEKTFFVLILLMYLVILLGNG/VLILMTVSNSHLHMPMYFFLGNLS
FLDICYTTSSVPLILDSFLTPRKTISFSACAVQMFLSFAMGATECVLLSMMAFDRYVAICNPLRYPVVM
SKAAYMPHKAAGSWVAGSTASMVQTSLAMRLPFCGDNIINHFTCEILAVLKLACADISVNVISMGVTNV
IFLGVPVLFISFSYVFIIATILRIPSAEGRKKAFSTCSAHLTVVVIFYGTILFMYGKPKSKDPLGADKQ
DLADKLISLFYGVVTPMLNPIIYSLRNKDVKAAVRDLIFQKCFA
GPCR1 a was initially identified on chromosome 9 with a TblastN analysis of a
proprietary sequence file for a G-protein coupled receptor probe or homolog,
which was run
against the Genomic Daily Files made available by GenBank. A proprietary
software program
(GenScanTM) was used to further predict the nucleic acid sequence and the
selection of exons.
The resulting sequences were further modified by means of similarities using
BLAST
searches. The sequences were then manually corrected for apparent
inconsistencies, thereby
obtaining the sequences encoding the full-length protein.
A region of the GPCR1 a nucleic acid sequence has 873 of 1016 bases (85%)
identical
to a sequence coding for a partial Mus musculus olfactory receptor mRNA (1731
bp), with an
E-value of 3.6e 160 (GENBANK-ID: Aj 133427). In all BLAST alignments herein,
the "E-
value" or "Expect" value is a numeric indication of the probability that the
aligned sequences
could have achieved their similarity to the BLAST query sequence by chance
alone, within the
database that was searched. For example, the probability that the subject
("Sbjct") retrieved
from the GPCRla BLAST analysis, e.g., the Mus musculus olfactory receptor,
matched the
Query GPCRIa sequence purely by chance is 3.6x10-160.
A BLASTX search was performed against public protein databases. The full amino
acid sequence of the protein of the invention was found to have 274 of 316
amino acid
residues (86%) identical to, and 295 of 316 residues (93%) positive with, the
319 amino acid
olfactory receptor protein from Mus musculus (ptnr:TREMBLNEW-ACC:CAB55592, E=
1.8
e-142). The disclosed GPCRIa protein (SEQ ID N0:2) has good identity with a
number of
olfactory receptor proteins. For example, GPCRla has 256/316 (81%) amino acids
identical
with the 319 amino acid Mus musculus olfactory receptor 37a protein, and
376/316 (87%)
amino acids identical, to (Expect = e-128, gig 11276075~ref~IVP-062346.1 ~)
olfactory receptor
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37a from Mus musculus. The disclosed protein is also similar to the olfactory
proteins
disclosed in Table 1C.
Table 1C. BLAST
results for
GPCRla
Gene Index/ Protein/ OrganismLength IdentityPositivesExpect
Identifier (aa)
gi1114649811refINP_Olfactory 319 255/318 275/318 e-127
062349.11 Receptor (OR) (80$) (86~)
37e
Mus musculus
gi1112760771refINPOR 37b 318 248/317 276/317 e-127
- Mus musculus (78~) (86~)
062347.11
gi1112760791refINP-OR 37c 318 251/319 274/314 e-127
062348.11 Mus musculus (79~) (86~)
gi1100926691refINPOR Family 2, 309 238/306 261/306 e-118
- Subfamily S, (77~) (84$)
063950.11
member 2
Homo sapiens
gi137696241gbIAA064OR 227 206/227 217/227 e-102
588.11 (AF091565)Rattus norvegicus (90~) (94g)
GPCR1 b
A disclosed GPCRIb (also referred to as ba32713 A) nucleic acid of 1015
nucleotides
is shown in Table 1D. An open reading frame was identified beginning with an
ATG
initiation codon at nucleotides 17-19 and ending with a TAG codon at
nucleotides 971-973. A
putative untranslated region upstream from the initiation codon and downstream
from the
termination codon is underlined in Table 1D, and the start and stop codons are
in bold letters.
Table 1D. GPCRlb Nucleic acid sequence (SEQ ID N0:3).
ACAGAGGAGATACACTATGGTAAGTGCCAATCAGACAGCCTCTGTGACCGAGTTTATTCTCCTGGGCCTC
TCTGCCCACCCAAAGCTGGAGAAAACGTTCTTTGTGCTCATCCTGCTGATGTACCTGGTGATCCTACTGG
GCAATGGGGTCCTCATCCTGATGACTGTGTCCAACTCCCACCTGCACATGCCCATGTACTTCTTCCTGGG
GAACCTCTCCTTCCTGGACATCTGCTATACAACATCCTCAGTCCCCCTCATCCTTGACAGCTTCTTGACC
CCCAGGAAAACCATCTCCTTCTCAGCCTGTGCAGTGCAGATGTTCCTCTCCTTTGCCATGGGAGCCACAG
AGTGTGTTCTCCTGAGCATGATGGCGTTTGATCGCTACGTGGCCATCTGCAACCCCCTTAGGTACCCTGT
GGTCATGAGCAAGGCTGCCTACATGCCCATAGCTGCCGGCTCCTGGGTAGCTGGAAGCACTGCTTCCATG
GTGCAGACATCCCTTGCAATGAGGCTGCCCTTCTGTGGAGACAACATCATCAACCACTTCACCTGTGAGA
TTCTGGCTGTCCTGAAGTTGGCCTGTGCTGATATCTCTGTCAATGTGATCAGTATGGGAGTGACCAATGT
GATCTTCCTGGGGGTCCCGGTTCTGTTCATCTCTTTCTCCTATGTCTTCATCATTGCCACCATCCTGAGG
ATCCCCTCAGCTGAGGGGAGGAAAAAGGCCTTCTCCACCTGCTCTGCCCACCTCACAGTCGTGGTCATCT
TCTATGGGACCATCCTCTTCATGTATGGGAAGCCCAAGTCTAAGGACCCGCTGGGGGCAGACAAGCAAGA
CCTTGCAGACAAACTCATTTCCCTTTTCTATGGGGTGGTGACCCCCATGCTCAACCCCATCATCTACAGC
CTGAGGAACAAGGATGTAAAGGCTGCTGTGAGGGACTTGATATTTCAGAAATGCTTTGCCTAGTGATGTT
TGGGGGAACAGATGTCCTCATAGCTCTTTGCCTCT
In a search of sequence databases, it was found, for example, that the nucleic
acid
sequence has 869 of 1015 bases (85%) identical to a 1731 by Mus musculus OR
37d
pseudogene (GENBANK-ID: MMU133427~acc:AJ133427, E = 5.5 e-161). It was also
found
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that the nucleic acid has 505 of 791 bases identical (63%) to a partial human
mRNA for
olfactory receptor protein (GENBANK-ID:HSOLFMF~acc:Y14442, E= 9.5 e-49).
The encoded protein having 318 amino acid residues is presented using the one-
letter
code in Table 1 E. The full amino acid sequence of the protein of the
invention was found to
have 274 of 315 amino acid residues (86%) identical to, and 296 of 315
residues (93%)
positive with, a 319 amino acid residue olfactory receptor protein from Mus
musculus (ptnr:
TREMBLNEW-ACC:CAB55592, E= 6.3 e-144.
The disclosed GPCRIb protein differs from the disclosed GPCRIa protein at only
two
positions. At positions 145 and 146, GPCRla has HK, while GPCRIb has a
deletion (~) and
an I.
Table 1E. Encoded GPCRlb protein sequence (SEQ ID N0:4).
MVSANQTASVTEFILLGLSAHPKLEKTFFVLILLMYLVILLGNG/VLILMTVSNSHLHMPMYFFLGNLSFL
DICYTTSSVPLILDSFLTPRKTISFSACAVQMFLSFAMGATECVLLSMMAFDRYVAICNPLRYPVVMSKA
AYMPIAAGSWVAGSTASMVQTSLAMRLPFCGDNIINHFTCEILAVLKLACADISVNVISMGVTNVIFLGV
PVLFISFSYVFIIATILRIPSAEGRKKAFSTCSAHLTVWIFYGTILFMYGKPKSKDPLGADKQDLADKL
ISLFYGWTPMLNPIIYSLRNKDVKAAVRDLIFQKCFA
A PSORT analysis predicts that the ba32713 A protein (GPCRIb) is localized in
the
plasma membrane with a certainty of 0.6000, or with lower certainty in the
mitochondrial
inner membrane, the mitochondrial intermembrane space or the Golgi body. It is
also
predicted that the protein has a signal peptide with the most likely cleavage
site between
residues 44 and 45: GNG-VL, indicated by a slash in Table 1E.
A BLASTX search was performed against public protein databases. The full amino
acid sequence of the protein of the invention was found to have 274 of 315
amino acid
residues (86%) identical to, and 296 of 315 residues (93%) positive with, the
319 amino acid
olfactory receptor protein from Mus musculus (ptnr:TREMBLNEW-ACC:CAB55592, E=
6.3
e-144). The disclosed GPCRIb protein (SEQ ID N0:4) has good identity with a
number of
olfactory receptor proteins, as shown in Table 1F.
Table BLAST
1F. results
for
GPCRlb
Gene Index/ Protein/OrganismLengthIdentityPositivesExpect
Identifier (aa) ($)
gi1112760751refINP-OR 37a 319 256/315277/315 e-129
062346.1 Mus sculus (81~) (87~)
mu
gi1119649811refINP-(OR) 37e 319 255/317276/317 e-128
062349.11 Mus (80~) (86~)
musculus
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gi~11276077~refINP_OR 37b 318 248/316 277/316 e-128
062347.11 Mus musculus (78~) (87~)
gi~112760791refINPOR 37c 318 251/313 275/313 e-128
_ Mus musculus (80~) (87~)
062348.11
gip0092669)refINP-OR Family 2, 309 238/305 262/305 e-119
063950.11 Subfamily S, (78~) (85~)
member 2
Homo sapiens
GPCR1 c
A disclosed GPCRIc (also referred to as ba113a10 C) nucleic acid of 1003
nucleotides is shown in Table 1G. An open reading frame was identified
beginning with an
ATG initiation codon at nucleotides 26-28 and ending with a TGA codon at
nucleotides 974-
976. Putative untranslated regions S' to the start codon and 3' to the stop
codon are underlined
in Table 1 G and the start and stop codons are in bold letters.
In a search of sequence databases, it was found, for example, that the nucleic
acid
sequence has 719 of 899 bases (79%) identical to a Mus musculus GPCR mRNA
(GENBANK-m: AJ133428, E = 1.9e-120).
Table 1G. GPCRIc Nucleic acid sequence (SEQ ID NO:S).
TTTGTACAAGTGACATAGAAACACCATGGTCAGTTCC
AATCAGACCTCCCCTGTGCTGGGGTTCCTTCTCCTGGGGCTCTCTGCCCATCCAAAGCTGGAGAAGACAT
TCTTCGTGCTCATCCTGCTGATGTACCTGGTGATCCTACTGGGCAATGGGGTCCTCATCCTGGTGACCAT
CCTTGACTCCCGCCTGGACACACCCATGTACTTCTTCCTGGGGAACCTCTCCTTCCTGGACATCTGCTAT
ACAACCTCCTCATCCTTGACAGCTTCCCTGACCCCCAGGAAAACCATCTCCTTCTCAGCCTGTGCAGTAC
AGATGTTCCTCTCCCTTGCCATGGGAGCCACAGAGTGTGTTCTCCTGAGCATGATGGCGTTTGATCGCTA
CGTGGCCATCTGCAACCCCCTTTGGTACCCTGAAGTCATGAACAAAGCTACTTATGTGCCCATGGCTGCT
GGCTCCTGGGTAGCTGGAAGCCTCACTGCCATGGTGCAGACACCCCTTGCATTGAGGCTGCCCTTCTGTG
GAGACAACATCATCAATCACTTCACCTGTGAGATTCTGGCTGTCCTGAAGTTGGCCTGTGCTGATATCTC
TGTCAATGTGATCAGTATGGGAGTGGCCAATGTGATCTTCCTGGGGGTCCCTGTTCTGTTCATCTCTTTC
TCCTATGTCTTCATCATTGCCACCATCCTGAGGATCCCCTCAGCTGAGGGGAGGAAAAAGGCCTTCTCCA
CCTGCTCTGCCCACCTCACTGTCGTGATCGTCTTCTACGGGACCATCCTCTTCATGTACGGGAAGCCCAA
GTCTAAGGACCCACTGGGAGCAGACAAACAGGACCTTGCAGACAAACTCATTTCCCTTTTCTATGGGGTG
GTGACCCCCATGCTCAACCCCATCATCTACAGCCTGAGGAACAAGGAAGTGAAGGCTGCTGTGAGGAACC
TGGTATTTCAGAAACGCTTCCTGCAGTGATGGTGGAGGGGTCCTGATGGCTCTGTG
The disclosed GPCRlc protein having 316 amino acid residues is presented using
the
one-letter code in Table 1H. An analysis using the PSORT program predicts that
the
ba113a10 C protein localizes in the plasma membrane with a certainty=0.6400;
it may also be
localized in the Golgi body with a moderate certainty. It is also predicted
that protein has a
signal peptide whose most likely cleavage site is between residues 44 and 45:
GNG-VL,
indicated by a slash in Table 1H.
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Table 1H. Encoded GPCRlc protein sequence (SEQ ID N0:6).
MVSSNQTSPVLGFLLLGLSAHPKLEKTFFVLILLMYLVILLGNG/VLILVTILDSRLDTPMYFFLGN
LSFLDICYTTSSSLTASLTPRKTISFSACAVQMFLSLAMGATECVLLSMMAFDRYVAICNPLWYPEVMNK
ATYVPMAAGSWVAGSLTAMVQTPLALRLPFCGDNIINHFTCEILAVLKLACADISVNVISMGVANVIFLG
VPVLFISFSYVFIIATILRIPSAEGRKKAFSTCSAHLTVVIVFYGTILFMYGKPKSKDPLGADKQDLADK
LISLFYGVVTPMLNPIIYSLRNKEVKAAVRNLVFQKRFLQ
A BLASTX search was performed against public protein databases. The full amino
acid sequence of the protein of the invention was found to have 270 of 317
amino acid
residues (85%) identical to, and 287 of 317 residues (90%) positive with, a
319 amino acid
residue OR from Mus musculus (ptnr:TREMBLNEW-ACC:CAB55596, E= 5.2 e-138). The
disclosed GPCRIb protein (SEQ ID N0:6) has good identity with a number of
olfactory
receptor proteins, as shown in Table 1I.
Table 1I. BLAST
results for
GPCRlc
Gene Index/ Protein/ LengthIdentityPositivesExpect
Identifier Organism (aa) (~k)
gi1114649811refINP-OR 37e 319 242/317 257/317 e-120
062349.11 Mus musculus (76~) (80~)
gi1112760751refINPOR 37a 319 291/319 256/319 e-119
- Mus musculus (75~) (87~)
062346.11
gi1112760771refINP-OR 37b 318 236/319 255/319 e-116
062347.11 Mus musculus (78~) (87g)
gi1112760791refINP-OR 37c 318 235/308 250/308 e-116
062348.11 Mus musculus (76~) (80g)
gi1100926691refINP_OR Family 309 220/309 242/309 e-108
2,
063950.11 Subfamily (71~) (78~)
S,
member 2
Homo Sapiens
The amino acids differences between the three GPCR1 proteins are shown in
Table 1J.
Deletions are marked by a delta (D). The differences between the three
proteins appear to be
localized to a few distinct regions. Thus, these proteins may have similar
functions, such as
serving as olfactory or chemokine receptors (see below).
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Table1J. for Proteins
Differences GPCRl
Position4 8 9 11 12 14 49 51 52 53 55 57 58
GPCRlaA A S T E I M V S N H H M
GPCRIbA A S T E I M V S N H H M
GPCRlcS S P L G L V I L D R D T
Position79 80 81 82 89 85 86 106132 135 138 191 193
GPCRlaV P L I D S F F R V S A M
GPCRlbV P L I D S F F R V S A M
GPCRlcS D D D T A S L W E N T V
Position145 146156 157 158 163 2D4251 252 304 311 313
166
GPCRlaH K T A S S M T V I D D I
GPCRlb0 I T A S S M T V I D D I
GPCR1CD M L T A P L A I V E N V
Position317 319320
GPCRIaC A D
GPCRlbC A D
GPCRlcR L Q
A ClustalW analysis comparing disclosed proteins of the invention with related
OR
protein sequences is given in Table 1K, with GPCRla shown on line 1, and
GPCRIc on line 2.
In the ClustalW alignment of the GPCR1 a protein, as well as all other
ClustalW
analyses herein, the black outlined amino acid residues indicate regions of
conserved sequence
(i.e., regions that may be required to preserve structural or functional
properties), whereas non-
highlighted amino acid residues are less conserved and can potentially be
mutated to a much
broader extent without altering protein structure or function. Unless
specifically addressed as
GPCRla GPCRlb, or GPCRIc, any reference to GPCR1 is assumed to encompass all
variants. Residue differences between any GPCRX variant sequences herein are
written to
show the residue in the "a" variant and the residue position with respect to
the "a" variant.
GPCR residues in all following sequence alignments that differ between the
individual GPCR
variants are highlighted with a box and marked with the (o) symbol above the
variant residue
in all alignments herein. For example, the protein shown in line 1 of Table 1K
depicts the
sequence for GPCRla, and the positions where GPCRlb differs are marked with a
(o) symbol
and are highlighted with a box. All GPCRl proteins have significant homology
to olfactory
receptor (OR) proteins: 37a, 37b, 37e, and 37c from Mus musculus and to a
human OR
protein member 2 from family 2, subfamily S (see also Tables 1C, 1F, and 1I).
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Table 1K. ClustalW Analysis of GPCRl
1) Novel GPCRIa (SEQ ID N0:2)
2) Novel GPCRIc (SEQ ID NO: 6)
3). gill 1276075~ref~NP_062346.1 ~ olfactory receptor 37a Mus musculus (SEQ ID
N0:39)
4) gig 11276077~ref~NP_062347.1 ~ olfactory receptor 37b Mus musculus (SEQ ID
N0:40)
5) gill 1464981 ~ref~NP_062349.1 ~ olfactory receptor 37e Mus musculus (SEQ 1D
N0:41)
6) gi~11276079~reflNP 062348.1 olfactory receptor 37c Mus musculus (SEQ ID
N0:42)
7) gi~10092669~reflNP 063950.1 OR, fam. 2, subfam. S, mem. 2 H. Sapiens (SEQ
ID N0:43)
10
20
30
40
5D
60
1
1
1
1
1
1
GPCRla . . I....I ....I....I. ... I. ...1. ... .... . ... .. . ... ..
..
S QTAS TEI ~ . . 5:
. ~
~
GPCRlc S QTSP L~ D '
gi1112760751iD ETAP S
i I 11276077E G -QST E~ D
I
g E ~ ~ SSI
gi1119699811 R- ~
KTTP[r
11112760791D V -QTT TE=' S S
u
g11100926691----------M - _
70 80 90
100
110
120
.I.. .I.. . .I. . I.. I.. . .I.. .I. ..I. ..I
GPCRla .I. .I.. ~S . . .I. S
~ ' ~
T
GPCRlc ~ S--- T L S
S
g11112760751 ~ ' y e S
g11112760771 ~ 3' ,
g11114649811 ~
g11112760791
g E ~.m nv ~a
11100926691 mw
130 190 150
160
170
180
.I.. .. . ...l..
.I .I. .I....oo .I..
.I..
.I..
.I..
.I..
.I
GPCRla ~ ' ~S n~ ~
~ ST
HK ~
~ ~T
GPCRlc ~ ' E ~ ~ SLT,:ATP
T ~ '
~
I
g11112760751~ ' ITN
v ~
S T
v
g11112760771~ ' S S GAN
~ ~_
w I
v
g11119649811~ ' S
1
SIT~T
I
~~
g1 I 112760791~ ' S '
q~ I
~
I
GAN
g11100926691~ ' ' "" S G
S I m
I HT
190 200 210
220
230
240
I
I
I
I
I
GPCRla .I.. .. .I. . . . I.. .I .. . .. .. ..
.I ~ ~ .I. .I. . . . . .
T . .
S
GPCRlc ~ ~
a
S
g11112760751 ~
v ~ r
F v
g11112760771 ~ F
g11114649811 ~ FV
g11112760791 ~ F
g11100926691 ~ E 5
T
250 260 270
I 280
290
300
I
I
I
I
I
I
I
GPCRld .I. . .. .I. . .. .. .. .. .. . ... ..
.I .I. .~y . . . w. . .
~I v v v
GPCRlc
g11112760751 ,.
g11112760771 ~ ~ ~ wr~W
g11114699811 : , , w
g11112760791 a
g1 I 100926691 ~ ~5'~ P
310 320
.I.. .. I
GPCRI a I .I CFA-
~
GPCRlc _ RFLQ
g11112760751 ~ HLTE
g11112760771 ~ ~ CLIQ
g11114649811 ~ HFKw
T
g11112760791 ~ SH CLTF
g11100926691 ~ RP GFTQ
The GPCR1 proteins also have regions of identity with a 321 amino acid human
transmembrane receptor (gi~6691937~emb~CAB65797.1 ~ bA150A6.2, novel 7
transmembrane
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receptor (rhodopsin family), olfactory receptor- like protein (hs6M1-21)), as
shown in line 3 of
in Table 1L (SEQ ID NO: 44).
Table 1L.
Alignment
of GPCRla
& c with
SEQ ID N0:44
10 20 90 50 60
30
.I....I....I....1....I....1.... 1....1....1....1....1....1
GPCRla ' S S".
GPCR1C S ~ ~P ~ L~ D
g1166919371 L NLN FCT ~ n
ER ~ FLL T P
~ T
F
70 80 100 110 120
90
.I.. I.. I.. .I.. .. I
GPCRIa .I.. I.. .I- .I.
. I.. I
GPCR1C ~ .I
gi166919371 .
~
fi~ t FFVG~a
'Li
D
S---
T
'S
Q"",
HL
K~
$
130 190 160 170 180
00
150
.I....I....I....I....I....1.... 1....1....1....1....1 ....1
GPCRla N' ~H T ~ S
GPCRlC N~ 'E T P
'T ~ '_
g1166919371 w n NA'' ~N F
' ~' H
N' ' TFC
S ~> '
LCNQ-" ~N
SC
190 200 220 230 240
210
.I....I....I....I....I....1.... 1....1....1....1....1 ....1
GPCRla
N
GPCR1C '
.v
gi166919371 N C
PPa I
S G
T E~LS
FIGWT
250 260 280 290 300
270
.I....I....I....I....I....1.... 1....1....1....1....1 ....1
GPCRIa
GPCRlC U
gi166919371 ~ F' 'AT ---- L
'I S
TYS
KK
310 320
.1....1....1....1....1....
GPCRla ~ D I C ---------
GPCRlc E LQ-_-_____-
gi166919371 n' E
T GS'~'PPISSLDSKLTY
The presence of identifiable domains in GPCR1, as well as all other GPCRX
proteins,
was determined by searches using software algorithms such as PROSITE, DOMAIN,
Blocks,
Pfam, ProDomain, and Prints, and then determining the Interpro number by
crossing the
domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/
interpro).
DOMAIN results, e.g., for GPCRIa as disclosed in Table 1M, were collected from
the
Conserved Domain Database (CDD) with Reverse Position Specific BLAST analyses.
This
BLAST analysis software samples domains found in the Smart and Pfam
collections. For
Table 1M and all successive DOMAIN sequence alignments, fully conserved single
residues
are indicated by black shading and "strong" semi-conserved residues are
indicated by grey
shading. The "strong" group of conserved amino acid residues may be any one of
the
following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY,
FYW.
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Table 1M lists the domain description from DOMAIN analysis results against
GPCRIa. The region from amino acid residue 53 through 239 (SEQ ID N0:2) most
probably
(E = 2e 19) contains a "seven transmembrane receptor (rhodopsin family)
fragment" domain,
aligned here with residues 12-180 of the 7tm 1 entry (TM7, SEQ ID N0:45, see
Table 1N for
the complete sequence) of the Pfam database. This indicates that the GPCRI
sequence has
properties similar to those of other proteins known to contain this domain as
well as to the 377
amino acid 7tm domain itself. GPCRIa also has identity to another region of
the TM7 protein.
The region from amino acid residue 226 through 298 (of SEQ ID N0:2) aligns
with amino
acid residues 310-377 of TM7 (E = 3e~). GPCRlb and GPCRIc also align to this
domain:
residues 53-238 of GPCRIb align with residues 12-180 of TM7 (E = 6e ~9) and
residues 225-
297 of GPCRIb align with residues 310-377 of TM7 (E = 3e~); residues 55-235 of
GPCRIc
align with residues 14-180 of TM7 (E = 2e 12) and residues 222-294 of GPCRIc
align with
residues 310-377 of TM7 (E = 2e~).
Table 1M. Domain Analysis of GPCRla
Sbjct: 7 transmembrane receptor Irhodopsin family) fragment
GnlIPfamIpfam00001; Length = 377
Score = 89.7 bits 221 Ex ect = 2e-19
....I....I....~....I....I....I....~....~....I....I....I....I
GPCRla -----------NSH HMPMYF~G~~FA~CY~SS~;.L, DSFPRKT~FSAQ
TM7 GNVLVCMAVSRE QTTTN L m LVp~L YLE GEWK~RIH D F
.I.. .I.. .I.. .I.. .I.. .I.. ~I~~ ~~~~ .I.. .1....I.. .I
TM7Rla ~T~DCTASIN~CPI~'.T CN~.L~PITRY S RRTVMIIS LSS I CPM
..l~.J.I....I....I....I....I....1....1....1....1....1....1....1
GPCRla TSLAMRLPFCGDNIINHFT~E~AVLKLACADI~V~I~G~IVFLGV~,FISF
TM7 LFGLNNTDQNE-------- I~NP-------- F Y~SI FY --- TLL
.I.. .1....I.. .I....I....I....I....~....I....I....I....I
GPCRla IAT L --IPSAE KKA-__________________--_-_________________
TM7 K~YIV~R~RRKRVNTK SSRAFRANLKAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRV
The representative member of the 7 transmembrane receptor family is the D2
dopamine receptor from Bos taurus (SWISSPROT: locus D2DR BOVIN, accession
P20288;
gene index 118205). The D2 receptor is an integral membrane protein and
belongs to Family
1 of G-protein coupled receptors. The activity of the D2 receptor is mediated
by G proteins
which inhibit adenylyl cyclase. Chio et al., Nature 343:255-269 (1990).
Residues 51-427 of
this 444 amino acid protein are considered to be the representative TM7
domain, shown in
Table 1N.
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Table 1N Amino Acid sequence for TM7 (SEQ ID N0:45)
GNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWVVYLEVVGEWKFSRIHCDIF
VTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMIAIVWVLSFTISCPM
LFGLNNTDQNECIIANPAFVVYSSIVSFYVPFIVTLLVYIKIYIVLRRRRKRVNTKRSSR
AFRANLKAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRVEAARRAQELEMEMLSSTSPP
ERTRYSPIPPSHHQLTLPDPSHHGLHSTPDSPAKPEKNGHAKTVNPKIAKIFEIQSMPNG
KTRTSLKTMSRRKLSQQKEKKATQMLAIVLGVFIICWLPFFITHILNIHCDCNIPPVLYS
AFTWLGYVNSAVNPIIY
The 7 transmembrane receptor family includes a number of different proteins,
including, for example, serotonin receptors, dopamine receptors, histamine
receptors,
andrenergic receptors, cannabinoid receptors, angiotensin II receptors,
chemokine receptors,
opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory
receptors (OR), and
the like. Some proteins and the Protein Data Base Ids/gene indexes include,
for example:
rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805,
231454,
1168221, 398971, 112806); G protein-coupled receptors (119130, 543823,
1730143, 132206,
137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors
(544463, 462208);
c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins
(129193,
129197, 129203); and olfactory receptor-like proteins (129091, 1171893,
400672, 548417);
Expression information for GPCRX RNA was derived using tissue sources
including,
but not limited to, proprietary database sources, public EST sources,
literature sources, andlor
RACE sources, as described in the Examples.
The nucleic acids and proteins of GPCR1 are useful in potential therapeutic
applications implicated in various GPCR- or olfactory receptor (OR)-related
pathologies
and/or disorders. For example, a cDNA encoding the G-protein coupled receptor-
like protein
may be useful in gene therapy, and the G-protein coupled receptor-like protein
may be useful
when administered to a subject in need thereof. The novel nucleic acid
encoding GPCRl
protein, or fragments thereof, may further be useful in diagnostic
applications, wherein the
presence or amount of the nucleic acid or the protein are to be assessed.
These materials are
further useful in the generation of antibodies that bind immunospecifically to
the novel
substances of the invention for use in therapeutic or diagnostic methods. The
GPCRX nucleic
acids and proteins are useful in potential diagnostic and therapeutic
applications implicated in
various diseases and disorders described below and/or other pathologies. For
example, the
compositions of the present invention will have efficacy for treatment of
patients suffering
from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects,
aortic stenosis,
atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus
arteriosus, pulmonary
stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases,
tuberous sclerosis,
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scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital
adrenal hyperplasia,
fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura,
immunodeficiencies, graft versus host disease, bronchial asthma, and other
diseases, disorders
and conditions of the like. By way of nonlimiting example, the compositions of
the present
invention will have efficacy for treatment of patients suffering from
neoplasm,
adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response,
AIDS, asthma,
Crohn's disease, multiple sclerosis, and Albright Hereditary Ostoeodystrophy.
Additional
GPCR-related diseases and disorders are mentioned throughout the
Specification.
Further, the protein similarity information, expression pattern, and map
location for
GPCR1 suggests that GPCR1 may have important structural and/or physiological
functions
characteristic of the GPCR family. Therefore, the nucleic acids and proteins
of the invention
are useful in potential diagnostic and therapeutic applications and as a
research tool. These
include serving as a specific or selective nucleic acid or protein diagnostic
and/or prognostic
marker, wherein the presence or amount of the nucleic acid or the protein are
to be assessed, as
well as potential therapeutic applications such as the following: (i) a
protein therapeutic, (ii) a
small molecule drug target, (iii) an antibody target (therapeutic, diagnostic,
drug
targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy
(gene delivery/gene
ablation), and (v) a composition promoting tissue regeneration in vitro and in
vivo (vi)
biological defense weapon.
These materials are further useful in the generation of antibodies that bind
immuno-
specifically to the novel GPCR1 substances for use in therapeutic or
diagnostic methods.
These antibodies may be generated according to methods known in the art, using
prediction
from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies"
section below.
GPCR2
An additional GPCR-like protein of the invention, referred to herein as GPCR2,
is an
Olfactory Receptor ("OR")-like protein. The novel nucleic acid of 1254
nucleotides
(11612531-1, SEQ >D N0:7) encoding a novel G-protein coupled receptor-like
protein is
shown in Table 2A. SeqCalling fragments for GPCR2 came from placenta,
indicating that it
may be expressed in tissues important for female health.
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Table 2A. GPCIt2 Nucleotide Sequence (SEQ ID NO:'n
ACGCGCTTCGACATACTATTCCTGGTGGGCTCTTCCTACATATCAGGTCGTTAAATAAGCTGCCAGATTTCTGCCTTTA
CAGCCCAA
GGAGCTTGTCATGGACCATGGGCATGGAGGGTCTTCTCCAGAACTCCACTAACTTCGTCCTCACAGGCCTCATCACCCA
TCCTGCCT
TCCCCGGGCTTCTCTTTGCAATAGTCTTCTCCATCTTTGTGGTGGCTATAACAGCCAACTTGGTCATGATTCTGCTCAT
CCACATGG
ACTCCCGCCTCCACACACCCATGTACTTCTTGCTCAGCCAGCTCTCCATCATGGATACCATCTACATCTGTATCACTGT
CCCCAAGA
TGCTCCAGGACCTCCTGTCCAAGGACAAGACCATTTCCTTCATGGGCTGTGCAGTTCAGATCTTCCTCTACCTGACCCT
GATTGGAG
GGGAATTCTTCCTGCTGGGTCTCATGGCCTATGACCGCTATGTGGCTGTGTGCAACCCTCTACGGTACCCTCTCCTCAT
GAACCGCA
GGGTTTGCTTATTCATGGTGGTCGGCTCCTGGGTTGGTGGTTCCTTGGATGGGTTCATGCTGACTCCTGTCACTATGAG
TTTCCCCT
TCTGTAGATCCCGAGAGATCAATCACTTTTTCTGTGAGATCCCAGCCGTGCTGAAGTTGTCTTGCACAGACACGTCACT
CTATGAGA
CCCTGATGTATGCCTGCTGCGTGCTGATTATCCCTCTATCTGTCATCTCTGTGTCCTACACGCACATCCTCCTGACTGT
CCACAGGA
TGAACTCTGCTGAGGGCCGGCGCAAAGCCTTTGCTACGTGTTCCTCCCACATTATGGTGGTGAGCGTTTTCTACGGGGC
AGCTTTCT
ACACCAACGTGCAGCCCCACTCCTACCACACTCCAGAGAAAGATAAAGTGGTGTCTGCCTTCTACACCATCCTCACCCC
CATGCTCA
ACCCACTCATCTACAGCTTGAGGAATAAAGATGTGGCTGCAGCTCTGAGGAAAGTACTAGGGAGATGTGGCTCCTCCCA
GAGCATCA
GGGTGATGACTGTGTGATCAGGAAGGACTAGCAGGGACTCCCAGAGTATCAGCGTGGTGACTATGATCAGGAAGGACTA
GTGGGGAC
TCCTAGAGCATCAGGGTGGCGACTGTGATCAGGAAGGACTAGCAAGGACTAGCGCAAACATCTGCGGTGCTGCGGCCAA
TAACGCAG
CTATTACAGAAAATATGTTATTGGTTCTGAAGAAGT
An open reading frame (ORF) for GPCR2 was identified from nucleotides 105 to
1058. The disclosed GPCR2 polypeptide (SEQ ID N0:8) encoded by SEQ ID N0:7 is
318
amino acid residues and is presented using the one-letter code in Table 2B.
The GPCR2
protein was analyzed for signal peptide prediction and cellular localization.
SignalPep results
predict that GPCR2 is cleaved between position 42 and 43 of SEQ ID N0:8, i.e.,
at the slash
in the amino acid sequence ITA-NL. Psort and Hydropathy profiles also predict
that GPCR2
contains a signal peptide and is likely to be localized at the plasma membrane
(certainty of
0.6400).
Table 2B. Encoded GPCIZ2 protein sequence (SEQ ID N0:8).
MGMEGLLQNSTNFVLTGLITHPAFPGLLFAIVFSIFVVAITA/NLVMILLIHMDSRLHTPMYFLLSQLS
IMDTIYICITVPKMLQDLLSKDKTISFMGCAVQIFLYLTLIGGEFFLLGLMAYDRYVAVCNPLRYPLLM
NRRVCLFMVVGSWVGGSLDGFMLTPVTMSFPFCRSREINHFFCEIPAVLKLSCTDTSLYETLMYACCVL
IIPLSVISVSYTHILLTVHRMNSAEGRRKAFATCSSHIMVVSVFYGAAFYTNVQPHSYHTPEKDKVVSA
FYTILTPMLNPLIYSLRNKDVAAALRKVLGRCGSSQSIRVMTV
The full amino acid sequence of the protein of the invention was found to have
151 of
216 amino acid residues (69%) identical to, and 177 of 216 residues (81 %)
positive with, a
216 amino acid residue olfactory receptor from Homo sapiens (ptnr:SPTREMBL-
ACC:
043869) (E = 2.2 a 6'). The protein encoded by GPCR2 (SEQ ID N0:7) has
significant
homology to olfactory, odorant, and taste chemoreceptors and belongs to the
family of G-
Protein coupled receptors (GPCRs). This family of genes has been used as a
target for small
molecule drugs and GPCRs are expressed on the plasma membrane and are also a
suitable
target for protein drugs like therapeutic antibodies, cytotoxic antibodies and
diagnostic
antibodies.
As shown in Table 2C, BLAST analysis shows that GPCR2 has significant homology
with a number of olfactory receptors.
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Table 2C. GPCR2 BLAST results
Smallest
Sum
ReadingHigh Prob.
Sequences producing High-scoring Segment Frame Score P(N)
Pairs:
Ptnr:SPTREMBL-ACC:093869 OLFACTORY RECEPTOR827 1.8e-821
- HOMO SAPIENS...
Ptnr:SWISSPROT-ACC:P23275 OLFACTORY RECEPTOR712 2.7e-701
15 (0R3) - M...
Ptnr:SPTREMBL-ACC:Q90808 OLFACTORY RECEPTOR708 7.3e-701
4 - GALLUS GALLUS...
Ptnr:TREMBLNEW-ACC:CAB55593 OLFACTORY RECEPTOR706 1.2e-691
- Mus musculus...
Ptnr:TREMBLNEW-ACC:CAB55599 OLFACTORY RECEPTOR704 1.9e-691
- Mus musculus...
Ptnr:SPTREMBL-ACC:Q63399 OL1 RECEPTOR - 702 3.1e-691
RATTUS NORVEGICUS...
Ptnr:TREMBLNEW-ACC:CAB55592 OLFACTORY RECEPTOR698 8.3e-691
- Mus musculus...
Ptnr:TREMBLNEW-ACC:CAB55596 OLFACTORY RECEPTOR697 1.1e-681
- Mus musculus...
Ptnr:SWISSPROT-ACC:Q95156 OLFACTORY RECEPTOR-LIKE695 1.7e-681
PROTEIN...
Ptnr:SWISSPROT-ACC:Q13606 OLFACTORY RECEPTOR-LIKE694 2.2e-681
PROTEIN...
Ptnr:SWISSPROT-ACC:Q13607 OLFACTORY RECEPTOR-LIKE689 7.5e-681
PROTEIN...
Ptnr:TREMBLNEW-ACC:AAC69376 OLFACTORY RECEPTOR-LIKE685 2.0e-671
PROTEIN...
Other BLAST results including the sequences used for ClustalW analysis is
presented
in Table 2D.
Table 2D. BLAST
results for
GPCRZ
Gene Index/ Protein/ Length IdentityPositivesExpect
Identifier Organism (aa) (~) (s)
gi139833821gbIAADI3OR E3 223 156/223 184/223 2e-78
319.1 (AF102527)Mus musculus (69~) (81~)
gi129216281gbIAAC39OR 216 151/216 177/216 5e-75
611.1 (U86215) Homo Sapiens (69~) (81g)
gi1120074231gb1~1AG4T2 OR 316 155/298 205/298 2e-72
5196.1 (AF321234)Mus musculus (52~) (68$)
gi1120074241gb~AAG9T3 OR 315 156/309 209/304 2e-72
5197.1 (AF321234)Mus musculus (51~) (68~)
gi1120074251gbIAAG4T4 OR 319 152/304 207/304 9e-72
5198.11 (AF321234)Mus musculus (50$) (68$)
gi112007422~gbIAAG4T1 OR 316 156/305 207/305 2e-67
5195.1( (AF321234)Mus musculus (51~) (67$)
This information is presented graphically in the multiple sequence alignment
given in
Table 2E (with GPCR2 being shown on line 1) as a ClustalW analysis comparing
GPCR2 with
related protein sequences.
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Table 2E. Information for the ClustalW proteins:
1) Novel GPCR2 (SEQ ID N0:8)
2) gi~3983382~gb~AAD13319.1 ~ olfactory receptor E3 Mus musculus (SEQ ID
N0:46)
3) gi~2921628~gb~AAC39611.1 ~ olfactory receptor Homo Sapiens (SEQ ID N0:47)
4) gi~12007423~gb~AAG45196.1 ~ T2 olfactory receptor Mus musculus (SEQ ID
N0:48)
5) gi~12007424~gb~AAG45197.1 ~ T3 olfactory receptor Mus musculus (SEQ ID
N0:49)
6) gig 12007425~gb~AAG45198.1 ~ T4 olfactory receptor Mus musculus (SEQ ID
NO:50)
7) gi~12007422~gb~AAG45195.1 ~ T1 olfactory receptor Mus musculus (SEQ ID
NO:51 )
1~
20 30 40 50 60
....I....1....I....I....I....1....1....1....1....1....1..._1
GPCR2 MGMEGLLQNSTN~T~i THPAE'~G~F'~IVF'$~~ :V~T~L . H~S~HTm
gi139833821 ____________________________________________________________
gi129216281 _____-_______ _______________ _ _ ____________
gi1120079231 -MEPWNSTLGTD I~DDSGS'E C~TFT '~ ~ I ~T ~ TY H '
gi1120074241 -MEVCNSTLRS ~I iLDDND ~'E C~TIT ~YIr-~ T~ G ~T T H ~'
gi1120074251 -MEFRNSTMGNGCdI ~ DDSG~D C~TIT T,~T~ G1»,T: R
gi1120074221 -MELWNSTLES..~~I 'i: GSSS~'E C~IVT L ~ I G;L"L Tk H.''~'
70 80 90 100 110 120
.I....I....I....I....I....1....1....1....1....1....1....1
GPCR2 ~F~S~ iWT~a IC~T L S~ ~ Y IG F GL
z
war ' v r v
gi139833821 ----SH ~~~, I T Q T ~ F GL
gi129216281 ________ y,;,.1 T -V~.;. DQ T Y ~ F GL
gi1120079231 ~m T T I ~ S' _ G ~ DL
d V
gi1120074241 L m LT ~T ~ m ~ ~ GT~ DL S
gi 1120074251 L ~'~ ~~M-LT T ~ ~ . ~ ~ E GS DL
gi I 120074221 L ~n=~.r T ";T ~~GS~DL F
130 140 150 160 170 180
.I....I....I....I....I....1....1....1....1....1....1....1
~ ~, ~ w nw . .~ . N.
GPCR2 ~' P 'LR L G G DGFML P S '
gi13983382/ n ' ~P L CR ~- FG DGFLL PI ~S '
. .
'I N N
gi129216281 m P L R miA FG DGFLL PI S
gi1120074231 ~' ' N d ~RPS ~~ L LGY TY Q S LL
v
gi I 12007429 I T L Q C~. ~ S SALGY~IHY Q L
gi I 120079221 m . APT GTI ITAVGI-IVY H ' LL
190 200 210 220 230 240
.I....I....I....I....I....1....1....1....1....1....1....1
GPCR2 - T~ L T ° CC -- ' S TH L H FA
'v41
gi I 3983382 I ~ L T ~ CC L -~ F 1~TI1~ Y S FA
gi129216281 L T CC ° .F~V - T QC S FA
~ y_- l _ h
gi1120074231 'P' L'4'e 5L L P N LV
gi1120074241 P' ' ~ ~L ~L FP SL L P ~LV
gi1120074251 'P' ' E~ T L G S fir' L P LV
giI120074221 LP $ ~ L T T '~' T TL S L P LV
250 260 270 280 290 300
.1.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .1.. .I.. .I
GPCR2 ~ S, ~ F~TN 'H ~ ~ E
r--
" ~ ".
gi139833821 ~T T.' ~~1'T 'H ' -' T ____________
v ~ v x ~'1111-
giI29216281 ~ ~~ H ~ ~I(~T,- = _______________
'N k: N V
giI120074231 °T G ~ T ~ TG
giI120074241 G ~ ~IV 'S ~ ' w S ~ TG
giI120074251 I G G SL 'SQ ' m ~ TG
gi1120074221 L G ~~T 'S~L K ~N t IG
310 320
.1....1....1....1
GPCR2 ~R~L~CGSSQSIRVMTV
gi139833821 _________-__________
gi129216281 ___________________
gi1120074231 I L YIVPAHPTL--
ex
gi1120074241 7L RLSVQSTF----
gi1120074251 LGPAHFLGSSF
gi1120074221 ~ ~L .YILPAHLTL---
DOMAIN results for GPCR2 were collected from the Conserved Domain Database
(CDD) with Reverse Position Specific BLAST. This BLAST samples domains found
in the
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Smart and Pfam collections. The 7tm_1, a seven transmembrane receptor
(rhodopsin family),
was shown to have two segments with homology to GPCR2. The region from amino
acid
residue 43 through 238 aligns with amino acids 2 through 181 of the "seven
transmembrane
receptor (rhodopsin family) fragment" domain(SEQ ID N0:45, E= 2e-22), and
GPCR2 amino
acids 226-289 aligned with residues 313-377 of the 7tm-1 entry (SEQ ID N0:45,
E= .008) of
the Pfam database. This indicates that the GPCR2 sequence has properties
similar to those of
other proteins known to contain this domain as well as to the 7tm_1 domain
itself.
The disclosed GPCR2 is expressed in tissues that are important in female
reproductive
health and hence GPCR2 may serve as a drug target for, e.g., premature labor,
endometriosis,
and in vitro fertilization. The homology to the olfactory receptors suggests
that an endogenous
small molecule ligand regulates this gene and hence drugs structurally similar
to the
endogenous ligand could serve as agonists and antagonists to regulate the
biological effects of
GPCR2.
The nucleic acids and proteins of the invention are useful in potential
therapeutic
applications implicated in various GPCR-related pathological disorders and/or
OR-related
pathological disorders, described further below. For example, a cDNA encoding
the olfactory
receptor -like protein may be useful in gene therapy, and the olfactory
receptor -like protein
may be useful when administered to a subject in need thereof. By way of
nonlimiting
example, the compositions of the present invention will have efficacy for
treatment of patients
suffering from neoplasm, adenocarcinoma, lymphoma, uterus cancer, immune
response,
AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright Hereditary
Ostoeodystrophy.
Other GPCR-related diseases and disorders are contemplated.
The novel nucleic acid encoding the GPCR-like protein of the invention, or
fragments
thereof, may further be useful in diagnostic applications, wherein the
presence or amount of
the nucleic acid or the protein are to be assessed. These materials are
further useful in the
generation of antibodies that bind immunospecifically to the novel substances
of the invention
for use in therapeutic or diagnostic methods. These antibodies may be
generated according to
methods known in the art, using prediction from hydrophobicity charts, as
described in the
"Anti-GPCRX Antibodies" section below. In one embodiment, a contemplated GPCR3
epitope is from about amino acids105 to 140. These novel proteins can be used
in assay
systems for functional analysis of various human disorders, which will help in
understanding
of pathology of the disease and development of new drug targets for various
disorders.
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GPC)t3
An additional GPCR-like protein of the invention, referred to herein as GPCR3,
is an
Olfactory Receptor ("OR")-like protein. The novel nucleic acid was identified
on
chromosome 6 by TblastN using CuraGen Corporation's sequence file for GPCR
probe or
homolog, run against the Genomic Daily Files made available by GenBank. The
nucleic acid
was further predicted by the program GenScanTM, including selection of exons.
These were
further modified by means of similarities using BLAST searches. The sequences
were then
manually corrected for apparent inconsistencies, thereby obtaining the
sequences encoding the
full-length protein. The novel nucleic acid of 957 nucleotides (ba145122 B,
SEQ ID N0:9)
encoding a novel olfactory receptor-like protein is shown in Table 3A. An open
reading frame
(ORF) was identified beginning with an ATG initiation codon at nucleotides 10-
12 and ending
with a TGA codon at nucleotides 955-957. Putative untranslated regions
upstream from the
initiation codon and downstream from the termination codon are underlined in
Table 3A, and
the start and stop codons are in bold letters.
Table 3A. GPCIR3 Nucleotide Sequence (SEQ ID N0:9)
AGATTAGTCATGAAGGCCAACTACAGCGCAGAGGAGCGCTTTCTCCTGCTGGGTTTCTCCGACTGGCCTT
CCCTGCAGCCGGTCCTCTTCGCCCTTGTCCTCCTGTGCTACCTCCTGACCTTGACGGGCAACTCGGCGCT
GGTGCTGCTGGCGGTGCGCGACCCGCGCCTGCACACGCCCATGTACTACTTCCTCTGCCACCTGGCCTTG
GTAGACGCGGGCTTCACTACTAGCGTGGTGCCGCCGCTGCTGGCCAACCTGCGCGGACCAGCGCTCTGGC
TGCCGCGCAGCCACTGCACGGCGCAGCTGTGCGCATCGCTGGCTCTGGGTTCGGCCGAATGCGTCCTCCT
GGCGGTGATGGCTCTGGACCGCGCGGCCGCAGTGTGCCGCCCGCTGCGCTATGCGGGGCTCGTCTCCCCG
CGCCTATGTCGCACGCTGGCCAGCGCCTCCTGGCTAAGCGGCCTCACCAACTCGGTTGCGCAAACCGCGC
TCCTGGCTGAGCGGCCGCTGTGCGCGCCCCGCCTGCTGGGCCACTTCATCTGTGAGCTGCCGGCGTTGCT
CAAGCTGGCCCGCGGAGGCGACGGAGACACTACCGAGAACCAGATGTTCGCCGCCCGCGTGGTCATCCTG
CTGCTGCCGTTTGCCGTCATCCTGGCCTCCTACGGTGCCGTGGCCCGAGCTGTCTGTTGTATGCGGTTCA
GCGGAGGCCGGAGGAGGGCGGTGGGCACGTGTGGGTCCCACCTGACAGCCGTCTGCCTGTTCTACGGCTC
GGCCATCTACACCTACCTGCAGCCCGCGCAGCGCTACAACCAGGCACGGGGCAAGTTCGTATCGCTCTTC
TACACCGTGGTCACACCTGCTCTCAACCCGCTCATCTACACCCTCAGGAATAAGAAAGTGAAGGGGGCAG
CGAGGAGGCTGCTGCGGAGTCTGGGGAGAGGCCAGGCTGGGCAGTGA
A putative splice site is located between nucleotides 15 and 16 in SEQ ID
N0:9. In
one embodiment, nucleotides 1-15 come from exon 1 and nucleotides 16-957 are
from exon 2.
The disclosed ba145122 B nucleic acid sequence has 592 of 934 bases (63%)
identical
to a GPCR mRNA (GENBANK-ID: HUMORLMHC~ acc: L35475) from Homo Sapiens (E=
2.4 a 51).
The disclosed GPCR3 polypeptide (SEQ ID NO:10) encoded by SEQ ID N0:9 is 315
amino acid residues and is presented using the one-letter code in Table 3B.
The first 70 amino
acids of the disclosed GPCR3 protein were analyzed for signal peptide
prediction and cellular
localization. SignalP results predict that GPCR3 is cleaved between position
46 and 47 of
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WO 01/64879 PCT/USO1/06474
SEQ m NO:10, i.e., at the slash in the amino acid sequence NSA-LV. Psort and
Hydropathy
profiles also predict that GPCR3 contains a signal peptide and is likely to be
localized at the
plasma membrane (certainty of 0.6000).
Table 3B. Encoded GPCR3 protein sequence (SEQ ID NO:10).
MKANYSAEERFLLLGFSDWPSLQPVLFALVLLCYLLTLTGNSA/LVLLAVRDPRLHTPMYYFLCHLALVDA
GFTTSVVPPLLANLRGPALWLPRSHCTAQLCASLALGSAECVLLAVMALDRAAAVCRPLRYAGLVSPRLC
RTLASASWLSGLTNSVAQTALLAERPLCAPRLLGHFICELPALLKLARGGDGDTTENQMFAARVVILLLP
FAVILASYGAVARAVCCMRFSGGRRRAVGTCGSHLTAVCLFYGSAIYTYLQPAQRYNQARGKFVSLFYTV
VTPALNPLIYTLRNKKVKGAARRLLRSLGRGQAGQ
A BLASTX search was performed against public protein databases. The full amino
acid sequence of the protein of the invention was found to have 253 of 308
amino acid
residues (82%) identical to, and 264 of 308 residues (85%) positive with, the
309 amino acid
residue MM17M1-6, 7 transmembrane receptor (OR-like protein) from Mus musculus
(ptnr:SPTREMBL-ACC:Q9WV09, SEQ ID N0:52) (E = 1.5 e'2$). The alignment of
these
proteins is shown in Table 3C.
Table 3C. Alignment of GPCR3 with QpWV09 (SEQ ID N0:52).
10 20 30 40 50 60
.I....I....I....I....I....1....1....1....1....1....1....1
GPCR3 M Y E ~ ~ ' !~ ~ S ~ ~ ~ ~ 60
Q9wv09 --~H~S v ~ ~. ~158
70 80 90 100 110 120
.I....I....I....I....I....1....1....1....1....1....1....1
GPCR3 ~PA~W~'~'-S T~~ ~ 120
Q9WV09 ~V~S S ~~ S 118
130 140 150 160 170 180
.I....I....1....1....1....1....1....1....1....1....1....1
GPCR3 ~R~G~S~S T V~~ ~ ~L~G~ 180
Q9WV09 N~~' TS L~G ~~ ~ C D~ 178
190 200 210 220 230 240
... .I....I....I....I....I....1....1....1....1....1....1....1
GPCR3 ~~R~DGD~ L : ~RF~C' ~ ~ V~ 2 4 0
Q9WV09 C GRS~ ~ S I~ HS S S 238
250 260 270 280 290 300
.1.. .1.. .1.. .1.. .1.. .I.. .1.. .1.. .1.. .1.. .I.. .~
GPCR3 I ~~ ' ~ ~ 300
Q9wv09 ~T ~ v~T 5 v ~ ~ 298
310
....i....1....1
GPCR3 GQAGQ 315
Q9WV09 ~L. .p____ 309
The disclosed GPCR3 protein (SEQ ID NO:10) also has good identity with a
number
of olfactory receptor proteins, as shown in Table 3D.
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Table 3D. BLAST
results for
GPCR3
Gene Index/ Protein/ LengthIdentityPositivesExpect
Identifier Organism (aa) (~)
gi(5051404(embICA573K1.15 309 229/296 240/296 e-108
-
B45012.11 mm17M1-6 (77~) (80$)
(novel
(AL078630) 7 transmembrane
receptor)
Mus musculus
gi1120593591embICOR 312 145/298 191/298 2e-69
AC20487.11 Homo Sapiens (48~) (63~)
(AJ302567)
(AJ02568)
gi1120543551embICOR 312 194/298 191/298 3e-69
AC20485.1~ Homo sapiens (48~) (63$)
(AJ302565)
(AJ302566)(AJ3025
69)(AJ302570)
gi1122310291spIQ1OR 2H3 316 143/294 185/294 3e-68
5062102H3 HUMANHomo Sapiens (48~) (62~)
gi197989201gb~AAFOR 303 142/287 183/287 5e-68
98752.1 AF211940_Homo Sapiens (99$) (63$)
1 (AF211940)
This information is presented graphically in the multiple sequence alignment
given in
Table 3E (with GPCR3 being shown on line 1) as a ClustalW analysis comparing
GPCR3 with
related protein sequences.
Table 3E. Information for the ClustalW proteins:
1) Novel GPCR3 (SEQ ID NO:10)
2) gi~5051404~emb~CAB45012.1~ 573K1.15 (mm17M1-6 (novel 7 transmembrane
receptor (rhodopsin family)
(olfactory receptor LIKE) protein)) Mus musculus (SEQ ID N0:53)
3) gi~12054359~emb~CAC20487.1 ~ olfactory receptor Homo Sapiens (SEQ ID N0:54)
1 ~ 4) gi~12054355~emb~CAC20485.1 ~ olfactory receptor Homo Sapiens (SEQ ID
N0:55)
5) gi~12231029~sp~Q15062~02H3_HUMAN OLFACTORY RECEPTOR 2H3 (OLFACTORY RECEPTOR-
LIKE
PROTEIN FAT11) (SEQ ID N0:56)
6) gi~9798920~gb~AAF98752.1 ~AF211940_1 olfactory receptor Homo Sapiens (SEQ
ID N0:57)
io-
-
zo
so
4o
so
so
....I....I....~....I....I....I....
GPCR3 --MK Y E ~ 'S~P T SA
Gi~5051404~---- H SA 5 vP T I
a
Gi~120543591MLMK EE H L~ fi ASH
Y
L
611120543551MLMK FE I 'H S ~ L 3 ASH
Y
GiI122310291---MD STPG H' RT FTS TL
y ~
Gi197989201----------- H RT FTS TL
70 80 90 100 110 120
.I.. .I.. .I.. .I.. .I.. .(.. .I.. .I.. .I.. .I.. .(.. .I
GPCR3 ~ C .L~~~ ~ 'P 1 ' LWP H~. ~ CAS ~. ~S
Gi~50514091 C L~:w G 'P SMLQ P CSS
Gi~12054359~ ~ v g 5:.~
Gi112054355~ ~ S~ F ~ ~
Gi1122310291 ~ C ' ~ ~LD ~ I T ~ T
Gi ~ 97989201 ~ C ' ~L LD 1u I s. T ~ T
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WO 01/64879 PCT/USO1/06474
130 140 150 160 170 180
.I.. .~.. .~.. .~.. .~.. .~.. .~.. .I.. .~.. .I.. .~.. .I
GPCR3 L~' S " ~T ~S. TN ~ ~LLAE ' P'L~ .
r ' ~ v
r
gi~5051404~ L~'TS ~S'L 'T ~G N LLAA ' '~ 1
gi~120543591 ~' T m 'L ~ S FTT H - a ' R ' n
gi~12054355~ n ' T 'L S FTT H , FW ' RH
gi~12231029~ n ' T I ' I E PS LH ' PD D
gi197989201 T ' I E PS LH ' PD' D
190 200 210 220 230 290
.I.. .1....I.. .I.. .I.~ ~I.. .I~~ .I.. .I.. .I~. .I.~ .I
GPCR3 ~'~ .~RGGDGDTT~ F~~ 'j ~ CC R
v Y v w
gi ~ 5051904 ~ ~' ~ ~~ RGGRSAT F ~ ~ , ~ S . I ~,~ H~S '
gi~12059359~ V -Q T , S~F, ~' I~ ~ T La L
gi~120543551 Q T S I' I ' S T Le L
gi~12231029~ ~ T' Ey SY I ~,,' ~a ~ T ~ ~~F
gi19798920~ ~ I' E SY ~ , ~ ~ TW~ N ~F
250 260 270 280 290 300
.I.. .I.. .I.. .I.~ .I~. .I.. .I.~ ~I.. ~I~~ .I.. .I.. .I
GPCR3 C ~ .YT t' Q
gi~5051404~ S C TYT ~'THSG
gi ~ 12054359 ~ ~ IP ~ C v P EN~~
gi~120543551 GIP x'C' 'P EN ~= I~,,
gi~12231029~ S S ~'K P ~ ~' F G ' T
gi197989201 S 5 P ~ ~ F G ~ T
310 320
.I.. .1....1....I
GPCR3 _ RSLGRGQAGQ--
gi150514041 ~ L' RSLGRP------
~w ' w w
gi~120543591 GM-------
gi~12054355~ GM-------
gi~12231029~ RDSRESWRAA
gi~97989201 ~F ' GLTQS----
DOMAIN results for GPCR3 were collected from the Conserved Domain Database
(CDD) with Reverse Position Specific BLAST. This BLAST samples domains found
in the
Smart and Pfam collections. The 7tm 1, a seven transmembrane receptor
(rhodopsin family),
was shown to have two segments with significant homology to GPCR3. The region
from
amino acid residue 40 through 226 aligns with amino acids 1 through 167 of the
"seven
transmembrane receptor (rhodopsin family) fragment" domain(SEQ ID N0:45, E=le-
15), and
GPCR3 amino acids 219-290 aligned with residues 305-377 of the 7tm_1 entry of
the Pfam
database (E=.004). This indicates that the GPCR3 sequence has properties
similar to those of
other proteins known to contain this domain as well as to the 7tm_1 domain
itself.
The nucleic acids and proteins of the invention are useful in potential
therapeutic
applications implicated in various GPCR-related pathological disorders and/or
OR-related
pathological disorders, described further below. For example, a cDNA encoding
the olfactory
receptor -like protein may be useful in gene therapy, and the olfactory
receptor -like protein
may be useful when administered to a subject in need thereof. By way of
nonlimiting
example, the compositions of the present invention will have efficacy for
treatment of patients
suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus
cancer, immune
response, AIDS, asthma, Crohn's disease, multiple sclerosis, and Albright
Hereditary
Ostoeodystrophy. Other GPCR-related diseases and disorders are contemplated.
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The novel nucleic acid encoding the GPCR-like protein of the invention, or
fragments
thereof, may further be useful in diagnostic applications, wherein the
presence or amount of
the nucleic acid or the protein are to be assessed. These materials are
further useful in the
generation of antibodies that bind immunospecifically to the novel substances
of the invention
S for use in therapeutic or diagnostic methods. These antibodies may be
generated according to
methods known in the art, using prediction from hydrophobicity charts, as
described in the
"Anti-GPCRX Antibodies" section below. These novel proteins can be used in
assay systems
for functional analysis of various human disorders, which will help in
understanding of
pathology of the disease and development of new drug targets for various
disorders.
GPCR4
GPCR4 includes a family of three nucleic acids disclosed below. The disclosed
nucleic acids encode a GPCR-like protein.
GPCR4a
The disclosed GPCR4a is encoded by three different nucleic acids, GPCR4a1
(dj408b20 C) GPCR4a2 (dj408b20 C dal), and GPCR4a3 (CG55358-03). A first
nucleic
acid, dj408b20 C (GPCR4a1), is 947 nucleotides long (SEQ ID NO:11). An open
reading
frame was identified beginning with an ATG initiation codon at nucleotides 3-5
and ending
with a TGA codon at nucleotides 939-941. Putative untranslated regions
upstream from the
initiation codon and downstream from the termination codon are underlined in
Table 4A, and
the start and stop codons are in bold letters. The encoded protein having 312
amino acid
residues is presented using the one-letter code in Table 4B (SEQ ID N0:12).
Table 4A. GPCR4a1 Nucleotide Sequence (SEQ ID NO:11).
_GTATGGAAAACGATAATACAAGTTCTTTCGAAGGCTTCATCCTGGTGGGCTTCTCTGATCGTCCCCACCT
AGAGCTGATCGTCTTTGTGGTTGTCCTCATCTTTTATCTGCTGACTCTTCTTGGCAACATGACCATTGTC
TTGCTTTCAGCTCTGGATTCCCGGCTGCACACACCAATGTATTTCTTTTTGGCAAACCTCTCATTCCTGG
ACATGTGTTTCACCACAGGTTCCATCCCTCAGATGCTCTACAACCTTTGGGGTCCAGATAAGACCATCAG
CTATGTGGGTTGTGCCATCCAGCTGTACTTTGTCCTGGCCCTGGGAGGGGTGGAGTGTGTCCTCCTGGCT
GTCATGGCATATGACCGCTATGCTGCAGTCTGCAAACCCCTGCACTACACCATCATCATGCACCCACGTC
TCTGTGGACAGCTGGCTTCAGTGGCATGGCTGAGTGGCTTTGGCAATTCTCTCATAATGGCACCCCAGAC
ATTGATGCTACCCCGCTGTGGGCACAGACGAGTTGACCACTTTCTCTGTGAGATGCCAGCACTAATTGGT
ATGGCCTGTGTAGACACCATGATGCTTGAGGCACTGGCTTTTGCCCTGGCAATCTTTATCATCCTGGCAC
CACTCATCCTCATTCTCATTTCTTATGGTTACGTTGGAGGAACAGTGCTTAGGATCAAGTCAGCTGCTGG
GCGAAAGAAAGCCTTCAACACTTGCAGCTCGCATCTAATTGTTGTCTCTCTCTTCTATGGTACAATCATA
TACATGTACCTCCAGCCAGCAAATACTTATTCCCAGGACCAGGGCAAGTTTCTTACCCTTTTCTACACAA
TTGTCACTCCCAGTGTTAACCCCCTGATCTATACACTAAGAAACAAAGATGTTAAAGAGGCCATGAAGAA
GGTGCTAGGGAAGGGGAGTGCAGAAATATAGTAAGGG
The disclosed nucleic acid GPCR4a1 sequence has 624 of 931 bases (67%)
identical
(with 624/931 positives, 67%) to a 939 by Homo Sapiens olfactory receptor-like
protein
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(OR2C1) gene (GENBANK-ID: AF098664) (E = 3.8e'2). In a search of sequence
databases,
partial matches were also identified, e.g., the minus strand of nucleotides
719-947 had 229 of
229 bases (100%) identical to a 1320 by synthetic GPCR mRNA (PATENT-ID:
T72050), the
sequence around marker 2B8 in HH region of chromosome 6p2.1, and the same
region of
S GPCR4a1 had 229 of 229 bases (100%) identical to a synthetic GPCR mRNA
(PATENT-ID:
T72050), also sequence around marker 2B8 in HH region of chromosome 6p2.1 (E
value in
both cases is 9.6e-47).
The GPCR4a polypeptide (SEQ ID N0:12) encoded by SEQ ID NO:11 is presented
using the one-letter amino acid code in Table 4B. The Psort profile for GPCR4a
predicts that
this sequence has a signal peptide and is likely to be localized at the plasma
membrane with a
certainty of 0.6000. The most likely cleavage site for a GPCR4a peptide is
between amino
acids 41 and 42, i.e., at the slash in the amino acid sequence LLG-NM, based
on the SignalP
result.
Table 4B. GPCR4a protein sequence (SEQ ID N0:12)
MENDNTSSFEGFILVGFSDRPHLELIVFVVVLIFYLLTLLG/NMTIVLLSALDSRLHTPMYFFLANLSF
LDMCFTTGSIPQMLYNLWGPDKTISYVGCAIQLYFVLALGGVECVLLAVMAYDRYAAVCKPLHYTIIMH
PRLCGQLASVAWLSGFGNSLIMAPQTLMLPRCGHRRVDHFLCEMPALIGMACVDTMMLEALAFALAIFI
ILAPLILILISYGYVGGTVLRIKSAAGRKKAFNTCSSHLIVVSLFYGTIIYMYLQPANTYSQDQGKFLT
LFYTIVTPSVNPLIYTLRNKDVKEAMKKVLGKGSAEI
1$
The predicted GPCR4a1 sequence, above, was subjected to the exon linking
process to
confirm the sequence. PCR primers were designed by starting at the most
upstream sequence
available, for the forward primer, and at the most downstream sequence
available for the
reverse primer. In each case, the sequence was examined, walking inward from
the respective
termini toward the coding sequence, until a suitable sequence that is either
unique or highly
selective was encountered, or, in the case of the reverse primer, until the
stop codon was
reached. Such suitable sequences were then used as the forward and reverse
primers in a PCR
amplification based on a wide range of cDNA libraries. The resulting amplicon
was gel
purified, cloned and sequenced to high redundancy, as described in the
Examples.
The cloned sequence is disclosed as an alternative embodiment of GPCR4a2 (SEQ
ID
N0:13), referred to herein as the GPCR4a2 and reported in Table 4C. This 945
nucleotide
sequence (GPCR4a2) is alternatively referred to herein as dj408b20 C dal .
This nucleic acid
is two nucleotides shorter than GPCR4a1 at the 5' UTR: However, CPCR4a2
encodes the
same 312 amino acid protein (GPCR4a, SEQ ID N0:12).
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Table 4C. GPCR4a2 Nucleotide Sequence (SEQ ID N0:13)
GTATGGAAAACGATAATACAAGTTCTTTCGAAGGCTTCATCCTGGTGGGCTTCTCTGATCGTCCCCACCTAGAGCTGAT
C
GTCTTTGTGGTTGTCCTCATCTTTTATCTGCTGACTCTTCTTGGCAACATGACCATTGTCTTGCTTTCAGCTCTGGATT
C
CCGGCTGCACACACCAATGTATTTCTTTTTGGCAAACCTCTCATTCCTGGACATGTGTTTCACCACAGGTTCCATCCCT
C
AGATGCTCTACAACCTTTGGGGTCCAGATAAGACCATCAGCTATGTGGGTTGTGCCATCCAGCTGTACTTTGTCCTGGC
C
CTGGGAGGGGTGGAGTGTGTCCTCCTGGCTGTCATGGCATATGACCGCTATGCTGCAGTCTGCAAACCCCTGCACTACA
C
CATCATCATGCACCCACGTCTCTGTGGACAGCTGGCTTCAGTGGCATGGCTGAGTGGCTTTGGCAATTCTCTCATAATG
G
CACCCCAGACATTGATGCTACCCCGCTGTGGGCACAGACGAGTTGACCACTTTCTCTGTGAGATGCCAGCACTAATTGG
T
ATGGCCTGTGTAGACACCATGATGCTTGAGGCACTGGCTTTTGCCCTGGCAATCTTTATCATCCTGGCACCACTCATCC
T
CATTCTCATTTCTTATGGTTACGTTGGAGGAACAGTGCTTAGGATCAAGTCAGCTGCTGGGCGAAAGAAAGCCTTCAAC
A
CTTGCAGCTCGCATCTAATTGTTGTCTCTCTCTTCTATGGTACAATCATATACATGTACCTCCAGCCAGCAAATACTTA
T
TCCCAGGACCAGGGCAAGTTTCTTACCCTTTTCTACACAATTGTCACTCCCAGTGTTAACCCCCTGATCTATACACTAA
G
The full amino acid sequence of the disclosed GPCR4a polypeptide has 197 of
305
amino acid residues (64%) identical to, and 242 of 305 residues (79%) positive
with, the 320
amino acid residue protein from Homo sapiens novel 7 transmembrane receptor
(rhodopsin
family, olfactory receptor-like protein HS6M1-15, ptnr:SPTREMBL-ACC:Q9Y3N9
DJ88J8.1, E = S.Oe'°g).
BLASTP (Non-Redundant Composite database) analysis of the best hits for
alignments
with GPCR4a are listed in Table 4D.
Table 4D.
BLASTP
results
for GPCR4a
Gene Index/Protein/ Organism LengthIdentity PositivesExpect
Identifier (aa)
ACC:Q9Y3N9Novel 7 Transmembrane320 197/305 242/305 9.4e-
DJ88J8.1 Receptor (Rhodopsin (64g) (79~) 109
Family, OR-Like)
Hs6m1-15 Homo Sapiens
ACC:P23275OLFACTORY RECEPTOR312 190/308 291/308 5.5e-
15
(OR3) Mus musculus (61$) (78$) 104
ACC:076001OR-LIKE PROTEIN 311 193/302 244/302 1.5e-
DJ80I19.7 (HS6M1-3) (63~) (80~) 103
Homo sapiens
A BLASTX was also performed to determine the proteins that have significant
identity
with GPCR4a. The BLASTX results are shown in Table 4E.
Table 4E. BLASTX results for GPCR4a
Smallest
Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(N) N
Ptnr:SPTREMBL-ACC:Q9Y3N9 DJ88J8.1 (NOVEL 7 TRANSMEMBRA... +3 1076 5.6e-108 1
Ptnr:SWISSPROT-ACC:P23275 OLFACTORY RECEPTOR 15 (0R3) ... +3 1031 3.3e-103 1
Ptnr:SPTREMBL-ACC:076001 DJ80I19.7 (OLFACTORY RECEPTOR... +3 1027 8.7e-103 1
Ptnr:SPTREMBL-ACC:095371 OLFACTORY RECEPTOR-LIKE PROTE... +3 992 9.5e-99 1
Ptnr:SPTREMBL-ACC:095918 DJ271M21.2 (HS6M1-12 (7 TRANS... +3 988 1.2e-98 1
Ptnr:SPTREMBL-ACC:Q9WV11 573K1.8 (MM17M1-2 (NOVEL 7 TR... +3 984 3.2e-98 1
Ptnr:SPTREMBL-ACC:Q9WV14 573K1.2 (MM17M1=3 (NOVEL 7 TR... +3 981 6.6e-98 1
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Possible SNPs found for GPCR4a2 are listed in Table4F.
Table 4F: SNPs
Base Base Base
Position Before After
44 T C(20
147 T C(2)
220 T C(2)
271 T C(3)
432 A G(2)
452 C T(2)
493 A G(2)
771 T C(3)
GPCR4b
The target sequence identified as dj408b20 C (GPCR4a1) was again subjected to
the
exon linking process to confirm the sequence. PCR primers were designed by
starting at the
most upstream sequence available, for the forward primer, and at the most
downstream
sequence available for the reverse primer. The cDNA coding for the sequence
was cloned by
polymerase chain reaction (PCR) using the following primers:
ATACAAGTTCTTTCGAAGGCTTCATCC (SEQ ID N0:14) and
CCCTTACTATATTTCTGCACTCCCCTT (SEQ ID NO:15) on pool 1 of human cDNAs
containing the following: Adrenal gland, bone marrow, brain - amygdala, brain -
cerebellum,
brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole,
fetal brain, fetal
kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary
gland, pancreas,
pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small
intestine, spinal cord,
spleen, stomach, testis, thyroid, trachea, uterus.
Primers were designed based on in silico predictions for the full length or
part (one or
more exons) of the DNA/protein sequence of the invention or by translated
homology of the
predicted exons to closely related human sequences or to sequences from other
species.
Usually multiple clones were sequenced to derive the sequence which was then
assembled
similar to the SeqCalling process. In addition, sequence traces were evaluated
manually and
edited for corrections if appropriate. The PCR product derived by exon linking
was cloned
into the pCR2.1 vector from Invitrogen. The bacterial clone
115843::DJ408B20 C.698322.D10 has an insert covering the entire open reading
frame
cloned into the pCR2.1 vector from Invitrogen.
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In each case, the sequence was examined, walking inward from the respective
termini
toward the coding sequence, until a suitable sequence that is either unique or
highly selective
was encountered, or, in the case of the reverse primer, until the stop codon
was reached. Such
primers were designed based on in silico predictions for the full length cDNA,
part (one or
more exons) of the DNA or protein sequence of the target sequence, or by
translated
homology of the predicted exons to closely related human sequences from other
species.
Typically, the resulting amplicons were gel purified, cloned and sequenced to
high redundancy
as described in the examples. The resulting sequences from all clones were
assembled with
themselves, with other fragments in CuraGen Corporation's database and with
public ESTs.
Fragments and ESTs were included as components for an assembly when the extent
of their
identity with another component of the assembly was at least 95% over 50 bp.
In addition,
sequence traces were evaluated manually and edited for corrections if
appropriate.
These procedures provided a third nucleic acid encoding a GPCR4 protein. The
nucleic acid is referred to as GPCR4a3 or CG55358-03. This nucleic acid is 932
nucleotides
long (SEQ ID N0:16, Table 4G) and is 16 nucleotides shorter in the S' UTR than
GPCR2al.
An open reading frame of the mature protein was identified beginning with an
ACA codon
which codes for the amino acid threonine at nucleotides 3-5 and ending with a
TAG codon at
nucleotides 924-926. Putative untranslated regions, if any, are found upstream
from the
initiation codon and downstream from the termination codon. One silent base
substitution is
present: C767 is T752 in GPCR4a3. GPCR4b, the protein encoded by GPCR4a3 is
identical
to GPCR4a, except for the 5 amino acid deletion at the N terminus, as shown in
Table 4H
(SEQ ID N0:17).
The GPCR Olfactory Receptor disclosed herein is expressed in at least the
following
tissues: Apical microvilli of the retinal pigment epithelium, arterial
(aortic), basal forebrain,
brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and
ventricle), caudate
nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon,
cortical neurogenic
cells, endothelial (coronary artery and umbilical vein) cells, palate
epithelia, eye, neonatal eye,
frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus,
leukocytes, liver,
fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult
lymphoid tissue, Those
that express MHC II and III nervous, medulla, subthalamic nucleus, ovary,
pancreas, pituitary,
placenta, pons, prostate, putamen, serum, skeletal muscle, small intestine,
smooth muscle
(coronary artery in aortic) spinal cord, spleen, stomach, taste receptor cells
of the tongue,
testis, thalamus, and thymus tissue. This information was derived by
determining the tissue
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sources of the sequences that were included in the invention including but not
limited to
SeqCalling sources, Public EST sources, Literature sources, andlor RACE
sources.
In a search of sequence databases, it was found, for example, that the
disclosed
GPCR4a3 nucleic acid sequence has 614 of 913 bases (67%) identical to a
gb:GENBANK-
ID:AF098664~acc:AF098664.1 mRNA from Homo Sapiens (Homo Sapiens olfactory
receptor-
like protein (OR2C 1 ) gene, complete cds, E= 2.1 e-71 ).
Table 4G. GPCR4a3 Nucleotide Sequence (SEQ ID N0:16)
TACAAGTTCTTTCGAAGGCTTCATCCTGGTGGGCTTCTCTGATCGTCCCCACCTAGAGC60
A
_ 120
TGATCGTCTTTGTGGTTGTCCTCATCTTTTATCTGCTGACTCTTCTTGGCAACATGACCA
TTGTCTTGCTTTCAGCTCTGGATTCCCGGCTGCACACACCAATGTATTTCTTTTTGGCAA180
ACCTCTCATTCCTGGACATGTGTTTCACCACAGGTTCCATCCCTCAGATGCTCTACAACC240
TTTGGGGTCCAGATAAGACCATCAGCTATGTGGGTTGTGCCATCCAGCTGTACTTTGTCC300
TGGCCCTGGGAGGGGTGGAGTGTGTCCTCCTGGCTGTCATGGCATATGACCGCTATGCTG360
CAGTCTGCAAACCCCTGCACTACACCATCATCATGCACCCACGTCTCTGTGGACAGCTGG420
CTTCAGTGGCATGGCTGAGTGGCTTTGGCAATTCTCTCATAATGGCACCCCAGACATTGA480
TGCTACCCCGCTGTGGGCACAGACGAGTTGACCACTTTCTCTGTGAGATGCCAGCACTAA540
TTGGTATGGCCTGTGTAGACACCATGATGCTTGAGGCACTGGCTTTTGCCCTGGCAATCT600
TTATCATCCTGGCACCACTCATCCTCATTCTCATTTCTTATGGTTACGTTGGAGGAACAG660
TGCTTAGGATCAAGTCAGCTGCTGGGCGAAAGAAAGCCTTCAACACTTGCAGCTCGCATC720
TAATTGTTGTCTCTCTCTTCTATGGTACAATTATATACATGTACCTCCAGCCAGCAAATA780
CTTATTCCCAGGACCAGGGCAAGTTTCTTACCCTTTTCTACACAATTGTCACTCCCAGTG840
TTAACCCCCTGATCTATACACTAAGAAACAAAGATGTTAAAGAGGCCATGAAGAAGGTGC900
TAGGGAAGGGGAGTGCAGAAATATAGTAAGGG 932
The SignalP, Psort and/or Hydropathy profile for the disclosed GPCR4b
Olfactory
Receptor-like protein predicts that this sequence has a signal peptide and is
likely to be
localized at the plasma membrane with a certainty of 0.6000. The SignalP shows
a signal
sequence is coded for in the first 36 amino acids with a cleavage site at
between amino acids
36 and 37, as indicated by a slash between LLG/NM in Table 4H. This is typical
of this type
of membrane protein.
Table 4H. GPCR4b Amino Acid Sequence (SEQ ID N0:17)
TSSFEGFILVGFSDRPHLELIVFVVVLIFYLLTLLG/NMTIVLLSALDSRLHTPMYFFLAN 60
LSFLDMCFTTGSIPQMLYNLWGPDKTISYVGCAIQLYFVLALGGVECVLLAVMAYDRYAA 120
VCKPLHYTIIMHPRLCGQLASVAWLSGFGNSLIMAPQTLMLPRCGHRRVDHFLCEMPALI 180
GMACVDTMMLEALAFALAIFIILAPLILiLISYGYVGGTVLRIKSAAGRKKAFNTCSSHL 240
IVVSLFYGTIIYMYLQPANTYSQDQGKFLTLFYTIVTPSVNPLIYTLRNKDVKEAMKKVL 300
GKGSAEI 307
The full amino acid sequence of the disclosed GPCR4b protein of the invention
was
found to have 195 of 299 amino acid residues (65%) identical to, and 239 of
299 amino acid
residues (79%) similar to, the 320 amino acid residue ptnr:SPTREMBL-ACC:Q9Y3N9
protein from Homo Sapiens (DJ88J8.1, NOVEL 7 TRANSMEMBRANE RECEPTOR
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(RHODOPS1N FAMILY) (OLFACTORY RECEPTOR LIKE) PROTEIN) (HS6M1-15), E=
1.6e-107).
In following positions, one or more consensus positions (Cons. Pos.) of the
GPCR4a3
nucleotide sequence have been identified as SNPs. "Depth" represents the
number of clones
covering the region of the SNP. The Putative Allele Frequency (Putative Allele
Freq.) is the
fraction of all the clones containing the SNP. A dash ("-"), when shown, means
that a base is
not present. The sign ">" means "is changed to": Cons. Pos.: 422 Depth: 18
Change: T > C,
Putative Allele Freq.: 0.333; Cons. Pos.: 546 Depth: 15 Change: T > C,
Putative Allele
Freq.: 0.133; Cons. Pos.: 753 Depth: 8 Change: C > T, Putative Allele Freq.:
0.250.
Unless specifically addressed as GPCR4a or GPCR4b, any reference to GPCR4 is
assumed to encompass all variants. Residue differences between any GPCRX
variant
sequences herein are written to show the residue in the "a" variant and the
residue position
with respect to the "a" variant. In all following sequence alignments, the
GPCR4a protein
sequence was used.
The disclosed GPCR4 protein (SEQ ID N0:12) also has good identity with a
number
of olfactory receptor proteins. The identity information used for ClustalW
analysis is
presented in Table 4I.
Table 4I. BLAST
results for
GPCR4
Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect
Identifier (aa) (~)
Gi16679170~ref(NPOR 15 (0R3) 312 174/308216/308 1e-93
03
_ Mus musculus (56~) (69g)
2788.1
Gi~4826521~emb~CAB92novel 7 tm 320 174/305213/305 2e-93
853.1 receptor protein (57~) (690)
(AL035402, dJ88J8.1)(rhodopsin fam.,
(AJ302594-99) OR-like)
(AJ302600-01) (hs6M1-15)
Homo Sapiens
Gi~120544311embICAC2OR 320 173/305213/305 3e-93
0523.11 (AJ302603)Homo Sapiens (56~) (690)
Gi~120544291embICAC2OR 320 173/305213/305 7e-93
0522.11 (AJ302602)Homo Sapiens (56~) (690)
Gi~12054347~embICAC2OR 311 170/302211/302 6e-90
0478.11 (AJ302558)Homo sapiens (56~) (69~)
This information is presented graphically in the multiple sequence alignment
given in
Table 4J (with GPCR4 being shown on line 1) as a ClustalW analysis comparing
GPCR4 with
related OR sequences.
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Table 4J Information for the ClustalW proteins:
1) GPCR4 (SEQ ID N0:12)
2) gi~6679170~ref~NP_032788.1 ~ olfactory receptor 15 Mus musculus (SEQ ID
N0:58)
3) gi~4826521 ~emb~CAB42853.1 ~ dJ88J8.1 (novel 7 transmembrane receptor
(rhodopsin family) (OR
like) protein) (hs6M1-15)) Homo Sapiens (SEQ ID N0:59)
4) gig 12054431 ~emb~CAC20523.1 ~ olfactory receptor Homo Sapiens (SEQ ID
N0:60)
5) gig 12054429~emb~CAC20522.1 ~ olfactory receptor Homo Sapiens (SEQ ID
N0:61)
6) gi~12054347~emb~CAC20478.1~ olfactory receptor Homo Sapiens (SEQ ID N0:62)
1D 20 30 90 50 60
.I....I....I....I....I....1....1....1....1....1....1....1
GPCR4 ~~ND---~T FE H~' F L T L
GiI66791701 n VD---N SGT H; T FF ALAS LT ~ L
Gi148265211 QS--- Y LH N ~ S I T L
Gi1120544311 ~ QS--- Y LH 5 I T L
Gi1120544291 QS--- Y LH K SG I T L
Gi I 120543971 ~DDGK EGY H ,V~ F L I L L Y
70 80 90 100 110 120
.I.. .I.. ~I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I
.. . v
GPCR9 W~ .GS '~ Y . '~~ ~ FV~
Gi166791701 S S n S~'~~ '~' T ~T
Gi198265211 ~ I '
Gi1120594311 ~ I ' w fi
Gi1120544291 ~ I ' '~ ~ a
Gi1120543471 S ~ S ' ~ ~ FV T
13D 190 150 160 170 180
.I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I
GPCR4 ~ ~ T~I ~S ~SGF ~ .P ~ ~ R ~
_ ~, a v w . w
Gi166791701 ~ y . ~. ' S LG ~QS ~RK~~
a ca
Gi148265211 n ' T T ~ L I I SIS TyC L N T NI~n
Gi I 12054431 I ~' T I L ',~I I SIS r' C L N 'T ~ NIi' ~
Gi 1120544291 m' T I' L~I , I SIS .~ C L N T ~ Ian
Gi1120543471 ~'~~T v ~'R HL S SGFT 5~ FW L ~R ~
190 200 210 220 230 240
.I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I
GPCR9 ~ ' G ' n _~" L E~I ~GGT ~ I G -
GiI66791701 ~'~_I ' ~ LN ~NG~CTFFT ~ S C 19I EG '
Gi198265211 I' ~ T= 8. ~T. -.T~. - S. '
Gi1120594311 ~,' ~ T '~- T' T S '
~ ;.,.~~a
Gi1120599291 ~ I~ ~ ,~T T' T K~S°'
M ~TTGL
Gill'20543471 ' S ~ HVN ' ITSS ~ I'
250 260 270 280 290 300
.I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. ~I.. .I.. .I
GPCR4 ~ S , I ~ ~ ~' TY ~ m I~"~l
a
Gi I 66791701 ~ L' KS ~Ii;~S~ ~;S I U
Gi148265211 T ~ v
Gi1120549311 T ~ m
Gi1120544291 T ~' ~~ 2 '
Gi1120543471 F ~ IPA~C 'PG
310 320
.I.. .I.. .I.. .I...
GPCR4 ~G--K~ ----
Gi166791701 ~SG,_=GKGRG "a-____--_
Gi148265211 HHK KRNCKS
Gi1120544311 i H KRNCKS
Gi1120544291 t HH KRNCKS
Gi1120543471 iG E------------
DOMAIN results for GPCR4 were collected from the Conserved Domain Database
(CDD) with Reverse Position Specific BLAST. This BLAST samples domains found
in the
Smart and Pfam collections. Two regions of GPCR4 have identity to the 377
amino acid 7TM
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domain, as described above. The 7tm 1, a seven transmembrane receptor
(rhodopsin family),
SEQ ID N0:45, above, was shown to have homology to GPCR4. Residues 1-120 of
7TM
align with residues 41-159 of GPCR4 (E= 7e-22, shown in Table 2K) and residues
224-290 of
GPCR4 have identity with residues 310-377 of 7TM (E=2e-04).
Table 4K: Domain Alignment between GPCR4 and 7TM.
TM7R4 ~LAV~RKA~Q~TTNL FLVA~LV~WEVVGWKF~RF
..I....I....~In....I....I....I....I....I....I....I....I....)
GPCR ~ D CTASI~I~V I'' T~ CK~ L~NTRYSS~RVTQI ? ~LSBTI~CP~
TM7
The nucleic acids and proteins of GPCR4 are useful in potential therapeutic
applications implicated in various GPCR-related pathological disorders and/or
OR-related
pathological disorders, described further below. For example, a cDNA encoding
the olfactory
receptor-like protein may be useful in gene therapy, and the olfactory
receptor-like protein
may be useful when administered to a subject in need thereof. The protein
similarity
information, expression pattern, and map location for the Olfactory Receptor-
like protein and
nucleic acid disclosed herein suggest that this Olfactory Receptor may have
important
structural and/or physiological functions characteristic of the Olfactory
Receptor family.
Therefore, the nucleic acids and proteins of the invention are useful in
potential diagnostic and
therapeutic applications and as a research tool. These include serving as a
specific or selective
nucleic acid or protein diagnostic and/or prognostic marker, wherein the
presence or amount
of the nucleic acid or the protein are to be assessed, as well as potential
therapeutic
applications such as the following: (i) a protein therapeutic, (ii) a small
molecule drug target,
(iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic
antibody), (iv) a
nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a
composition
promoting tissue regeneration in vitro and in vivo (vi) biological defense
weapon.
The nucleic acids and proteins of the invention are useful in potential
diagnostic and
therapeutic applications implicated in various diseases and disorders
described below and/or
other pathologies. For example, the compositions of the present invention will
have efficacy
for treatment of patients suffering from: developmental diseases, MHCII and
III diseases
(immune diseases), taste and scent detectability Disorders, Burkitt's
lymphoma,
corticoneurogenic disease, signal transduction pathway disorders, retinal
diseases including
those involving photoreception, cell growth rate disorders; cell shape
disorders, feeding
disorders; control of feeding; potential obesity due to over-eating; potential
disorders due to
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starvation (lack of appetite), noninsulin-dependent diabetes mellitus
(NIDDM1), bacterial,
fungal, protozoal and viral infections (particularly infections caused by HIV-
1 or HIV-2), pain,
cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma;
prostate cancer;
uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart
failure, hypotension,
hypertension, urinary retention, osteoporosis, Crohn's disease; multiple
sclerosis; and
treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial
infarction,
ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and
neurological
disorders, including anxiety, schizophrenia, manic depression, delirium,
dementia, severe
mental retardation, dentatorubro-pallidoluysian atrophy (DRPLA)
hypophosphatemic rickets,
autosomal dominant (2) acrocallosal syndrome and dyskinesias, such as
Huntington's disease
or Gilles de la Tourette syndrome and/or other pathologies and disorders of
the like. The
polypeptides can be used as immunogens to produce antibodies specific for the
invention, and
as vaccines. They can also be used to screen for potential agonist and
antagonist compounds.
For example, a cDNA encoding the OR -like protein may be useful in gene
therapy, and the
OR-like protein may be useful when administered to a subject in need thereof.
By way of
nonlimiting example, the compositions of the present invention will have
efficacy for
treatment of patients suffering from bacterial, fungal, protozoal and viral
infections
(particularly infections caused by HIV-1 or HIV-2), pain, cancer (including
but not limited to
neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia,
bulimia,
asthma, Parkinson's disease, acute heart failure, hypotension, hypertension,
urinary retention,
osteoporosis, Crohn's disease; multiple sclerosis; and treatment of Albright
Hereditary
Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma,
allergies, benign
prostatic hypertrophy, and psychotic and neurological disorders, including
anxiety,
schizophrenia, manic depression, delirium, dementia, severe mental retardation
and
dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome.
Other GPCR-4
diseases and disorders are contemplated.
The novel nucleic acid encoding OR-like protein, and the OR-like protein of
the
invention, or fragments thereof, may further be useful in diagnostic
applications, wherein the
presence or amount of the nucleic acid or the protein are to be assessed.
These materials are
further useful in the generation of antibodies that bind immu~ospecifically to
the novel
substances of the invention for use in therapeutic or diagnostic methods and
other diseases,
disorders and conditions of the like. These materials are further useful in
the generation of
antibodies that bind immunospecifically to the novel substances of the
invention for use in
therapeutic or diagnostic methods. These antibodies may be generated according
to methods
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known in the art, using prediction from hydrophobicity charts, as described in
the "Anti-
GPCRX Antibodies" section below. In one embodiment, a contemplated GPCR4
epitope is
from about amino acids 65 to 85. In another embodiment, a GPCR4 epitope is
from about
amino acids 115 to 130. In additional embodiments, GPCR4 epitopes are from
amino acids
155 to 175, from 215 to 240, from 250 to 275 and from amino acids 280 to 310.
These novel
proteins can also be used to develop assay system for functional analysis.
GPCRS
GPCRS includes a family of three similar nucleic acids and three similar
proteins
disclosed below. The disclosed nucleic acids encode GPCR, OR-like proteins.
GPCRSa
The disclosed novel GPCRSa nucleic acid of 1003 nucleotides (also referred to
as 115-
a-12-A) is shown in Table 5A. An ORF begins with an ATG initiation codon at
nucleotides 6-
8 and ends with a TAA codon at nucleotides 999-1001. A putative untranslated
region
upstream from the initiation codon and downstream from the termination codon
is underlined
in Table 5A, and the start and stop codons are in bold letters.
Table 5A. GPCRSa Nucleotide Sequence (SEQ ID N0:18)
AGACAATGAGTCCTGATGGGAACCACAGTAGTGATCCAACAGAGTTCGTCCTGGCAGGGCTCCCAAATCT
CAACAGCGCAAGAGTGGAATTATTTTCTGTGTTTCTTCTTGTCTATCTCCTGAATCTGACAGGCAATGTG
TTGATTGTGGGGGTGGTAAGGGCTGATACTCGACTACAGACCCCTATGTACTTCTTTCTGGGTAACCTGT
CCTGCCTAGAGATACTGCTCACTTCTGTCATCATTCCAAAGATGCTGAGCAATTTCCTCTCAAGGCAACA
CACTATTTCCTTTGCTGCATGTATCACCCAATTCTATTTCTACTTCTTTCTCGGGGCCTCCGAGTTCTTA
CTGTTGGCTGTCATGTCTGCGGATCGCTACCTGGCCATCTGTCATCCTCTGCGCTACCCCTTGCTCATGA
GTGGGGCTGTGTGCTTTCGTGTGGCCTTGGCCTGCTGGGTGGGGGGACTCGTCCCTGTGCTTGGTCCCAC
AGTGGCTGTGGCCTTGCTTCCTTTCTGTAAGCAGGGTGCTGTGGTACAGCACTTCTTCTGCGACAGTGGC
CCACTGCTCCGCCTGGCTTGCACCAACACCAAGAAGCTGGAGGAGACTGACTTTGTCCTGGCCTCCCTCG
TCATTGTATCTTCCTTGCTGATCACTGCTGTGTCCTACGGCCTCATTGTGCTGGCAGTCCTGAGCATCCC
CTCTGCTTCAGGCCGTCAGAAGGCCTTCTCTACCTGTACCTCCCACTTGATAGTGGTGACCCTCTTCTAT
GGAAGTGCCATTTTTCTCTATGTGCGGCCATCGCAGAGTGGTTCTGTGGACACTAACTGGGCAGTGACAG
TAATAACGACATTTGTGACACCACTGTTGAATCCATTCATCTATGCCTTACGTAATGAGCAAGTCAAGGA
AGCTTTGAAGGACATGTTTAGGAAGGTAGTGGCAGGCGTTTTAGGGAATCTTTTACTTGATAAATGTCTC
AGTGAGAAAGCAGTAAAGTAAAA
The GPCRSa protein encoded by SEQ 1D N0:18 has 331 amino acid residues and is
presented using the one-letter code in Table 5B. The Psort profile for GPCRSa
predicts that
this sequence has a signal peptide and is likely to be localized at the plasma
membrane with a
certainty of 0.6000, it may also localize to the Golgi body. The most likely
cleavage site for a
peptide is between amino acids 54 and 55, i.e., at the slash in the amino acid
sequence VRA-
DT (shown as a slash in TableSB) based on the SignalP result.
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Table SB. Encoded GPCRSa protein sequence (SEQ ID N0:19)
MSPDGNHSSDPTEFVLAGLPNLNSARVELFSVFLLVYLLNLTGNVLIVGVVRA/DTRLQTPMYFF
LGNLSCLEILLTSVIIPKMLSNFLSRQHTISFAACITQFYFYFFLGASEFLLLAVMSADRYLAIC
HPLRYPLLMSGAVCFRVALACWVGGLVPVLGPTVAVALLPFCKQGAVVQHFFCDSGPLLRLACTN
TKKLEETDFVLASLVIVSSLLITAVSYGLIVLAVLSIPSASGRQKAFSTCTSHLIVVTLFYGSAI
FLYVRPSQSGSVDTNWAVTVITTFVTPLLNPFIYALRNEQVKEALKDMFRKVVAGVLGNLLLDKC
LSEKAVK
The disclosed nucleic acid sequence for GPCRS has 604 of 934 bases (64%)
identical
to and 604 of 934 bases (64%) positive with Rattus norvegicus olfactory
receptor protein
mRNA (936 bp) (GENBANK-ID: RATOLFPROD~ acc:M64378) (E= 1.1 a 45).
The full GPCRS amino acid sequence has 149 of 304 amino acid residues (49 %)
identical to, and 201 of 304 residues (66%) positive with, the 313 amino acid
residue olfactory
receptor from Mus musculus (ptnr: SPTREMBL-ACC: Q9Z1V0) (E= 2.6e'4).
GPCRSb
GPCRSa (115-a-12-A) was subjected to an exon linking process to confirm the
sequence. PCR primers were designed by starting at the most upstream sequence
available,
for the forward primer, and at the most downstream sequence available for the
reverse primer.
In each case, the sequence was examined, walking inward from the respective
termini toward
the coding sequence, until a suitable sequence that is either unique or highly
selective was
encountered, or, in the case of the reverse primer, until the stop codon was
reached. Such
suitable sequences were then employed as the forward and reverse primers in a
PCR
amplification based on a wide range of cDNA libraries. The resulting amplicon
was gel
purified, cloned and sequenced to high redundancy to provide GPCRSb, which is
also referred
to as 115-a-12-B.
The nucleotide sequence for GPCRSb (1004 bp, SEQ ID N0:20) is presented in
Table
5C. The nucleotide sequence differs from GPCRSa by the addition of a T between
AS and A6,
and by 6 nucleotide changes (numbered with respect to GPCRSa) C131 >T; T186>C;
6472>A; T579>A; A687>T; C799>T.
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Table 5C. GPCRSb Nucleotide Sequence (SEQ ID N0:20)
AGACATATGAGTCCTGATGGGAACCACAGTAGTGATCCAACAGAGTTCGTCCTGGCAGGGCTCCCAA
ATCTCAACAGCGCAAGAGTGGAATTATTTTCTGTGTTTCTTCTTGTCTATCTCCTGAATCTGATAGG
CAATGTGTTGATTGTGGGGGTGGTAAGGGCTGATACTCGACTACAGACCCCCATGTACTTCTTTCTG
GGTAACCTGTCCTGCCTAGAGATACTGCTCACTTCTGTCATCATTCCAAAGATGCTGAGCAATTTCC
TCTCAAGGCAACACACTATTTCCTTTGCTGCATGTATCACCCAATTCTATTTCTACTTCTTTCTCGG
GGCCTCCGAGTTCTTACTGTTGGCTGTCATGTCTGCGGATCGCTACCTGGCCATCTGTCATCCTCTG
CGCTACCCCTTGCTCATGAGTGGGGCTGTGTGCTTTCGTGTGGCCTTGGCCTGCTGGGTGGGGGGAC
TCATCCCTGTGCTTGGTCCCACAGTGGCTGTGGCCTTGCTTCCTTTCTGTAAGCAGGGTGCTGTGGT
ACAGCACTTCTTCTGCGACAGTGGCCCACTGCTCCGCCTGGCATGCACCAACACCAAGAAGCTGGAG
GAGACTGACTTTGTCCTGGCCTCCCTCGTCATTGTATCTTCCTTGCTGATCACTGCTGTGTCCTACG
GCCTCATTGTGCTGGCTGTCCTGAGCATCCCCTCTGCTTCAGGCCGTCAGAAGGCCTTCTCTACCTG
TACCTCCCACTTGATAGTGGTGACCCTCTTCTATGGAAGTGCCATTTTTCTCTATGTGCGGTCATCG
CAGAGTGGTTCTGTGGACACTAACTGGGCAGTGACAGTAATAACGACATTTGTGACACCACTGTTGA
ATCCATTCATCTATGCCTTACGTAATGAGCAAGTCAAGGAAGCTTTGAAGGACATGTTTAGGAAGGT
AGTGGCAGGCGTTTTAGGGAATCTTTTACTTGATAAATGTCTCAGTGAGAAAGCAGTAAAGTAAAA
The encoded GPCRSb protein is presented in Table SD. The disclosed protein is
331
amino acids long and is denoted by SEQ ID N0:21. GPCRSb differs from GPCRSa by
3
amino acid residues: T42>I; V 151>I; P265>S. Like GPCRSa, the Psort profile
for GPCRSb
predicts that this sequence has a signal peptide and is likely to be localized
at the plasma
membrane with a certainty of 0.6000. The most likely cleavage site for a
peptide is between
amino acids 54 and 55, i.e., at the slash in the amino acid sequence VRA-DT
(shown as a slash
in TableSD) based on the SignalP result.
Table SD. Encoded GPCRSb protein sequence (SEQ ID N0:21)
MSPDGNHSSDPTEFVLAGLPNLNSARVELFSVFLLVYLLNLIGNVLIVGVVRA/DTRLQTPMYFFLGN
LSCLEILLTSVIIPKMLSNFLSRQHTISFAACITQFYFYFFLGASEFLLLAVMSADRYLAICHPLRYP
LLMSGAVCFRVALACWVGGLIPVLGPTVAVALLPFCKQGAVVQHFFCDSGPLLRLACTNTKKLEETDF
VLASLVIVSSLLITAVSYGLIVLAVLSIPSASGRQKAFSTCTSHLIVVTLFYGSAIFLYVRSSQSGSV
DTNWAVTVITTFVTPLLNPFIYALRNEQVKEALKDMFRKVVAGVLGNLLLDKCLSEKAVK
BLASTP (Non-Redundant Composite database) analysis of the best hits for
alignments
with GPCRSb are listed in Table SE.
Table SE.
BLASTP
results
for GPCRSb
Gene Index/Protein/ OrganismLength IdentityPositivesExpect
Identifier (aa) (~) ($)
SPTREMBL- OLFACTORY RECEPTOR313 150/304 203/304 9.4e-76
ACC:Q9Z1V0 C6 Mus musculus (49$) (66$)
SWISSPROT- OLFACTORY RECEPTOR-311 152/301 199/301 5.2e-75
ACC:P23267 LIKE PROTEIN (50$) (66$)
F6
Rattus norvegicus
SPTREMBL- HS6M1-17 (NOVEL 306 195/301 197/301 1.1e-67
7 TM
ACC:Q9Y3P5 (RHODOPSIN FAMILY) (48$) (65$)
DJ994E9.5 (OR LIKE PROTEIN)
Homo sapiens
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A BLASTX was also performed to determine the proteins that have significant
identity
with GPCR4a. The BLASTX results are shown in Table SF.
Table SF. BLASTX results for GPCRSb
Smallest
Sum
Reading High Prob
Sequences producing High-scoring Segment Pairs: Frame Score P(N) N
Ptnr:SPTREMBL-ACC:Q9Z1V0 OLFACTORY RECEPTOR C6 - Mus m... +1 769 5.6e-75 1
ptnr:SWISSPROT-ACC:P23267 OLFACTORY RECEPTOR-LIKE PROT... +1 757 3.1e-79 1
ptnr:SWISSPROT-ACC:P23270 OLFACTORY RECEPTOR-LIKE PROT... +1 701 2.7e-68 1
ptnr:SPTREMBL-ACC:Q9Y3P5 DJ994E9.5 (HS6M1-17 (NOVEL 7 ... +1 688 6.3e-67 1
ptnr:SPTREMBL-ACC:070271 OLFACTORY RECEPTOR-LIKE PROTE... +1 688 6.3e-67 1
ptnr:SPTREMBL-ACC:095007 WUGSC:H_DJ0669B10.3 PROTEIN -... +1 680 9.5e-66 1
ptnr:SPTREMBL-ACC:013036 CHICK OLFACTORY RECEPTOR 7 - ... +1 675 1.5e-65 1
ptnr:SPTREMBL-ACC:095222 OLFACTORY RECEPTOR - Homo sap... +1 672 3.1e-65 1
ptnr:SPTREMBL-ACC:070269 OLFACTORY RECEPTOR-LIKE PROTE... +1 670 5.1e-65 1
ptnr:SPTREMBL-ACC:057597 CHICK OLFACTORY RECEPTOR 7 - ... +1 669 6.5e-65 1
ptnr:SPTREMBL-ACC:070270 OLFACTORY RECEPTOR-LIKE PROTE... +1 668 8.4e-65 1
ptnr:TREMBLNEW-ACC:AAF65961 OLFACTORY RECEPTOR P2 - Mu... +1 666 1.9e-69 1
ptnr:SPTREMBL-ACC:Q9WU86 ODORANT RECEPTOR S1 - Mus mus... +1 665 1.7e-64 1
ptnr:SPTREMBL-ACC:Q90808 OLFACTORY RECEPTOR 9 - Gallus... +1 665 1.7e-69 1
ptnr:SWISSPROT-ACC:P37071 OLFACTORY RECEPTOR-LIKE PROT... +1 646 1.8e-62 1
GPCRSC
Another nucleotide sequence resulted when GPCRSa (115-a-12-A) was subjected to
an
exon linking process to confirm the sequence. PCR primers were designed by
starting at the
most upstream sequence available, for the forward primer, and at the most
downstream
sequence available for the reverse primer. In each case, the sequence was
examined, walking
inward from the respective termini toward the coding sequence, until a
suitable sequence that
is either unique or highly selective was encountered, or, in the case of the
reverse primer, until
the stop codon was reached. Such suitable sequences were then employed as the
forward and
reverse primers in a PCR amplification based on a wide range of cDNA
libraries. The
resulting amplicon was gel purified, cloned and sequenced to high redundancy
to provide the
sequence reported below, which is designated as Accession Number 115 A 12 A
dal, or
GPCRSc.
Human tissues providing SeqCalling Fragments of the clone include Pool One:
adrenal
gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus,
brain -
substantia nigra, brain - thalamus, brain - whole, fetal brain, fetal kidney,
fetal liver, fetal
lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary
gland, placenta,
prostate, salivary gland, skeletal muscle, small intestine, spinal cord,
spleen, stomach, testis,
thyroid, trachea, uterus. The tissue origin of the clone is
RACE(asm:126603384).
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The nucleotide sequence for GPCRSc (1005 bp, SEQ ID N0:22) is presented in
Table
SG. The GPCRSc nucleotide sequence differs from GPCRSa by having an extra T at
the 5'
end, an A at the 3' end, and 6 nucleotide changes: (numbered with respect to
GPCRSa)
T123>C; C131>T; T186>C; G472>A; T579>A; A687>T.
Table SG. GPCRSc Nucleotide Sequence (SEQ ID N0:22)
TAGACAATGAGTCCTGATGGGAACCACAGTAGTGATCCAACAGAGTTCGTCCTGGCAGGGCTCCCAAATC
TCAACAGCGCAAGAGTGGAATTATTTTCTGTGTTTCTTCTTGTCTATCTCCCGAATCTGATAGGCAATGT
GTTGATTGTGGGGGTGGTAAGGGCTGATACTCGACTACAGACCCCCATGTACTTCTTTCTGGGTAACCTG
TCCTGCCTAGAGATACTGCTCACTTCTGTCATCATTCCAAAGATGCTGAGCAATTTCCTCTCAAGGCAAC
ACACTATTTCCTTTGCTGCATGTATCACCCAATTCTATTTCTACTTCTTTCTCGGGGCCTCCGAGTTCTT
ACTGTTGGCTGTCATGTCTGCGGATCGCTACCTGGCCATCTGTCATCCTCTGCGCTACCCCTTGCTCATG
AGTGGGGCTGTGTGCTTTCGTGTGGCCTTGGCCTGCTGGGTGGGGGGACTCATCCCTGTGCTTGGTCCCA
CAGTGGCTGTGGCCTTGCTTCCTTTCTGTAAGCAGGGTGCTGTGGTACAGCACTTCTTCTGCGACAGTGG
CCCACTGCTCCGCCTGGCATGCACCAACACCAAGAAGCTGGAGGAGACTGACTTTGTCCTGGCCTCCCTC
GTCATTGTATCTTCCTTGCTGATCACTGCTGTGTCCTACGGCCTCATTGTGCTGGCTGTCCTGAGCATCC
CCTCTGCTTCAGGCCGTCAGAAGGCCTTCTCTACCTGTACCTCCCACTTGATAGTGGTGACCCTCTTCTA
TGGAAGTGCCATTTTTCTCTATGTGCGGCCATCGCAGAGTGGTTCTGTGGACACTAACTGGGCAGTGACA
GTAATAACGACATTTGTGACACCACTGTTGAATCCATTCATCTATGCCTTACGTAATGAGCAAGTCAAGG
AAGCTTTGAAGGACATGTTTAGGAAGGTAGTGGCAGGCGTTTTAGGGAATCTTTTACTTGATAAATGTCT
CAGTGAGAAAGCAGTAAAGTAAAAA
The coding region of GPCRSc is from nucleotide 7 to 1000, giving the encoded
GPCRSc protein, as presented in Table SH. The disclosed protein is 331 amino
acids long and
is denoted by SEQ ID NO: 23. GPCRSc differs from GPCRSa by 3 amino acid
residues:
L39>P; T42>I; and V151>I. Like GPCRSa, the Psort profile for GPCRSc predicts
that this
sequence has a signal peptide and is likely to be localized at the plasma
membrane with a
certainty of 0.6000. The most likely cleavage site for a peptide is between
amino acids 54 and
55, i.e., at the slash in the amino acid sequence VRA-DT (shown as a slash in
TableSH) based
on the SignalP result.
Table SH. Encoded GPCRSb protein sequence (SEQ ID N0:23)
MSPDGNHSSDPTEFVLAGLPNLNSARVELFSVFLLVYLPNLIGNVLIVGVVRA/DTRLQTPMYFFLGNLSC
LEILLTSVIIPKMLSNFLSRQHTISFAACITQFYFYFFLGASEFLLLAVMSADRYLAICHPLRYPLLMSG
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that differ between the individual GPCR variants are highlighted with a box
and marked with
the (o) symbol above the variant residue in all alignments herein. For
example, the protein
shown in line 1 of Table SJ depicts the sequence for GPCRSa, and the positions
where
GPCRSb or GPCRSc differs are marked with a (o) symbol and are highlighted with
a box. All
GPCRS proteins have significant homology to olfactory receptor (OR) proteins:
Table SI. BLAST
results for
GPCRS
Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect
Identifier (aa) ($) ($)
Gi11290911spIP23267OR 15 (0R3) 311 194/301 189/301 7e-67
IOLF6 RAT Rattus norvegicus (47$) (61$)
Gi167549321refINP_0OR 99, ORC6 313 195/305 192/305 3e-66
35121.11 (AF102523)Mus musculus (47$) (62$)
Gi172421651refINP_0OR 41, ORI7 234 136/304 181/304 9e-61
35113.11 (AF106007)Mus musculus (44$) (58$)
(AF321233)
Gi173634371refINP_0OR, family 306 139/301 186/301 2e-59
10,
39229.11 subfamily C, (46$) (61$)
member 1
Homo Sapiens
Gi1120074311gbIAAG4M50 OR 316 129/303 181/303 4e-59
5202.11AF321236Mus musculus (42$) (59$)
1
(AF321236)
This information is presented graphically in the multiple sequence alignment
given in
Table SJ (with GPCRS being shown on line 1) as a ClustalW analysis comparing
GPCRS with
related protein sequences.
Table SJ Information for the ClustalW proteins:
1) GPCRS (SEQ ID N0:19)
1 S 2) gi~129091~sp~P23267~OLF6_RAT OLFACTORY RECEPTOR-LIKE PROTEIN F6 (SEQ ID
N0:63)
3) gi~6754932~ref~NP_035121.1 ~ olfactory receptor 49 Mus musculus (SEQ ID
N0:64)
4) gi~7242165~ref~NP_035113.1 ~ olfactory receptor 41 Mus musculus (SEQ ID
N0:65)
5) gi~7363437~ref~NP 039229.1 olfactory receptor, family 10, subfamily C,
member 1 Homo Sapiens
(SEQ ID N0:66)
6) gi~12007431~gb~AAG45202.1~AF321236_1 m50 olfactory receptor Mus musculus
(SEQ ID N0:67)
10 20 30 40 50 60
.I....I....I....I....I....I....I...ol.o..l....l....l....l
GPCR5 -MSPDGN S DP ' LNSA E S~ ~ N T G RADT Q
Gi11290911 MAWSTGQ L TPGP I 'GPRS-R L= rv T' S GAHRC Q
Gi I 67549321 ---- S T': LSDACE~~_ ~ L T I L ~FLVDR Y
Gi172421651 --MERRN TGR~IS PAP FS _ TE F~T~RNHPT HK
Gi17363437W- - ~ SHLAD - S. TI ~ T STD Q
Gi1120074311 ----=MEI~N S 'TAP S FT ;FV~LE ' LT VTGS HK
70 80 90 100 110 120
I....I....I....I I....1....1....1....1....1....1....1
GPCR5 ~G~IN~SRQH----T~F I . FF~~FL
Gi I 129091 I ~C ~ ACS T 'T PRGG--- L ~'~ FS~C~YF
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GiI67549321 F - I TNI 'TGHK----T LL FL ~~L F T FF
1
Gi172421651 ~ ~VT T-' GSEENHGQL FE ~~ L C CV
Gi173634371 RTE G T 'L~ HHL TGRR----H RS L~~ LF CC
Gi1120079311 , ST I T ' ~ ~°FRPN----T FL S C CV
130 140 150 160 170 180
.I....I....I....I....I....I....I....lo...l....l....l....l
GPCR5 t~ H' '~ G F$tV~LAC LVPV,~~,GPTV WAL P KQGA
Gi11290911 ~' L' ' GG PG ~L 'C FSAITVPATL S S-R
GiI67599321 ~S m ~.,. K ~ FC _S LLLI PSSI QP P-
Gi172921651 W H' H S ~~' FG S KVFL S- P-
Gi173634371 ~ E' ' L~ H '~ ~GS C VL~ ~GHTP FS P P-
Gi1120074311 ~' '~TG TIS S FTS KVY R~ N-
190 200 210 220 230 240
.1....1....1....1....1.. .1....1....1 ...1....1....1....1
GPCR5 ~s~a~ ~S ' ~ T KKL ETD ''SL a 'SS~LL L S ' S
_ Cdr
gi 1 1290911 S -' T~ Q~ LVS ~' FC -' GSCG L . Y T I~~ ~ R
giI67599321 N '~E I FL ~ FS' GT TC H Y~» H ' K
7
gi172421651 T ~ S'm N ~ T~ T LTD ~-~~I GP S G TG .
gi173639371 T P E ~ N LQIITAL 1 CPFGIL A
gi1120074311 ~" S'~TVD~IVFP~SADVL S ' T
250 260 270 280 290 300
.I....I....I....I....I....o....l....l....l....l....l....l
v _ ~ . v.
' V 1' W
GPCR5 I T ' '~ '-SQSG ~ - R-TT '~
giI 1290911 'Hrv,~~ ~ T L s T T-SVES LILT ~ '~' NTs' T
~+
I~ 'a ~ N
gi 1 67549321 E' S I 5 = ~ ,S GQG ~H ~ ~H - T 'T T
gi172421651 H ~ T I S ~ LSn ~ V Y ~ 'I C
gi173634371 ~ I S' ~ x.._" SYDPP~.P ,y.- FY ~ 'I S
gi1120074311 T m IAS ~I Y ~ 'I C
310 320 330
.I.. .1....1....1....1....1....1..
GPCRS ' E DMFRKVVAGVLGNLLLDKCLSEKAVK
gi11290911 ' _ ~ 'R~7KGK-___________________
gi167549321 'EH SKFQKFSQT-------------
=vr
gi172421651 HLAQGQDANTKKSSRDG------
gi173634371 ' T QKTVPMEI---------------
gi1120D74311 D~~'~~ ~ GGRAPALGESIS----------
DOMAIN results for GPCRS were collected from the Conserved Domain Database
(CDD) with Reverse Position Specific BLAST. This BLAST samples domains found
in the
Smart and Pfam collections. The results are listed in Table SK with the
statistics and domain
description.
Residues 1-115 of 7tm_1 (SEQ ID N0:45) are aligned with GPCRS 43-156 (E = 1e-
20), in Table SK. Residues 314-377 of 7tm 1 also have identity with residues
231-293
(E=2e-OS) of GPCRS.
Table SK. DOMAIN results for GPCRS
gnllPfamlpfam00001, 7tm 1, 7 transmembrane receptor (rhodopsin
family) (SEQ ID NO: 45) Length = 377
Statistics for GPCRS: Score = 93.6 bits (231), Expect = 1e-20
1....1....1....1 ...1....I... I ...1....1....1....1....1
GPCRS ~RF~'~PMY~F~G_ CLI T SNFR~HT~FA~ITQ
TM7 "~C~S K TTN L l1YLE= G~WK~RIH DIF
.I....I....I....I....I....1....1....1..../....1....1....1
GPCRS FYFYF~EF~~L~<pCH' R~PLLM-~GAVCFRALA~GGLI-----
TM7 VTLDV T SI~~C ° I ~' T ~ L~TRYS~KRRVTV IAI~LSFTISCPM
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The nucleic acids and proteins of GPCRS are useful in potential therapeutic
applications implicated in various GPCR-related pathological disorders and/or
OR-related
pathological disorders, described further below. For example, a cDNA encoding
the GPCR
(or olfactory-receptor) like protein may be useful in gene therapy, and the
receptor -like
protein may be useful when administered to a subject in need thereof. The
nucleic acids and
proteins of the invention are also useful in potential therapeutic
applications used in the
treatment of infections such as bacterial, fungal, protozoal and viral
infections (particularly
infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited
to neoplasm;
adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia,
asthma,
Parkinson's disease, acute heart failure, hypotension, hypertension, urinary
retention,
osteoporosis, Crohn's disease; multiple sclerosis; and treatment of Albright
Hereditary
Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma,
allergies, benign
prostatic hypertrophy, and psychotic and neurological disorders, including
anxiety,
schizophrenia, manic depression, delirium, dementia, severe mental retardation
and
dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome
and/or other
pathologies and disorders. Other GPCR-related diseases and disorders are
contemplated.
The polypeptides can be used as immunogens to produce antibodies specific for
the
invention, and as vaccines. They can also be used to screen for potential
agonist and
antagonist compounds. For example, a cDNA encoding the GPCR-like protein may
be useful
in gene therapy, and the GPCR-like protein may be useful when administered to
a subject in
need thereof. By way of nonlimiting example, the compositions of the present
invention will
have efficacy for treatment of patients suffering from bacterial, fungal,
protozoal and viral
infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer
(including but not
limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus
cancer), anorexia,
bulimia, asthma, Parkinson's disease, acute heart failure, hypotension,
hypertension, urinary
retention, osteoporosis, Crohn's disease; multiple sclerosis; and treatment of
Albright
Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers,
asthma, allergies,
benign prostatic hypertrophy, and psychotic and neurological disorders,
including anxiety,
schizophrenia, manic depression, delirium, dementia, severe mental retardation
and
dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome
and/or other
pathologies and disorders. The novel nucleic acid encoding GPCR-like protein,
and the
GPCR-like protein of the invention, or fragments thereof, may further be
useful in diagnostic
applications, wherein the presence or amount of the nucleic acid or the
protein are to be
assessed. These materials are further useful in the generation of antibodies
that bind
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immunospecifically to the novel substances of the invention for use in
therapeutic or
diagnostic methods. These antibodies may be generated according to methods
known in the
art, using prediction from hydrophobicity charts, as described in the "Anti-
GPCRX
Antibodies" section below. This novel protein also has value in development of
powerful
assay system for functional analysis of various human disorders, which will
help in
understanding of pathology of the disease and development of new drug targets
for various
disorders.
GPCR6
The disclosed novel GPCR6 nucleic acid of 948 nucleotides (also referred to as
6-L-19-C) is shown in Table 6A. An open reading begins with an ATG initiation
codon at
nucleotides 7-9 and ends with a TAG codon at nucleotides 940-942. A putative
untranslated
region upstream from the initiation codon and downstream from the termination
codon are
underlined in Table 6A, and the start and stop codons are in bold letters.
Table 6A. GPCR6 Nucleotide Sequence (SEQ ID N0:24)
GGAGACATGGGCAAGGAAAACTGCACCACTGTGGCTGAGTTCATTCTCCTTGGACTATCAGATGTCCCTG
AGTTGAGAGTCTGCCTCTTCCTGCTGTTCCTTCTCATCTATGGAGTCACGTTGTTAGCCAACCTGGGCAT
GATTGCACTGATTCAGGTCAGCTCTCGGCTCCACACCCCCATGTACTTTTTCCTCAGCCACTTGTCCTCT
GTAGATTTCTGCTACTCCTCAATAATTGTGCCAAAAATGTTGGCTAATATCTTTAACAAGGACAAAGCCA
TCTCCTTCCTAGGGTGCATGGTGCAATTCTACTTGTTTTGCACTTGTGTGGTCACTGAGGTCTTCCTGCT
GGCCGTGATGGCCTATGACCGCTTTGTGGCCATCTGTAACCCTTTGCTATACACAGTCACCATGTCTTGG
AAGGTGCGTGTGGAGCTGGCTTCTTGCTGCTACTTCTGTGGGACGGTGTGTTCTCTGATTCATTTGTGCT
TAGCTCTTAGGATCCCCTTCTATAGATCTAATGTGATTAACCACTTTTTCTGTGATCTACCTCCTGTCTT
AAGTCTTGCTTGCTCTGATATCACTGTGAATGAGACACTGCTGTTCCTGGTGGCCACTTTGAATGAGAGT
GTTACCATCATGATCATCCTCACCTCCTACCTGCTAATTCTCACCACCATCCTGAAGATGGGCTCTGCAG
AGGGCAGGCACAAAGCCTTCTCCACCTGTGCTTCCCACCTCACAGCTATCACTGTCTTCCATGGAACAGT
CCTTTCCATTTATTGCAGGCCCAGTTCAGGCAATAGTGGAGATGCTGACAAAGTGGCCACCGTGTTCTAC
ACAGTCGTGATTCCTATGCTGAACTCTGTGATCTACAGCCTGAGAAATAAAGATGTGAAAGAAGCTCTCA
GAAAAGTGATGGGCTCCAAAATTCACTCCTAGGGAAGA
The disclosed nucleic acid sequence has 617 of 915 bases (67%) identical to a
G.
gallus cor4 olfactory receptor 4 DNA (GENBANK-ID: GGCOR4GEN~acc:X94744) (E
value
= 8.7e-65).
The GPCR6 protein encoded by SEQ ID N0:24 has 318 amino acid residues, and is
presented using the one-letter code in Table 6B (SEQ ID N0:25). The SignalP,
Psort and/or
Hydropathy profile for GPCR6 predict that GPCR6 has a signal peptide and is
likely to be
localized at the plasma membrane with a certainty of 0.6000. The SignalP shows
a signal
sequence is coded for in the first 41 amino acids, i.e., with a cleavage site
at the slash in the
sequence TLL-AN, between amino acids 40 and 41. This is typical of this type
of membrane
protein.
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Table 6B. Encoded GPCR6 protein sequence (SEQ ID N0:25).
MGKENCTTVAEFILLGLSDVPELRVCLFLLELLIYGVTLL/ANLGMIALIQVSSRLHTPMYFFLSHLSSVD
FCYSSIIVPKMLANIFNKDKAISFLGCMVQFYLFCTCVVTEVFLLAVMAYDRFVAICNPLLYTVTMSWKV
RVELASCCYFCGTVCSLIHLCLALRIPFYRSNVINHFFCDLPPVLSLACSDITVNETLLFLVATLNESVT
IMIILTSYLLILTTILKMGSAEGRHKAFSTCASHLTAITVFHGTVLSIYCRPSSGNSGDADKVATVFYTV
VIPMLNSVIYSLRNKDVKEALRKVMGSKIHS
The full amino acid sequence of the protein of the invention was found to have
166 of
307 amino acid residues (54%) identical to, and 217 of 307 residues (70%)
positive with, the
314 amino acid residue human olfactory receptor-like protein OLF1
(ptnr:SWISSPROT-
ACC:Q13606) (E value = 5.8e-86)
The GPCR6 target sequence identified previously (6 L-19 C) was subjected to
the
exon linking process to confirm the sequence. PCR primers were designed by
starting at the
most upstream sequence available, for the forward primer, and at the most
downstream
sequence available for the reverse primer. In each case, the sequence was
examined, walking
inward from the respective termini toward the coding sequence, until a
suitable sequence that
is either unique or highly selective was encountered, or, in the case of the
reverse primer, until
the stop codon was reached.
The cDNA coding for the sequence was cloned by polymerase chain reaction (PCR)
using the following primers: CACTGTGGCTGAGTTCATTCTCCTT (SEQ ID N0:26) and
TCTTCCCTAGGAGTGAATTTTGGAGC (SEQ ID N0:27) on the following pool of human
cDNAs: Pool 1 - Adrenal gland, bone marrow, brain - amygdala, brain -
cerebellum, brain -
hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal
brain, fetal kidney,
fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland,
pancreas, pituitary
gland, placenta, prostate, salivary gland, skeletal muscle, small intestine,
spinal cord, spleen,
stomach, testis, thyroid, trachea, uterus. Primers were designed based on in
silico predictions
for the full length or part (one or more exons) of the DNA/protein sequence of
the invention or
by translated homology of the predicted exons to closely related human
sequences or to
sequences from other species. Usually multiple clones were sequenced to derive
the sequence
which was then assembled similar to the SeqCalling process. In addition,
sequence traces
were evaluated manually and edited for corrections if appropriate.
The PCR product derived by exon linking was cloned into the pCR2.1 vector from
Invitrogen. The bacterial clone 55446::6 L-19 C.698018.M1 has an insert
covering the
entire open reading frame cloned into the pCR2.1 vector from Invitrogen.
Usually the resulting amplicons were gel purified, cloned and sequenced to
high
redundancy. The resulting sequences from all clones were assembled with
themselves, with
other fragments in CuraGen Corporation's database and with public ESTs.
Fragments and
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ESTs were included as components for an assembly when the extent of their
identity with
another component of the assembly was at least 95% over 50 bp. In addition,
sequence traces
were evaluated manually and edited for corrections if appropriate. These
procedures provide
the sequence reported below, which is designated Accession Number CG50383_O1
which
does not differ from GPCR6 (6 L-19 C) in amino acid or nucleotide sequence.
The disclosed GPCR6-Olfactory Receptor-like protein is expressed in at least
the
following tissues: Apical microvilli of the retinal pigment epithelium,
arterial (aortic), basal
forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria
and ventricle),
caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex,
colon, cortical
neurogenic cells, endothelial (coronary artery and umbilical vein) cells,
palate epithelia, eye,
neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus,
hypothalamus,
leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid
tissue, adult
lymphoid tissue, tissues that express MHC II and III, nervous tissue, medulla,
subthalamic
nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum,
skeletal muscle,
small intestine, smooth muscle (coronary artery in aortic) spinal cord,
spleen, stomach, taste
receptor cells of the tongue, testis, thalamus, and thymus tissue. This
information was derived
by determining the tissue sources of the sequences that were included in the
invention
including but not limited to SeqCalling sources, Public EST sources,
Literature sources, and/or
RACE sources.
The disclosed GPCR6 protein (SEQ ID N0:25) has good identity with a number of
olfactory receptor proteins. The identity information used for ClustalW
analysis is presented
in Table 6C. The GPCR6 protein has significant identity to olfactory receptor
(OR) proteins:
Table 6C. BLAST
results for
GPCR6
Gene Index/ Protein/ OrganismLengthIdentityPositivesExpect
Identifier (aa)
Gi157299601ref~NPOR fam. 5, 314 159/307 200/307 2e-72
0
_ subfam. I, (50~) (64~)
06628.11 mem 1
Homo Sapiens
Gi124950541spIQ9515OR-like prt 311 152/308 207/308 8e-70
OLF2
510LF2 CANFA Canis familiaris (99~) (66$)
Gi1116925191gbIAAG3OR 41, K11 314 150/308 199/308 8e-69
9B56.11AF282271 Mus musoulus (98~) (63~)
1
(AF282271)
Gi137464431gbIAAC63OR, OR93ch 314 151/306 199/306 1e-68
969.1 (AF045577)Pan troglodytes (99~) (64$)
Gi137464481gbIAAC63OR OR93Gib 313 149/305 198/305 3e-68
971.11 (AF095580)Hylobates (98~) (64~)
lar
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This information is presented graphically in the multiple sequence alignment
given in
Table 6D (with GPCR6 being shown on line 1) as a ClustalW analysis comparing
GPCR6
with related protein sequences.
Table 6D Information for the ClustalW proteins:
1) GPCR6 (SEQ ID N0:25)
2) gi~5729960~ref]NP_006628.1 ~ OR, family 5, subfamily I, member 1 Homo
Sapiens (SEQ ID N0:25)
3) gi~2495054~sp~Q95155~OLF2_CANFA OR-LIKE PROTEIN OLF2 (SEQ ID N0:25)
4) gi~11692519Igb~AAG39856.1~AF282271_1 odorant receptor K11 Mus musculus (SEQ
ID N0:25)
5) gi~3746443~gb~AAC63969.1~ olfactory receptor OR93Ch [Pan troglodytes] (SEQ
ID N0:25)
6) gi~3746448~gb~AAC63971.1~ olfactory receptor OR93Gib [Hylobates lar] (SEQ
ID N0:25)
10 20 30 40 50 60
.. .I_.__I....1....I....1....1....1....1....1....1....1....1
GPCR6 ---~GK C T L ~'D ~~ ~ C GI', _VS
g1157299601 -MEFTD L L FPT T-~ ISI ~ LL~'~ 3 PH Q
g1124950591 --- DG CAS N ~N G~ T T I ~ I~V
g1111692519 MND TS~ CT ~E'~~ P G T GS H
g1137464931 -- N~ FT~YN~TT~T
g1 l 37464481 -- N FT YNIIa T- Si~~T
70 80 90 100 110 120
.1.. .1.. .1.. .I.. .I.. .1.. .I.. .1.. .I.. .1.. .I.. .I
GPCR6 Sn m~ Iw ~ ~IFND ~L ~ F L.CTC ~T
g1157299601 L ~ D V~ V LS ~Y ~ F CT DT S
g1124950541 S IAN ~ P YS ~, L CT D L
g11116925191 S ~ T~ T KNI P T L LI I
g1137464431 ~~ ~ SF~ L ~ Q G
g1137464481 I S~ T~ F L N Q G
130 140 150 160 170 180
.I....I....1....1....1....1....1....1....1....1....1....1
1'
GPCR6 ~ ~ m T C ~R~E SCC FC aC s' I LCL ~ L P YRS
g1 1 57299601 ~ ~ RG~G LS = S ' S ~ I K , D
g1124950541 m~ S ~ F Si S~tSL "~ ~ D.'SV ILT - C~ ES
g1 I 116925191 ~ ~ T Y ~Yr ~ S ~ C' S ' GFM ~' KL
g1137464431 ,~ ~ Q P :~~~TN~ ' P GP
g1137464981 ~~ S Q "jV P ~T TN~ - P GL
190 20D 210 220 230 290
.1.. .1.. .I.. .I.. .1....1.. .I.. .1.. .1.. .I.. .I.. .I
GPCR6 ~ WP S g ~ S IT ~ T '~~LATLNES.aT.II~ TT ~_-,~ ~ ~H
g1157299601 ~ P~ N T~ T~' ~.STYGSSVE°CF I a:= F, $. ~ FS
g1124950541 ~ P~ L S S~ Q 'V'~T~FGFIE-I SG~3 C~, T~A~~ N
g11116925191 m L~ ~ ~N Y~; LFFGTLNI ~-PI T ~I~ - ~ T S
g1 I 37464931 n S S - ~ I~AGAAG~ ~~~.,; ~ ~ C
1137464481 n"S S I GAVG G ~ C
250 260 270 280 290 300
.1.. .1.. .I.. .I.. .1.. .I.. .1.. .I.. .I.. .I.. .1.. .I
GPCR6 ~T ,, H , LS ~C~ ~GN v ~T ~ S
g1157299601 T~. S T~L S~~ yL' P.T. T . T
g1 124950541 L ~ S W - I~I ~ S
g1 l 116925191 S L~ ~,~ S $ L~~ ~ VS n S ~ T
g1137464431 T S yLY S ~ L
g1137464981 T S FLY 5 P~ L7 ~,
310
.I.. .I.. .I..
GPCR6 ~~ GS IHS-
Y
&~
g1157299601 E :--RS DSS-
gi124950541 rv~wKN y H--
gi I 116925191 ~~~~K~ E
g1137469431 H T T CKA
g1 I 37464981 H T ~T,' CKA
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The presence of identifiable domains in GPCR6 was determined by searches using
algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then
determining the
Interpro number by crossing the domain match (or numbers) using the Interpro
website
(http:www.ebi.ac.uk/interpro~.
DOMAIN results for GPCR6 were collected from the Conserved Domain Database
(CDD) with Reverse Position Specific BLAST. This BLAST samples domains found
in the
Smart and Pfam collections. The results are listed in Table 6E with the
statistics and domain
description. The results indicate that this protein contains the 7tm-1
(InterPro) 7
transmembrane receptor (rhodopsin family) (as defined by Interpro) at residues
42-203, which
align with residues 2-158 of the 7TM domain. This indicates that the sequence
of GPCR6 has
properties similar to those of other proteins known to contain this/these
domains) and similar
to the properties of these domains.
Table 6E. DOMAIN results for GPCR6
gnl~Pfamlpfam00001, 7tm 1, 7 transmembrane receptor (rhodopsin
family) Length = 377 (SEQ ID N0:45).
Score = 86.3 bits (212), Expect = 2e-18
.. .I.. .1....1.. .I.. .1.. .I.. .1.. .1.. .I.. .I.. .1.. .I
TM7R6 G~~L~CQREKA~Q~TTN~L~...SS~, 9~LLY,S~I~I3~ ~ ~YLE~V~VG~WKf~R~H~D~Q
.I....I....1....1....1....1....1....1....1....1....1....1
GPCR6 FY.FCTCVVTE'FL Y ' ~,RCN ,~ TVT E~AS-CC FCGTVC LIH
TM7 VT~DVMMCTAS LNBC ~~IT~ TRY S R~' T'r~~TMIAI LSFTI~CPM
..I....I....I....I....1....~....1.. .I.. .1....I.. .1....1
GPCR6 .CL-ALRIPFYR--------SNVINHFFCDLPPV-- L LA .DITVNETLL~ A ---
TM7 ~FGLNNTDQNE--------CIIANP--------AFVVY~SIV~FYV----PFI TLLVYI
The similarity information for the GPCR6 protein and nucleic acid disclosed
herein
suggest that GPCR6 may have important structural and/or physiological
functions
characteristic of the Olfactory Receptor family and the GPCR family.
Therefore, the nucleic
acids and proteins of the invention are useful in potential diagnostic and
therapeutic
applications and as a research tool. These include serving as a specific or
selective nucleic
acid or protein diagnostic and/or prognostic marker, wherein the presence or
amount of the
nucleic acid or the protein are to be assessed, as well as potential
therapeutic applications such
as the following: (i) a protein therapeutic, (ii) a small molecule drug
target, (iii) an antibody
target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a
nucleic acid useful in
gene therapy (gene delivery/gene ablation), and (v) a composition promoting
tissue
regeneration in vitro and in vivo (vi) biological defense weapon. The novel
nucleic acid
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encoding GPCR6, and the GPCR6 protein of the invention, or fragments thereof,
may further
be useful in diagnostic applications, wherein the presence or amount of the
nucleic acid or the
protein are to be assessed.
The nucleic acids and proteins of the invention are useful in potential
diagnostic and
therapeutic applications implicated in various diseases and disorders
described below and/or
other pathologies. For example, the compositions of the present invention will
have efficacy
for treatment of patients suffering from: developmental diseases, MHCII and
III diseases
(immune diseases), taste and scent detectability disorders, Burkitt's
lymphoma,
corticoneurogenic disease, signal transduction pathway disorders, retinal
diseases including
those involving photoreception, cell growth rate disorders; cell shape
disorders, feeding
disorders; control of feeding; potential obesity due to over-eating; potential
disorders due to
starvation (lack of appetite), noninsulin-dependent diabetes mellitus
(NIDDM1), bacterial,
fungal, protozoal and viral infections (particularly infections caused by HIV-
1 or HIV-2), pain,
cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma;
prostate cancer;
uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart
failure, hypotension,
hypertension, urinary retention, osteoporosis, Crohn's disease; multiple
sclerosis; and
treatment of Albright hereditary ostoeodystrophy, angina pectoris, myocardial
infarction,
ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and
neurological
disorders, including anxiety, schizophrenia, manic depression, delirium,
dementia, severe
mental retardation dentatorubro-pallidoluysian atrophy(DRPLA) hypophosphatemic
rickets,
autosomal dominant (2) acrocallosal syndrome and dyskinesias, such as
Huntington's disease
or Gilles de la Tourette syndrome and/or other pathologies and disorders of
the like.
The polypeptides can be used as immunogens to produce antibodies specific for
the
invention, and as vaccines. They can also be used to screen for potential
agonist and
antagonist compounds. For example, a cDNA encoding the OR -like protein may be
useful in
gene therapy, and the OR-like protein may be useful when administered to a
subject in need
thereof. By way of nonlimiting example, the compositions of the present
invention will have
efficacy for treatment of patients suffering from bacterial, fungal, protozoal
and viral
infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer
(including but not
limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus
cancer), anorexia,
bulimia, asthma, Parkinson's disease, acute heart failure, hypotension,
hypertension, urinary
retention, osteoporosis, Crohn's disease; multiple sclerosis; and treatment of
Albright
hereditary ostoeodystrophy, angina pectoris, myocardial infarction, ulcers,
asthma, allergies,
benign prostatic hypertrophy, and psychotic and neurological disorders,
including anxiety,
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schizophrenia, manic depression, delirium, dementia, severe mental retardation
and
dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome
and/or other
pathologies and disorders.
The novel nucleic acid encoding OR-like protein, and the OR-like protein of
the
invention, or fragments thereof, may further be useful in diagnostic
applications, wherein the
presence or amount of the nucleic acid or the protein are to be assessed.
These materials are further useful in the generation of antibodies that bind
immuno-
specifically to the novel GPCR6 substances for use in therapeutic or
diagnostic methods.
These antibodies may be generated according to methods known in the art, using
prediction
from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies"
section below. In
one embodiment, a contemplated GPCR6 epitope is from about as 225 to 240. In
another
embodiment, a GPCR6 epitope is from about as 255 to 275. In additional
embodiments,
GPCR6 epitopes are from as 280 to 310.
GPCR7
A novel GPCR nucleic acid was identified by TblastN using CuraGen
Corporation's
sequence file for GPCR probes or homologs, and run against the Genomic Daily
Files made
available by GenBank. The nucleic acid was further predicted by the program
GenScanTM,
including selection of exons. These were further modified by means of
similarities using
BLAST searches. The sequences were then manually corrected for apparent
inconsistencies,
thereby obtaining the sequences encoding the full-length protein. The
disclosed novel GPCR7
nucleic acid of 1013 nucleotides (also referred to as dj313i6 D) is shown in
Table 7A. An
open reading begins with an ATG initiation codon at nucleotides 5-7 and ends
with a TAG
codon at nucleotides 997-999. A putative untranslated region upstream from the
initiation
codon and downstream from the termination codon are underlined in Table 7A,
and the start
and stop codons are in bold letters.
Table 7A. GPCR7 Nucleotide Sequence (SEQ ID N0:28)
TACAATGGAAAGAGCTAACGACAGCACCTTCTCTGGATTCATCCTCCTGGGCTTCTCCAACAGGCCTCAGCTGGAAAC
AGCTCTCTTTGTGGTCATCTTGATCATCTACTTTCTGAGCTTTCTGGGCAATGGCACCATTATACTTTTATCCATTGT
AGATCCTCGCCTCCATACCCCTATGTATTTCTTCCTCTCCAATCTCTCTTTTATGGATCTTTGTTTGACCACTTGTAC
TGTCCCTCAGACACTGGTCAACTTTAAGGGGAAGGACAAGACCATCACCTATGGTGGCTGCGTGACCCAGCTATTCAT
TGCCTTGGGACTCGGGGGGGGAGTGGAGTGTGTCTTATTGTCTGCCATGGCCTATGACCGCTATGCAGCCGTCTGCCG
CCCACTCCACTACATGGTGAGCATGCATCCCCAACTTTGCTTGCAGTTGGTTGTAACCACTTGGCTCACAGGGTTTGG
CAATTCTGTGATACAGACAGCATTGACCATGACTCTCCCCCTCTGTGATAAAAACCAAGTGGATCATTTCTTCTGTGA
AGTTCCAGTGATGCTGAAACTGTCCTGCACCAACACCTCCATCAACGAGGCTGAAATCTTTGCTGTCAGTGTCTTCTT
CTTGGTGGTGCCTCTCTCACTCATCTTAGCATCCTATGGTCACATTACTCATGCAGTCCTGAAGATAAAGTCAGCTCA
AGGGAGGCAGAAGGCTTTTGGAACCTGTGGTTCTCACCTCCTGGTAGTGATCATTTTCTTTGGGACACTCATCTCCAT
GTACCTCCAGCCTCCCTCCAGTTATTCACAGGATGTGAACAAAAGCATTGCACTCTTCTATACTCTGGTGACTCCTCT
ACTGAATCCCCTAATTTACACTCTGAGGAACAAGGAAGTCAAAGGGGCAACTAAGAAGACTAGTGGGGAGGACCATAG
ATGCATGAGAAAGTTAACGCAGGGTTTGCAGTTCCAAACATTTGTGCACTAGAAGACTGCTGAGAAGCTACAAACTA
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The disclosed nucleic acid sequence has 615 of 939 bases (65%) identical to a
Homo
Sapiens olfactory receptor-like protein (OR2C1) gene (GENBANK-ID: AF098664) (E
value =
1.7e-6').
The GPCR7 protein encoded by SEQ ID N0:28 has 327 amino acid residues, and is
presented using the one-letter code in Table 7B (SEQ ID N0:29). The SignalP,
Psort and/or
Hydropathy profile for GPCR7 predict that GPCR7 has a signal peptide and is
likely to be
localized at the plasma membrane with a certainty of 0.6000. The SignalP shows
a signal
sequence is coded for in the first 44 amino acids, i.e., with a cleavage site
at the slash in the
sequence GNG-TI, between amino acids 43 and 44. This is typical of this type
of membrane
protein.
Table 7B. Encoded GPCR7 protein sequence (SEQ ID N0:29).
MERANDSTFSGFILLGFSNRPQLETALFVVILIIYFLSFLGNG/TIILLSIVDPRLHTPMYFFLSNL
SFMDLCLTTCTVPQTLVNFKGKDKTITYGGCVTQLFIALGLGGGVECVLLSAMAYDRYAAVCRPLHY
MVSMHPQLCLQLVVTTWLTGFGNSVIQTALTMTLPLCDKNQVDHFFCEVPVMLKLSCTNTSINEAEI
FAVSVFFLVVPLSLILASYGHITHAVLKIKSAQGRQKAFGTCGSHLLVVIIFFGTLISMYLQPPSSY
SQDVNKSIALFYTLVTPLLNPLIYTLRNKEVKGATKKTSGEDHRCMRKLTQGLQFQTFVH
The full amino acid sequence of the protein of the invention was found to have
183 of
304 amino acid residues (60%) identical to, and 229 of 304 residues (75%)
positive with, the
313 amino acid residue OL1 receptor protein from Rattus norvegicus
(ptnr:SPTREMBL-
ACC:Q63394) (E value = 2.6e-9'). Further BLAST analysis produced the
significant results
listed in Table 7C. The disclosed GPCR7 protein (SEQ m N0:29) has good
identity with a
number of olfactory receptor proteins.
Table 7C. BLAST
results for
GPCR7
Gene Index/ Protein/ LengthIdentityPositivesExpec
Identifier Organism (aa) (~) (~) t
gi1111779061refINPOR 313 168/304 209/304 2e-86
0
68632.11 (L34079)Rattus (55~) (680)
norve icus
gi1109445161embICACInovel 7 TM 313 169/304 207/309 2e-85
-OR
4158.11 (AL133267)family (hS6M1- (55$) (67~)
dJ408B20.2 32) Homo Sapiens
gi1120544111embICAC2OR 41, K11 357 166/304 206/309 1e-83
0513.11 (AJ302593)Homo Sapiens (54~) (67~)
gi1120543931embICAC2OR 357 165/304 206/304 4e-83
0504.11 (AJ302589Homo Sapiens (54~) (67$)
-
592)
gi130804671embICABI1OR 310 165/304 206/304 4e-83
427.11 (Z98744) Homo Sapiens (54~) (67~)
52
<IMG>
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The presence of identifiable domains in GPCR7 was determined by searches using
algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then
determining the
Interpro number by crossing the domain match (or numbers) using the Interpro
website
(http:www.ebi.ac.uk/interpron.
DOMAIN results for GPCR7 were collected from the Conserved Domain Database
(CDD) with Reverse Position Specific BLAST. This BLAST samples domains found
in the
Smart and Pfam collections. The results are listed in Table 7E with the
statistics and domain
description. The results indicate that this protein contains the following
protein domains (as
defined by Interpro) at the indicated positions: domain name 7tm 1 (InterPro)
7
transmembrane receptor (rhodopsin family). Residues 61-142 of 7tm_1 (SEQ ID
N0:45) are
aligned with GPCR7 41-180 (E = Se-18) in Table 7E. Residues 307-377 of 7tm-1
also have
identity with residues 222-291 (E=0.001) of GPCR7. This indicates that the
sequence of
GPCR7 has properties similar to those of other proteins known to contain
this/these domains)
and similar to the properties of these domains.
Table 7E. DOMAIN results for GPCR7
gnl~Pfamlpfam00001, 7tm 1, 7 transmembrane receptor (rhodopsin
family) Length = 377 (SEQ ID N0:45).
Score = 85.1 bits (209), Expect = Se-18
.I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I
TM7R~ ~1L~CAVSRKA~Q~TTN L~5 ~V ~ L'T LV~VYLEVVGWKE'~RIF~VDIF
.1....I....1....1....I....I....1....1....1....1.._..1....1
TM7R7 T ~ D :GjtCTASILN~C,~~DRYT~VAMPNI~NTRYSSKP - TW~FTI~CP
.I....I....I....I....I....1....1....1....1....1....1....1
GPCR7 QT. T LP--L--------CD QVDHFFC-----------------------------
TM7 LFG~NN~DQNE--------CIIA~P--------AFVVYSSIVSFYV----PFIVTLLVYI
The similarity information for the GPCR7 protein and nucleic acid disclosed
herein
suggest that GPCR7 may have important structural and/or physiological
functions
characteristic of the Olfactory Receptor family and the GPCR family.
Therefore, the nucleic
acids and proteins of the invention are useful in potential diagnostic and
therapeutic
applications and as a research tool. These include serving as a specific or
selective nucleic
acid or protein diagnostic and/or prognostic marker, wherein the presence or
amount of the
nucleic acid or the protein are to be assessed, as well as potential
therapeutic applications such
as the following: (i) a protein therapeutic, (ii) a small molecule drug
target, (iii) an antibody
target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a
nucleic acid useful in
gene therapy (gene delivery/gene ablation), and (v) a composition promoting
tissue
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regeneration in vitro and in vivo (vi) biological defense weapon. The novel
nucleic acid
encoding GPCR7, and the GPCR7 protein of the invention, or fragments thereof,
may further
be useful in diagnostic applications, wherein the presence or amount of the
nucleic acid or the
protein are to be assessed.
The nucleic acids and proteins of the invention are useful in potential
therapeutic
applications implicated in used in the treatment of infections such as
bacterial, fungal,
protozoa) and viral infections (particularly infections caused by HIV-1 or HIV-
2), pain, cancer
(including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate
cancer; uterus
cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure,
hypotension,
hypertension, urinary retention, osteoporosis, Crohn's disease; multiple
sclerosis; and
treatment of Albright hereditary ostoeodystrophy, angina pectoris, myocardial
infarction,
ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and
neurological
disorders, including anxiety, schizophrenia, manic depression, delirium,
dementia, severe
mental retardation and dyskinesias, such as Huntington's disease or Gilles de
la Tourette
syndrome andlor other pathologies and disorders.
The disclosed GPCR7 polypeptides can be used as immunogens to produce
antibodies
specific for the invention, and as vaccines. They can also be used to screen
for potential
agonist and antagonist compounds. For example, a cDNA encoding the GPCR-like
protein
may be useful.in gene therapy, and the GPCR-like protein may be useful when
administered to
a subject in need thereof. By way of nonlimiting example, the compositions of
the present
invention will have efficacy for treatment of patients suffering from
bacterial, fungal,
protozoa) and viral infections (particularly infections caused by HIV-1 or HIV-
2), pain, cancer
(including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate
cancer; uterus
cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure,
hypotension,
hypertension, urinary retention, osteoporosis, Crohn's disease; multiple
sclerosis; and
treatment of Albright hereditary ostoeodystrophy, angina pectoris, myocardial
infarction,
ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and
neurological
disorders, including anxiety, schizophrenia, manic depression, delirium,
dementia, severe
mental retardation and dyskinesias, such as Huntington's disease or Gilles de
la Tourette
syndrome and/or other pathologies and disorders. The novel nucleic acid
encoding GPCR-like
protein, and the GPCR-like protein of the invention, or fragments thereof, may
further be
useful in diagnostic applications, wherein the presence or amount of the
nucleic acid or the
protein are to be assessed. These materials are further useful in the
generation of antibodies
that bind immunospecifically to the novel substances of the invention for use
in therapeutic or
CA 02401453 2002-08-26
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diagnostic methods. These antibodies may be generated according to methods
known in the
art, using prediction from hydrophobicity charts, as described in the "Anti-
GPCRX
Antibodies" section below.
GPCR8
A novel nucleic acid was identified on chromosome 6 by TblastN using CuraGen
Corporation's sequence file for GPCR probes or homologs and , run against the
Genomic
Daily Files made available by GenBank. The nucleic acid was further predicted
by the
program GenScanTM, including selection of exons. These were further modified
by means of
similarities using BLAST searches. The sequences were then manually corrected
for apparent
inconsistencies, thereby obtaining the sequences encoding the full-length
protein. The
disclosed novel GPCR8 nucleic acid of 958 nucleotides (also referred to as
dj408b20 A) is
shown in Table SA. An open reading begins with an ATG initiation codon at
nucleotides 4-6
and ends with a TGA codon at nucleotides 955-957. A putative untranslated
region upstream
from the initiation codon and downstream from the termination codon are
underlined in Table
8A, and the start arid stop codons are in bold letters.
Table 8A. GPCR8 Nucleotide Sequence (SEQ ID N0:30)
_GCAATGGAAAAATCCAATGTCAGCTCAGTGTATGGTTTTATCTTGGTGGGTTTCTCTGATCGTCCCAAGCTG
GAGATGGTGCTCTTTACAGTAAATTTTATTCTGTATTCAGTGGCTGTGCTGGGAAATTCAACCATAATCC
TTGTGTGTATATTAGACTCTCAACTTCATACCCCAATGTACTTCTTTCTGGCAAATCTTTCCTTTCTAGA
TCTCTGCTTCAGTACTAGTTGCATCCCACAAATGCTGGTAAACCTCTGGGGCCCTGACAAGACTATTAGC
TGTGCTGGCTGTGTTGTCCAGCTTTTCTCTTTCCTTTCTGTCAGGGGAATTGAGTGCATCCTTCTGGCTG
TCATGGCCTATGACAGCTATGCTGCAGTCTGCAAACCGTTGCGCTATCTGGTCATTATGCACCTCCAGCT
GTGTCTAGGACTGATGGCTGCAGCCTGGGGGAGTGGACTGGTCAATGCCGTTGTCATGTCACCACTAACA
ATGACCCTCTCCAGAAGTGGCCGCCGCCGAGTTAACCATTTCCTCTGTGAAAAGCCAGCACTGATCAAGA
TGGCTTGTTTGGATGTTCGTGCAGTGGAAATGCTGGCTTTTGCTTTTGCCGTTCTCATTGTCCTACTGCC
CCTCACTCTTATTCTTGTCTCCTACGGCTACATTGCTGCAGCTGTGCTAAGCATCAAGTCAGCTGCCAGG
CAATGGAAGGCCTTCCATACCTGTAGCTCTCACCTCACAGTGGTCTCCCTGTTTTATGGGAGCATCATCT
ATATGTATATGCAGCCAGGAAACAGTTCTTCCCAAGACCAAGGCAAGTTTCTCACTCTCTTCTACAACCT
GGTGACTCCTATGTTGAATCTGCTCATCTATACTTTAAGGAATAAGGAGGTGAAAGGAGCACTGAAGAAG
GTTTTGGGGAGGCAAAATGAACTGGAGAAATATGATAAGTTGTGAA
The disclosed nucleic acid sequence has 768 of 1148 bases (66%) identical to a
Homo
Sapiens OR-like (gb:GENBANK-ID:HS88J8~acc:AL035402 Human DNA sequence from
clone 88J8 on chromosome 6p21.31-21.33. Contains a gene for a novel 7
transmembrane
receptor (rhodopsin family) (olfactory receptor like) protein, pseudogene
similar to olfactory
receptor genes and a GTP binding protein SARA (mouse) pseudogene. Contains
ESTs, an
STS and GSS, complete sequence - Homo Sapiens, 47216 bp.) (E value = 1.3e 81).
The GPCR8 protein encoded by SEQ ID N0:30 has 317 amino acid residues, and is
presented using the one-letter code in Table 8B (SEQ ID N0:31). The SignalP,
Psort and/or
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Hydropathy profile for GPCR8 predict that GPCR8 has a signal peptide and is
likely to be
localized at the plasma membrane with a certainty of 0.6850. The SignalP shows
a signal
sequence is coded for in the first 42 amino acids, i.e., with a cleavage site
at the slash in the
sequence VLG-NS, between amino acids 41 and 42. This is typical of this type
of membrane
protein.
Table 8B. Encoded GPCR8 protein sequence (SEQ ID N0:31).
MEKSNVSSVYGFILVGFSDRPKLEMVLFTVNFILYSVAVLG/NSTIILVCILDSQLHTPMYFFLANLSF
LDLCFSTSCIPQMLVNLWGPDKTISCAGCVVQLFSFLSVRGIECILLAVMAYDSYAAVCKPLRYLVIMH
LQLCLGLMAAAWGSGLVNAVVMSPLTMTLSRSGRRRVNHFLCEKPALIKMACLDVRAVEMLAFAFAVLI
VLLPLTLILVSYGYIAAAVLSIKSAARQWKAFHTCSSHLTWSLFYGSIIYMYMQPGNSSSQDQGKFLT
LFYNLVTPMLNLLIYTLRNKEVKGALKKVLGRQNELEKYDKL
The full amino acid sequence of the protein of the invention was found to have
187 of
305 amino acid residues (61%) identical to, and 239 of 305 residues (78%)
positive with, the
320 amino acid residue novel transmembrane receptor (rhodopsin family, OR-
like, HS6M1-
15) protein from Homo Sapiens (ptnr:SPTREMBL-ACC:Q9Y3N9) (E value = 2.4e-log).
Further BLAST analysis produced the significant results listed in Table 8C.
The disclosed
GPCR8 protein (SEQ ID N0:31) has good identity with a number of olfactory
receptor
proteins.
Table 8C. BLAST
results for
GPCR8
Gene Index/ Protein/ OrganismLengthIdentitPositivesExpect
Identifier (aa) y ($) ($)
Gi148265211embICAB42novel 7 tm 320 177/305226/305 5e-92
853.11 receptor protein (58~) (79~)
(AL035402, dJ88J8.1)(rhodopsin
fam.,
(AJ302594-99) OR-like)
(AJ302600-O1) (hs6Ml-15)
Homo Sapiens
Gi112059431)embICAC2OR 320 176/305226/305 1e-91
0523.11 (AJ302603)Homo Sapiens (57~) (73$)
Gi1120544291embICAC2OR 320 177/305225/305 1e-91
0522.11 (AJ302602)Homo Sapiens (58~) (73~)
03 OR 15 (0R3) 312 166/307211/307 2e-81
Gi166791701refINP
_ Mus musculus (54~) (68~)
2788.11
Gi1122310291spIQ1506OR 2H3 316 163/306208/306 5e-81
2102H3 HUMAN Homo Sapiens (53$) (67~)
This information is presented graphically in the multiple sequence alignment
given in
Table 8D (with GPCR8 being shown on line 1) as a ClustalW analysis comparing
GPCR8
with related protein sequences.
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Table 8D. Information for the ClustalW proteins:
1) GPCRB (SEQ ID N0:31)
2) gi~4826521 ~emb~CAB42853.1 ~ dJ88J8. l (novel 7 TM receptor (rhodopsin
family) (OR like) protein)
(hs6M1-15)) Homo Sapiens (SEQ ID N0:59)
3) gi~12054431~emb~CAC20523.1~ olfactory receptor Homo Sapiens (SEQ ID N0:60)
4) gig 12054429~emb~CAC20522.1 ~ olfactory receptor Homo Sapiens (SEQ ID
N0:61)
5) gi~6679170~ref~NP_032788.1 ~ olfactory receptor 15 Mus musculus (SEQ ID
N0:58)
6) gi~12231029~sp~Q15062~02H3 HUMAN OR 2H3 (OR-LIKE PRT FAT11) (SEQ ID N0:56)
20 30 40 50 60
.I....I....I....I....I....1....1....1....1....1....1....1
V
GPCRB FT L S Y ~T CST ~
~i V !h Y
Gi l 48265211 A~~ Y °' Y S
Gi1120544311 _~ Y ~ ' ~ S
GiI120544291 ~~~ Y _ S
Gi166791701 D N SGT H~ FF LAS LT L ~
Gi1122310291 -MD TP E G~ RT F FTS L L ~P S~
70 80 90 100 110 120
.I....I....I....I....I....I....I....I....I.~..I....I....1
GPCRB ~ S C ~ '~ C '~'~ ~S y S,, R I
L
Gi148265211 I ~ ~ I
gi1120544311 ~ I '~ ISO
gi1120544291 ~ I - .~ ;~ ° ,
gi166791701 S S ~ S'~ '~ ~ ~ = T
gi I 122310291 S ~ C ~ S IE'~i. = S T T
130 190 150 160 170 180
.I....I....I..~.I....I...~1....1....1....1....1....1....1
GPCR8 S ' L L G GS SP ~'T SRS RR
1148265211 ~ T z rn~H I~~~I I S S ~C T '"NI°'~~
9
g11120544311 T ~ ~H I I 5 S ~C 'T Ie
g11120544291 ~ ~T ~ 'H n I I S~S C 'T ~NI'~
g1166791701 '...~ ~ ~' L G, S LG QS - t
g11122310291 V T S, E QTPS PRQ D
190 200 210 220 230 240
.I....I....I....I....I....1....1.~..1....1....1....1....1
GPCR8 K~~ I R~ y L ~F L' T ~ SI
g1198265211 A Z ~ T 1S G ;I T~ I I~ ~_ 'T KeS '
gi112054431j I ~ T 5 GI T~ I 3 ~;~ T K~S '
g11120549291 I ~ 'T S G T I ~ ' 'T S
g1166791701 I ~ ~ ~LN VIG CTFFT ~'S-' C ~ ~_ h EG '
g11122310291 ' I ~~ E ~YN Q~AVAS' S TW~ IN KG ' F
250 260 270 280 290 300
.1.. .I.. .I.. .I.. .I.. .I.. .I.. .1.. ~I.. .I.. ~I.. .I
GPCR8 ~ S ~ S ~ w ~ M.- ~ L
g1 1 9826521: .- . >, m T ~ " D
g11120549311 ~~ ~ ~~~~ ~ I ~ ~~ D
g11120549291 m I, D
g1166791701 F= L~ KS S 5
g11122310291 S Sft- K~PY - F TR
310 320
.I.. .I.. .1....I
GPCR8 GRQ: LE~YD~,L---
gi148265211 ~ RFH' T I,'~~1~,',', CKS
g11120549311 RFH T IKNCKS
g1 1120549291 ~ FH T I.N.ItliINCKS
91166791701 G~ GKGRG------AS--
gi1122310291 F,~ = KERD RCS AA--
I~
The presence of identifiable domains in GPCR8 was determined by searches using
algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then
determining the
Interpro number by crossing the domain match (or numbers) using the Interpro
website
(http:www.ebi.ac.uklinterpro~.
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DOMAIN results for GPCR8 were collected from the Conserved Domain Database
(CDD) with Reverse Position Specific BLAST. This BLAST samples domains found
in the
Smart and Pfam collections. The results are listed in Table 8E with the
statistics and domain
description. The results indicate that GPCR8 contains the 7tm_1 (InterPro) 7
transmembrane
receptor (rhodopsin family)domain (as defined by Interpro) at amino acid
positions residues
41-170. This indicates that the sequence of GPCR8 has properties similar to
those of other
proteins known to contain this/these domains) and similar to the properties of
these domains.
Table 8E. DOMAIN results for GPCR8
gnllPfamlpfam00001, 7tm 1, 7 transmembrane receptor (rhodopsin
family) Length = 377 (SEQ ID N0:95).
Score = 85.9 bits (211), Expect = 3e-18
.1....1....1....1 .1....1..._.1....1 ...1....1....1....1
TM7R$ ~e VL C~VSR~KA~Q~TTNL L~ LV~LVW _ YLEVVG~WKF~RIH~D~F
~....1....1....1....1....1....1....1 I....I....1....~
TM7R$ TL~CTASIN~CAIBS T~ C ~ L~NTRYSSKRVTG ~'I GLSFTICP~
....I....i....I....I....I....1....1....1....1....1....1....1
GPCRB SPL-T LSRSG-_____-_________________-_______________-_____--
TM7 LFGLNN DQNE--------CIIANP--------AFVVYSSIVSFYV----PFIVTLLVYI
The similarity information for the GPCR8 protein and nucleic acid disclosed
herein
suggest that GPCR8 may have important structural and/or physiological
functions
characteristic of the Olfactory Receptor family and the GPCR family.
Therefore, the nucleic
acids and proteins of the invention are useful in potential diagnostic and
therapeutic
applications and as a research tool. These include serving as a specific or
selective nucleic
acid or protein diagnostic and/or prognostic marker, wherein the presence or
amount of the
nucleic acid or the protein are to be assessed, as well as potential
therapeutic applications such
as the following: (i) a protein therapeutic, (ii) a small molecule drug
target, (iii) an antibody
target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a
nucleic acid useful in
gene therapy (gene delivery/gene ablation), and (v) a composition promoting
tissue
regeneration in vitro and in vivo (vi) biological defense weapon. The novel
nucleic acid
encoding GPCRB, and the GPCR8 protein of the invention, or fragments thereof,
may further
be useful in diagnostic applications, wherein the presence or amount of the
nucleic acid or the
protein are to be assessed.
The disclosed GPCR8 nucleic acids and proteins of the invention are useful in
potential
therapeutic applications implicated in neoplasm; adenocarcinoma; lymphoma;
prostate cancer;
uterus cancer; immune response; AIDS; asthma; Crohn's disease; multiple
sclerosis; and
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treatment of Albright hereditary ostoeodystrophy and/or other pathologies and
disorders. For
example, a cDNA encoding the GPCR-like protein may be useful' in gene therapy,
and the
GPCR-like protein may be useful when administered to a subject in need
thereof. By way of
nonlimiting example, the compositions of the present invention will have
efficacy for
treatment of patients suffering from neoplasm; adenocarcinoma; lymphoma;
prostate cancer;
uterus cancer; immune response; AIDS; asthma; Crohn's disease; multiple
sclerosis; and
treatment of Albright hereditary ostoeodystrophy. The novel nucleic acid
encoding GPCR-
like protein, and the GPCR-like protein of the invention, or fragments
thereof, may further be
useful in diagnostic applications, wherein the presence or amount of the
nucleic acid or the
protein are to be assessed. These materials are further useful in the
generation of antibodies
that bind immuno-specifically to the novel GPCR8 substances for use in
therapeutic or
diagnostic methods. These antibodies may be generated according to methods
known in the
art, using prediction from hydrophobicity charts, as described in the "Anti-
GPCRX
Antibodies" section below.
GPCR9
A novel nucleic acid was identified on chromosome 11 by TblastN using CuraGen
Corporation's sequence file for GPCR probe or homolog, run against the Genomic
Daily Files
made available by GenBank. The nucleic acid was further predicted by the
program
GenScanTM, including selection of exons. These were further modified by means
of
similarities using BLAST searches. The sequences were then manually corrected
for apparent
inconsistencies, thereby obtaining the sequences encoding the full-length
protein. The
disclosed novel GPCR9 nucleic acid of 946 nucleotides (also referred to as 6-L-
19-B) is
shown in Table 9A. An open reading begins with an ATG initiation codon at
nucleotides 5-7
and ends with a TAA codon at nucleotides 932-934. A putative untranslated
region upstream
from the initiation codon and downstream from the termination codon are
underlined in Table
9A, and the start and stop codons are in bold letters.
In a search of sequence databases, it was found, for example, that the nucleic
acid
sequence has 563 of 902 bases (62%) identical to (E= 8.5e-47) a Mus musculus
gene for
odorant receptor A16 (GENBANK-ID: GENBANK-ID:AB030896~acc:AB030896). In a
search of sequence databases, partial matches (353 of 373 bases, 94%
identical) were also
identified with the nucleotides 1- 372 of GPCR9 identical with nucleotides 306-
678 of a
Homo Sapiens GPCR EST (GENBANK-ID: GENBANK-ID: AW182678~acc:AW182678;
xj45d11.x1 Soares NFL T GBC S1 Homo Sapiens cDNA clone IMAGE:2660181 3'
similar
CA 02401453 2002-08-26
WO 01/64879 PCT/USO1/06474
to TR:Q9Z1V0 Q9Z1V0 OLFACTORY RECEPTOR C6). This 94% match (E=l.le-69)
between regions of the public sequence and regions of the present invention
(gene) suggests
that the present invention (gene) could be a splice variant of the public GPCR
EST (partial
mRNA). This also supports identification of GPCR9 as a GPCR. In a search of
sequence
databases, partial matches (94 of 100 bases, 94% identical) of nucleotides 893-
794 of GPCR9
with nucleotides 251-348 of a Homo Sapiens GPCR EST (GENBANK-ID:
AA206680~acc:AA206680: zq51c1l.rl Stratagene neuroepithelium (#937231) Homo
Sapiens
cDNA clone IMAGE:645140 5' similar to contains Ll.b2 L1 repetitive element).
This 94%
match between nucleotides of the public sequence and nucleotides of the GPCR9
sequence
suggests that GPCR9 may be a splice variant of the public GPCR EST (partial
mRNA).
Table 9A. GPCR9 Nucleotide Sequence (SEQ ID N0:32)
GTTCATGGAAAATAGGAATATTGTCACTGTCTTTATTCTCCTGGGACTTTCTCAAAACAAGAACATTGAA
GTTTTTTGGTTTGTATTATTTGTATTTTGCTACATTGCTATTTGGATGGAAAACTTCATCATAATGATTT
CTATCATGTACATTTGGCTAATTGACCAACCCATGTATTTCTTCCTTAATTACCTCGCACTCTCAGATCT
TTGCTACATATCCACTGTGGCCCCCAAGCTAATGATTGACCTACTAACAGAAAGGAAGATCGTTTCCTAT
AATAACTGCATGATACAGCTATTTATCACTCACTTCCTTGGAGACATTGAGATCTTCATACTCAAAGCAA
TGGCCTATGACCACTACATAGCCATCTGCAAGCACCTGCACTACACCATCATCACGACCAAGCAAAGCTG
TAACACCATCATCATAGCTTGTTGTACTGGGGGATTTATACACTCTGCCAGTCAGTTTCTTCTTACCATC
TTCTTACCGTTCTGTGGTCTTAATGAGATAGATCAGTACTTCTGCTATGTGTATCCTCTGCTGAAGTTGG
CTCGCATTGATATATACAGAATTGGTTTCTTGGTAATTGTTAATTCAGGCCTGATTTCTTTGTTGGCTTT
TGTGATTTTGATGGTGTCTTATTATTTGATATTATCCACCATCAGGGTTTACTCTGCTGAGAGTCATACC
AAAGCTCTTTCAACCTGTAGCTCTCACATAATAGTTGTGGTCCTATTCTTTGTGCCTGCCCTCTTCATTT
ACATCAGACCAGCCATAACTTTTCCAGAAGATAAAGTGTTTGTTCTCTTCTGTGCCATCATTGCTCCCAT
GTTCAGTCTTCTTATCTACATGCTGAGAAAGGTGGAGATGAAGAACGCTGTAAGGAAAATGTGGTGTCAT
CAATTGCTTCTGGCAAGGAAGTAACTTGTATGAAAG
The GPCR9 protein encoded by SEQ ID N0:32 has 309 amino acid residues, and is
presented using the one-letter code in Table 9B (SEQ ID N0:33). The SignalP,
Psort and/or
Hydropathy profile for GPCR9 predict that GPCR9 has a signal peptide and is
likely to be
localized at the endoplasmic reticulum membrane with a certainty of 0.6850 or
to the plasma
membrane with a certainty of 0.6400. The SignalP predicts a cleavage site at
the sequence
IWM-EN between amino acids 38 and 39 as indicated by the slash in Table 9B.
Table 9B. Encoded GPCR9 protein sequence (SEQ ID N0:33)
MENRNIVTVFILLGLSQNKNIEVFWFVLFVFCYIAIWM/ENFIIMISIMYIWLIDQPMYFFLNYLALSDLC
YISTVAPKLMIDLLTERKIVSYNNCMIQLFITHFLGDIEIFILKAMAYDHYIAICKHLHYTIITTKQSCN
TIIIACCTGGFIHSASQFLLTIFLPFCGLNEIDQYFCYVYPLLKLARIDIYRIGFLVIVNSGLISLLAEV
ILMVSYYLILSTIRVYSAESHTKALSTCSSHIIVWLFFVPALFIYIRPAITFPEDKVFVLFCAIIAPMF
SLLIYMLRKVEMKNAVRKMWCHQLLLARK
The full amino acid sequence of the protein of the invention was found to have
140 of
300 amino acid residues (46%) identical to, and 193 of 300 residues (64%)
positive with, the
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302 amino acid residue odorant receptor A16 protein from Mus musculus
(ptnr:TREMBLNEW-ACC:BAA86127) (E value = 1.0e-72). Further BLAST analysis
produced the significant results listed in Table 9C. The disclosed GPCR8
protein (SEQ ID
N0:33) has good identity with a number of olfactory receptor proteins.
S
Table 9C. BLAST
results for
GPCR9
Gene Index/ Protein/ LengthIdentityPositivesExpect
Identifier Organism (aa) (~)
Gi1114962491refINP_odorant receptor308 140/300 185/300 1e-59
067343.11 MOR18 Mus (46~) (61~)
(AB030895) musculus
Gi1114649951refINP_odorant receptor302 137/300 185/300 2e-59
065261.11 AB030896)A16 Mus musculus (45~ (61~)
Gi14237021pirIIS297olfactory 307 142/303 186/303 6e-57
receptor OR1B (46~) (60~)
-
rat
Gi1114699931refINP_odorant receptor308 133/297 182/297 7e-55
065260.11 MOR83 Mus (44~) (60~)
(AB030894) musculus
Gi1106495191gbIAAG2odorant receptor264 124/262 169/262 6e-51
1324.11AF271051Mus musculus (47~) (64$)
1
(AF271051)
This information is presented graphically in the multiple sequence alignment
given in
Table 9D (with GPCR9 being shown on line 1) as a ClustalW analysis comparing
GPCR9
with related protein sequences.
Table 9D. Information for the ClustalW proteins:
1) GPCR9 (SEQ ID N0:33)
2) gi~11496249~ref~NP_067343.1~ odorant receptor 16 Mus musculus (SEQ ID
N0:78)
3) gi~11464995~ref~NP_065261.1~ gene for odorant receptor A16 Mus musculus
(SEQ ID N0:79)
4) giI423702~pir~~S29710 olfactory receptor OR18 - rat (SEQ ID N0:80)
5) gig 11464993~ref~NP_065260.1 ~ gene for odorant receptor MOR83 Mus musculus
(SEQ ID N0:81)
6) gi~10644519~gb~AAG21324.1~AF271051_1 odorant receptor Mus musculus (SEQ ID
N0:70)
10 20 30 40 50 6
.I....I....I....I....I....1....1....1....1....1....1....1
GPCR9 --~ENRNI I~ KN V FC IW E ~S" ' IWL D '
1'
Gi I 114962491 --i iEIPHN ~ RPE ~YL ~';~ C ~rt S
Gi 1119649951 -- DSPRN . P L G r"°tl I~T ~ P-.
6i14237021 -- GENNN= I PDGRKAL ~ ~, L ~I P-I~
Gi 1119699931 MGALNQT I mD VIP T T y ~~P;~H
Gi1106445191 _________________________~C ~ :P ~ ~ I K
70 80 90 100 110 120
.I....I....I....I....I....1....1....1....1....1....1....1
GPCR9 ~ Y _LS~L I T I~ T I NN 'I~ IT FL DI Fg~L
Gi1114962491 FW T S ~S Y GT ° ,"~~ G FL G
Gi1114649951 aY F T C T' S H~G" LL GT
Gi14237021 S L L. S~ ~ Y~_ IE L G
611106945191 Y L~~IL~ TV~ FTC N~I P~~~~ FL FFF~VT m
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130 140 150 160 170 180
.I....I....I....I....I....1....1....1....1....1....1....1
GPCR9 . H ~ I H IT~QS T '~,~~IACCT ~ S FL TL E ~0- Y
gi1114962491 ~ TT ~ H V y L IF
v
gi I 119649951 TT YI~H" ' V _~ I ' T
gi14237021 ~' L - ~T IF T T ~ FVYN ~ ~ ~I
gi1106945191 ~~ ' I T ~~PL H~~~ T~T~P ~ ~ ;S
190 200 210 220 230 240
.I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .1....I.. .I
GPCR9 ~RI~I1 ~ S~ ~ ~Y_ S ~ ~ SHT
N
gi1114962991 .,Y~ ~ m - _ ~ C c~ I H~c~S.
gi1114699951 ~ ' E ' ~ ~ ~ C N I ~S
gi I 423702 I , V ' ~ ~ T I~',, "~NCi ~I T ~ G'T
gi1119699931 P~ ~~ ' ~ T I~IVS T~S "-C T T ~K ~ w
gi 1106995191 ~ ~ ~ :FU~~4: F ~FSFS 'S~I Y a ~
250 260 270 280 290 300
.I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I.. .I
I ~ ~ ~i ~ v N v
GPCR9 ~~I' I ' I~F~PE~ FVv' C ' FL K ~_ ~,~;K
gi1114962491 G FT T S ''ST LP ~ ~ D~ K
U N
gi1114649951 G' FT I ~~~ LS ~ V C " ~ N
gi14237021 ~ I' I , ~ ' YN P C T T ~ ~ S ~ SC K
gi1114649931 ~ F T m T~'D 6. 5 ~ T -- !7 T~ H
gi1106445191 ~5 I~= ~ ~ ~ TE T~
310
.1....I.
GPCR9 ~,,CHQLLLARK
gi1114962491 ~SEVVGA
gi1114699951 ----
gi14237021 C LHAD-
gi1119649931 R . RICS--
gi1106445191 ----------
The presence of identifiable domains in GPCR9 was determined by searches using
algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then
determining the
Interpro number by crossing the domain match (or numbers) using the Interpro
website
(http:www.ebi.ac.uk/interpro~.
DOMAIN results for GPCR9 were collected from the Conserved Domain Database
(CDD) with Reverse Position Specific BLAST. This BLAST samples domains found
in the
Smart and Pfam collections. The results are listed in Table 9E with the
statistics and domain
description. The results indicate that GPCR9 contains the 7tm 1 (InterPro) 7
transmembrane
receptor (rhodopsin family)domain (as defined by Interpro) at amino acid
positions residues
56-234. This indicates that the sequence of GPCR9 has properties similar to
those of other
proteins known to contain this/these domains) and similar to the properties of
these domains.
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Table 9E. DOMAIN results for GPCR9
gnl~Pfam~pfam00001, 7tm 1, 7 transmembrane receptor (rhodopsin
family) Length = 377 (SEQ ID N0:45).
Score = 73.6 bits (179), Expect = 1e-14
....1....1....1....1.. .1.. .1....1.. .I.. .I....1.. .1.. .I
GPCR9 -----------PMYF~NY~CYI~TIDLERKI~YN~Q
TM7 GNVLVCMAVSREKALQTTTNY~L S LV L Ta YLE GEWK RI D F
....I....I....I....1....I....1....1....1....1....1....1....1
TM7R9 T DVI~CTAS~LN~CI~tFt~T CKPL~I~RYRVTVAIVWVLS~TI~CPM
.I....I....I....1....1....1....1....1....1....1....1....1
GPCR9 FLL-TIFLPFCGLNEIDQYFCYVY~LLKLARIDIRGFL---VI~SGLIS- F ~ L
TM7 LFGLNNTDQNE--------CIIAN~--------A V YSSIVSFY ----PF TL ,
.1....I.. .I.. .1....1....1....1....1....1....1..../....1
GPCR9 VS~Y ~ LSTI~VY~ASH;,,~-________________________________-_____
TM7 KI I ~-RRR RV T RS FRANLKAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRV
The similarity information for the GPCR9 protein and nucleic acid disclosed
herein
suggest that GPCR9 may have important structural and/or physiological
functions
characteristic of the Olfactory Receptor family and the GPCR family.
Therefore, the nucleic
acids and proteins of the invention are useful in potential diagnostic and
therapeutic
applications and as a research tool. These include serving as a specific or
selective nucleic
acid or protein diagnostic and/or prognostic marker, wherein the presence or
amount of the
nucleic acid or the protein are to be assessed, as well as potential
therapeutic applications such
as the following: (i) a protein therapeutic, (ii) a small molecule drug
target, (iii) an antibody
target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a
nucleic acid useful in
gene therapy (gene delivery/gene ablation), and (v) a composition promoting
tissue
regeneration in vitro and in vivo (vi) biological defense weapon. The novel
nucleic acid
encoding GPCR9, and the GPCR9 protein of the invention, or fragments thereof,
may further
be useful in diagnostic applications, wherein the presence or amount of the
nucleic acid or the
protein are to be assessed.
The nucleic acids and proteins of the invention are useful in potential
therapeutic
applications implicated in used in the treatment of infections such as
bacterial, fungal,
protozoal and viral infections (particularly infections caused by HIV-1 or HIV-
2), pain, cancer
(including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate
cancer; uterus
cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure,
hypotension,
hypertension, urinary retention, osteoporosis, Crohn's disease; multiple
sclerosis; and
treatment of Albright hereditary ostoeodystrophy, angina pectoris, myocardial
infarction,
ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and
neurological
disorders, including anxiety, schizophrenia, manic depression, delirium,
dementia, severe
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mental retardation and dyskinesias, such as Huntington's disease or Gilles de
la Tourette
syndrome and/or other pathologies and disorders.
The polypeptides can be used as immunogens to produce antibodies specific for
the
invention, and as vaccines. They can also be used to screen for potential
agonist and
antagonist compounds. For example, a cDNA encoding the GPCR-like protein may
be useful
in gene therapy, and the GPCR-like protein may be useful when administered to
a subject in
need thereof. By way of nonlimiting example, the compositions of the present
invention will
have efficacy for treatment of patients suffering from bacterial, fungal,
protozoal and viral
infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer
(including but not
limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus
cancer), anorexia,
bulimia, asthma, Parkinson's disease, acute heart failure, hypotension,
hypertension, urinary
retention, osteoporosis, Crohn's disease; multiple sclerosis; and treatment of
Albright
hereditary ostoeodystrophy, angina pectoris, myocardial infarction, ulcers,
asthma, allergies,
benign prostatic hypertrophy, and psychotic and neurological disorders,
including anxiety,
schizophrenia, manic depression, delirium, dementia, severe mental retardation
and
dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome
andlor other
pathologies and disorders. The novel nucleic acid encoding GPCR-like protein,
and the
GPCR-like protein of the invention, or fragments thereof, may further be
useful in diagnostic
applications, wherein the presence or amount of the nucleic acid or the
protein are to be
assessed.
These materials are further useful in the generation of antibodies that bind
immuno-
specifically to the novel GPCR9 substances for use in therapeutic or
diagnostic methods.
These antibodies may be generated according to methods known in the art, using
prediction
from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies"
section below.
GPCR10
GPCR10 includes a family of three similar nucleic acids and three similar
proteins
disclosed below. The disclosed nucleic acids encode GPCR, OR-like proteins.
GPCRIOa
The disclosed novel nucleic acid was identified on chromosome 11 by TblastN
using
CuraGen Corporation's sequence file for GPCR probe or homolog, run against the
Genomic
Daily Files made available by GenBank. The nucleic acid was further predicted
by the
program GenScanTM, including selection of exons. These were further modified
by means of
similarities using BLAST searches. The sequences were then manually corrected
for apparent
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inconsistencies, thereby obtaining the sequences encoding the full-length
protein. GPCRIOa is
a 948 by long nucleic acid (also referred to as 6-L-19-A) as shown in Table
10A (SEQ ID
N0:34). An ORF begins with an ATG initiation codon at nucleotides 7-9 and ends
with a
TAA codon at nucleotides 934-936. A putative untranslated region upstream from
the
initiation codon and downstream from the termination codon is underlined in
Table 10A, and
the start and stop codons are in bold letters.
Table 10A. GPCRIOa Nucleotide Sequence (SEQ ID N0:34)
TGAGAAATGGAAAATCAAAACAATGTGACTGAATTCATTCTTCTGGGTCTCACAGAGAACCTGGAGCTGT
GGAAAATATTTTCTGCTGTGTTTCTTGTCATGTATGTAGCCACAGTGCTGGAAAATCTACTTATTGTGGT
AACTATTATCACAAGTCAGAGTCTGAGGTCACCTATGTATTTTTTTCTTACCTTCTTGTCCCTTTTGGAT
GTCATGTTCTCATCTGTCGTTGCCCCCAAGGTGATTGTAGACACCCTCTCCAAGAGCACTACCATCTCTC
TCAAAGGCTGCCTCACCCAGCTGTTTGTGGAGCATTTCTTTGGTGGTGTGGGGATCATCCTCCTCACTGT
GATGGCCTATGACCGCTACGTGGCCATCTGTAAGCCCCTGCACTACACGATCATCATGAGTCCACGGGTG
TGCTGCCTAATGGTAGGAGGGGCTTGGGTGGGGGGATTTATGCACGCAATGATACAACTTCTCTTCATGT
ATCAAATACCCTTCTGTGGTCCTAATATCATAGATCACTTTATATGTGATTTGTTTCAGTTGTTGACACT
TGCCTGCACGGACACCCACATCCTGGGCCTCTTAGTTACCCTCAACAGTGGGATGATGTGTGTGGCCATC
TTTCTTATCTTAATTGCGTCCTACACGGTCATCCTATGCTCCCTGAAGTCTTACAGCTCTAAAGGGCGGC
ACAAAGCCCTCTCTACCTGCAGCTCCCACCTCACGGTGGTTGTATTGTTCTTTGTCCCCTGTATTTTCTT
GTACATGAGGCCTGTGGTCACTCACCCCATAGACAAGGCAATGGCTGTGTCAGACTCAATCATCACACCC
ATGTTAAATCCCTTGATCTATACACTGAGGAATGCAGAGGTGAAAAGTGCCATGAAGAAACTCTGGATGA
AATGGGAGGCTTTGGCTGGGAAATAACTGCAATGCTGA
The GPCRIOa protein encoded by SEQ ID N0:34 has 309 amino acid residues, and
is
presented using the one-letter code in Table lOB (SEQ ID N0:35). The SignalP,
Psort and/or
Hydropathy profile for GPCRIOa predict that GPCRlOa has a signal peptide and
is likely to be
localized at the plasma membrane with a certainty of 0.6000. The SignalP
predicts a cleavage
site at the sequence VLE-NL, between amino acids 39 and 40, as indicated by
the slash in
Table 10B.
In a search of sequence databases, it was found, for example, that the nucleic
acid
sequence has 625 of 908 bases (68%) identical to a 909 by Mus musculus gene
for odorant
receptor A16 mRNA (GENBANK-ID: AB030896~acc:AB030896) (E= 3.0e-75).
Table 10B. Encoded GPCRIOa protein sequence (SEQ ID N0:35).
MENQNNVTEFILLGLTENLELWKIFSAVFLVMYVATVLE/NLLIVVTIITSQSLRSPMYFFLTFLSLLDVM
FSSWAPKVIVDTLSKSTTISLKGCLTQLFVEHFFGGVGIILLTVMAYDRYVAICKPLHYTIIMSPRVCC
LMVGGAWVGGFMHAMIQLLFMYQIPFCGPNIIDHFICDLFQLLTLACTDTHILGLLVTLNSGMMCVAIFL
ILIASYTVILCSLKSYSSKGRHKALSTCSSHLTVVVLFFVPCIFLYMRPVVTHPIDKAMAVSDSIITPML
NPLIYTLRNAEVKSAMKKLWMKWEALAGK
The full amino acid sequence of the protein of the invention was found to have
183 of
302 amino acid residues (60%) identical to, and 232of 302 residues (76%)
positive with, the
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307 amino acid residue OR18 odorant receptor protein from Rattus sp.(ptnr:
TREMBLNEW-
ACC:G264618).
GPCRIOb
GPCRIOa (6-L-19-A) was subjected to an exon linking process to confirm the
sequence. PCR primers were designed by starting at the most upstream sequence
available,
for the forward primer, and at the most downstream sequence available for the
reverse primer.
In each case, the sequence was examined, walking inward from the respective
termini toward
the coding sequence, until a suitable sequence that is either unique or highly
selective was
encountered, or, in the case of the reverse primer, until the stop codon was
reached. Such
suitable sequences were then employed as the forward and reverse primers in a
PCR
amplification based on a wide range of cDNA libraries. The resulting amplicon
was gel
purified, cloned and sequenced to high redundancy to provide GPCRIOb, which is
also
referred to as 6-L-19-A 1.
The nucleotide sequence for GPCRIOb (948 bp, SEQ ID N0:36) is presented in
Table
l OC. The nucleotide sequence differs from GPCRIOa by one nucleotide change
(numbered
with respect to GPCRIOa) T404 >C.
Table 10C. GPCRIOb Nucleotide Sequence (SEQ ID N0:36)
TGAGAAATGGAAAATCAAAACAATGTGACTGAATTCATTCTTCTGGGTCTCACAGAGAACCTGGAGCTGTGGAAAATAT
T
TTCTGCTGTGTTTCTTGTCATGTATGTAGCCACAGTGCTGGAAAATCTACTTATTGTGGTAACTATTATCACAAGTCAG
A
GTCTGAGGTCACCTATGTATTTTTTTCTTACCTTCTTGTCCCTTTTGGATGTCATGTTCTCATCTGTCGTTGCCCCCAA
G
GTGATTGTAGACACCCTCTCCAAGAGCACTACCATCTCTCTCAAAGGCTGCCTCACCCAGCTGTTTGTGGAGCATTTCT
T
TGGTGGTGTGGGGATCATCCTCCTCACTGTGATGGCCTATGACCGCTACGTGGCCATCTGTAAGCCCCTGCACTACACG
A
TCACCATGAGTCCACGGGTGTGCTGCCTAATGGTAGGAGGGGCTTGGGTGGGGGGATTTATGCACGCAATGATACAACT
T
CTCTTCATGTATCAAATACCCTTCTGTGGTCCTAATATCATAGATCACTTTATATGTGATTTGTTTCAGTTGTTGACAC
T
TGCCTGCACGGACACCCACATCCTGGGCCTCTTAGTTACCCTCAACAGTGGGATGATGTGTGTGGCCATCTTTCTTATC
T
TAATTGCGTCCTACACGGTCATCCTATGCTCCCTGAAGTCTTACAGCTCTAAAGGGCGGCACAAAGCCCTCTCTACCTG
C
AGCTCCCACCTCACGGTGGTTGTATTGTTCTTTGTCCCCTGTATTTTCTTGTACATGAGGCCTGTGGTCACTCACCCCA
T
AGACAAGGCAATGGCTGTGTCAGACTCAATCATCACACCCATGTTAAATCCCTTGATCTATACACTGAGGAATGCAGAG
G
TGAAAAGTGCCATGAAGAAACTCTGGATGAAATGGGAGGCTTTGGCTGGGAAATAACTGCAATGCTGA
The encoded GPCRIOb protein is presented in Table l OD. The disclosed protein
is
309 amino acids long and is denoted by SEQ ID N0:37. GPCRIOb differs from
GPCRlOa by
one amino acid residues: I133>T. Like GPCRIOa, the Psort profile for GPCRIOb
predicts
that this sequence has a signal peptide and is likely to be localized at the
plasma membrane
with a certainty of 0.6000. The most likely cleavage site for a peptide is
between amino acids
39 and 40, i.e., at the slash in the amino acid sequence VLE-NL (shown as a
slash in
TablelOD) based on the SignalP result.
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Table 10D. Encoded GPCRIOb protein sequence (SEQ ID N0:37)
MENQNNVTEFILLGLTENLELWKIFSAVFLVMYVATVLE/NLLIWTIITSQSLRSPMYFFLTFLSLLD
VMFSSWAPKVIVDTLSKSTTISLKGCLTQLFVEHFFGGVGIILLTVMAYDRYVAICKPLHYTITMSPR
VCCLMVGGAWVGGFMHAMIQLLFMYQIPFCGPNIIDHFICDLFQLLTLACTDTHILGLLVTLNSGMMCV
AIFLILIASYTVILCSLKSYSSKGRHKALSTCSSHLTVWLFFVPCIFLYMRPVVTHPIDKAMAVSDSI
ITPMLNPLIYTLRNAEVKSAMKKLWMKWEALAGK
Table 10E presents the BLASTP results for GPCRIOb.
Table 10E. BLASTP Results for GPCRIOb
Score = 999 (351.7 bits), Expect= 1.2e-100, P = 1.2e-100 Identities = 182/302
(60%), Positives =
231/302 (76%) with PIR-ID:S29710 olfactory receptor OR18 - rat
Score = 757 (266.5 bits), Expect = 5.2e-75, P = 5.2e-75 Identities = 144/298
(48%), Positives =
200/298 (67%) with ACC:095013 WiJGSC:H_DJ0855D21.1 PROTEIN - Homo Sapiens
lHumanl. 312 aa.
Score = 757 (266.5 bits), Expect = 5.2e-75, P = 5.2e-75 Identities = 144/298
(48%), Positives =
200/298 (67%) with ACC:095013 WUGSC:H DJ0855D21.1 PROTEIN - Homo Sapiens
(Human), 312 aa.
Score = 667 (234.8 bits), Expect = 1.1e-64, P = 1.1e-64 Identities = 131/300
(43%), Positives =
194/300 (64%), Frame =+1 with ACC:043749 OLFACTORY RECEPTOR - Homo Sapiens
(Human), 312 aa.
GPCRIOc
Another nucleotide sequence resulted when GPCRIOa (6-L-19-A) was subjected to
an
exon linking process to confirm the sequence. PCR primers were designed by
starting at the
most upstream sequence available, for the forward primer, and at the most
downstream
sequence available for the reverse primer. In each case, the sequence was
examined, walking
inward from the respective termini toward the coding sequence, until a
suitable sequence that
is either unique or highly selective was encountered, or, in the case of the
reverse primer, until
the stop codon was reached. Such suitable sequences were then employed as the
forward and
reverse primers in a PCR amplification based on a wide range of cDNA
libraries.
These primers were then employed in PCR amplification based on the following
pool
of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain -
cerebellum, brain -
hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal
brain, fetal kidney,
fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland,
pancreas, pituitary
gland, placenta, prostate, salivary gland, skeletal muscle, small intestine,
spinal cord, spleen,
stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons
were gel purified,
cloned and sequenced to high redundancy. The resulting sequences from all
clones were
assembled with themselves, with other fragments in CuraGen Corporation's
database and with
public ESTs. Fragments and ESTs were included as components for an assembly
when the
extent of their identity with another component of the assembly was at least
95% over 50 bp.
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In addition, sequence traces were evaluated manually and edited for
corrections if appropriate.
The resulting amplicon was gel purified, cloned and sequenced to high
redundancy to provide
the sequence reported below, which is designated as Accession Number 6-L-19-A-
dal, or
GPCRl Oc.
The nucleotide sequence for GPCRIOc (943 bp, SEQ ID N0:38) is presented in
Table
IOF. The GPCRIOc nucleotide sequence differs from GPCRIOa by having six fewer
nucleotides at the 5' end and two nucleotide changes: (numbered with respect
to GPCRIOa) .
6466>A and C834>T.
Table 10F. GPCRIOc Nucleotide Sequence (SEQ ID N0:38)
ATGGAAAATCAAAACAATGTGACTGAATTCATTCTTCTGGGTCTCACAGAGAACCTGGAGCTGTGGAAAA
TATTTTCTGCTGTGTTTCTTGTCATGTATGTAGCCACAGTGCTGGAAAATCTACTTATTGTGGTAACTAT
TATCACAAGTCAGAGTCTGAGGTCACCTATGTATTTTTTTCTTACCTTCTTGTCCCTTTTGGATGTCATG
TTCTCATCTGTCGTTGCCCCCAAGGTGATTGTAGACACCCTCTCCAAGAGCACTACCATCTCTCTCAAAG
GCTGCCTCACCCAGCTGTTTGTGGAGCATTTCTTTGGTGGTGTGGGGATCATCCTCCTCACTGTGATGGC
CTATGACCGCTACGTGGCCATCTGTAAGCCCCTGCACTACACGATCATCATGAGTCCACGGGTGTGCTGC
CTAATGGTAGGAGGGGCTTGGGTGGGGGGATTTATGCACACAATGATACAACTTCTCTTCATGTATCAAA
TACCCTTCTGTGGTCCTAATATCATAGATCACTTTATATGTGATTTGTTTCAGTTGTTGACACTTGCCTG
CACGGACACCCACATCCTGGGCCTCTTAGTTACCCTCAACAGTGGGATGATGTGTGTGGCCATCTTTCTT
ATCTTAATTGCGTCCTACACGGTCATCCTATGCTCCCTGAAGTCTTACAGCTCTAAAGGGCGGCACAAAG
CCCTCTCTACCTGCAGCTCCCACCTCACGGTGGTTGTATTGTTCTTTGTCCCCTGTATTTTCTTGTACAT
GAGGCCTGTGGTCACTCACCCCATAGACAAGGCAATGGCTGTGTCAGACTCAATCATTACACCCATGTTA
AATCCCTTGATCTATACACTGAGGAATGCAGAGGTGAAAAGTGCCATGAAGAAACTCTGGATGAAATGGG
AGGCTTTGGCTGGGAAATAACTGCAATGCTGA
The coding region of GPCRIOc is from nucleotide 1 to 928, giving the encoded
GPCRIOc protein, as presented in Table 10G. The disclosed protein is 309 amino
acids long
and is denoted by SEQ ID NO: 83. GPCRIOc differs from GPCRIOa by one amino
acid
residue: A154>T. Like GPCRIOa, the Psort profile for GPCRSc predicts that this
sequence
1 S has a signal peptide and is likely to be localized at the plasma membrane
with a certainty of
0.6000. The most likely cleavage site for a peptide is between amino acids 39
and 40, i.e., at
the slash in the amino acid sequence VLE-NL (shown as a slash in TablelOG)
based on the
SignalP result.
Table 10G. Encoded GPCRIOc protein sequence (SEQ ID N0:83)
MENQNNVTEFILLGLTENLELWKIFSAVFLVMYVATVLE/NLLIVVTIITSQSLRSPMYFFLTFLSLLDVM
FSSVVAPKVIVDTLSKSTTISLKGCLTQLFVEHFFGGVGIILLTVMAYDRYVAICKPLHYTIIMSPRVCC
LMVGGAWVGGFMHTMIQLLFMYQIPFCGPNIIDHFICDLFQLLTLACTDTHILGLLVTLNSGMMCVAIFL
ILIASYTVILCSLKSYSSKGRHKALSTCSSHLTVVVLFFVPCIFLYMRPVVTHPIDKAMAVSDSIITPML
NPLIYTLRNAEVKSAMKKLWMKWEALAGK
Possible SNPs found for GPCR10 are listed in TablelOH.
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Table IOH:
SNPs
Base Base Base
Position Before After
65 T A(2)
120 T Gap(2)
147 T C(2)
234 A G(3)
412 T C(7)
471 G A(2)
814 A G(3)
The disclosed GPCR10 protein (SEQ ID N0:35) has good identity with a number of
olfactory receptor proteins. The identity information used for ClustalW
analysis is presented
in Table 10I. Unless specifically addressed as GPCRIOa GPCRIOb, or GPCRIOc,
any
reference to GPCR10 is assumed to encompass all variants. Residue differences
between any
GPCRX variant sequences herein are written to show the residue in the "a"
variant and the
residue position with respect to the "a" variant. GPCR residues in all
following sequence
alignments that differ between the individual GPCR variants are highlighted
with a box and
marked with the (o) symbol above the variant residue in all alignments herein.
For example,
the protein shown in line 1 of Table l OJ depicts the sequence for GPCRIOa,
and the positions
where GPCRIOb or GPCRIOc differs are marked with a (o) symbol and are
highlighted with a
box. All GPCR10 proteins have significant homology to olfactory receptor (OR)
proteins:
Table 10I. BLAST
results for GPCR10
Gene Index/ Protein/ Length IdentityPositivesExpect
Identifier Organism (aa) (~) ($)
Gi111496249~refINP_0Odorant 308 184/306 291/306 6e-91
67343.11 (AB030895)receptor (60~) (78~)
MOR18
Mus musculus
Gi1423702~pirIIS2971OR OR18 307 183/302 232/302 2e-88
- rat
0 (60$) (760)
Gi1119649951ref~NP_0Odorant 302 175/302 232/302 8e-86
65261.11 AB030896)receptor (57~) (75~)
A16
Mus musculus
Gi1114649931refINPOdorant 308 157/297 208/297 3e-72
0
_ receptor (52~) (69~)
65260.11 (AB030894)MOR83
Mus musculus
Gi1106445171gbIAAG21Odorant 264 155/260 202/260 2e-71
323.1~AF271050_1 receptor (59~) (77~)
(AF271050) Rattus
norvegicus
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This information is presented graphically in the multiple sequence alignment
given in
Table lOJ (with GPCR10 being shown on line 1) as a ClustalW analysis comparing
GPCR10
with related protein sequences.
Table lOJ Information for the ClustalW proteins:
1) GPCR10 (SEQ ID N0:35)
2) gi~11496249~ref)NP_067343.1~ odorant receptor 16 Mus musculus (SEQ ID
N0:78)
3) gi~423702~pir~~S29710 olfactory receptor OR18 - rat (SEQ ID N0:80)
4) gig 11464995 ~ref)NP_065261.1 ~ gene for odorant receptor A 16 Mus musculus
(SEQ ID N0:79)
. 5) gi~11464993~ref)NP_065260.1 ~ gene for odorant receptor MOR83 Mus
musculus (SEQ ID N0:81)
6) gi~10644517~gb~AAG21323.1~AF271050_1 odorant receptor Rattus norvegicus
(SEQ ID N0:82)
10 20 30 40 50 60
..I.. .I.. .I.. .I.. .1....I.. .1.. .1.. .1.. .I.. .1.. .I
GPCR10 .ENQ I' ~T~ LE - FS ~E 7 IT .
gi1119962491 iEIP QRPEQ - L ~ I . C ~ T S
gi I 423702 I GEN t - I T(;~ PDG L I F T~ ~ i I P
_ . I,
gi I 119699951 iDSP - F~,PQ~Q z L G ~~r'I,VS ~ ~~ 'T~P
gi 1119699931 GAL QT I D VEI3 P T T ~~~ V~P
gi1106995171 _________________________'-C ~ P', NI
70 BO 90 100 110 120
.I....I....I....I....I....1....1....1....1....1....1....1
GPCR10 L m ~T S~ ST L~T~ E 3
,.~
gi1119962491 .~N F~ TC'- S ~ ~S ~:~GT ' Y ~' G FL
gi19237021 S L=~ L- S' ~ ~L ; Q~,/ ~5~ IE L
gi 111464995 I Y F ~TCC~T' ,~ ~S ~~ R~~~~LL T~ I ~
gi1114649931 m IC T EGL L DN I~~~ FL L CS I ~
gi1106445171 Y L. IL T T~L I, L F T
130 190 150 160 170 180
.I....I....o....I....I....I....lo...l....l....l....l....l
GPCR10 IP CL~ G ' I L Y~ T
gi 1114962491 ~ ~ . TT~ H' ~ ~ ~ L - F,
gi14237021 - ~' LAT -I T T V YN
gi I 114649951 ~' TTi F~~H I ' T FaL9L> '
gi 111464993,1 ~ I' ~N"~ KAL ~ T~ TF,~~TI ' ~ ~Sj,
gi1106445171 ~ T ~~P H GTIP ~ ' I
190 200 210 220 230 240
.I....I....I....I....I....1....1....1....1....1....1....1
GPCR10 ~ F ~ T Hy~ TL ~ I I ,I°"~ T ,~ ,_ 'H
gi111496249j Y' ~ I
gi14237021 S' ~ ~ T ~ Tp G
gi1114649951 Y ~ ~ I ~ L I ~ ~
gi I 114649931 P ~ T ~ ,~~ T~S~; C , T T K
gi1106945171 ~H ~ F x F S FP ~ I
250 260 270 280 290 300
.I....i....I....I....I....l....l....l..._1....1....1....1
GPCR10 ~~~LT ' ~ HP ~ SDI ' S K
gi l 119962491 G ~ FT T S ~ "SLP ~ ~ ~ ' E D~ t~K
gi14237021 ~ IL Iv ' YNFP ~ C~T ~ ' ' S ~ SC~ K
gi1114649951 G FT ~ ~':SLS ~ I ~ C ' N
gi1114649931 F T ~ T' D FS yY~ ' E T~ H
gi1106945171 ~ I - BYTE L ~~ ~ __________
310
.I....1.
GPCR10 ~~~-;WEALAGK
gi1119962491 T SEVVGA-
gi14237021 ~C~~ LHAD--
gi1114649951 ------
gi1114649931 RQ~RICS---
gi1106445171 --- -------
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The presence of identifiable domains in GPCR10 was determined by searches
using
algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then
determining the
Interpro number by crossing the domain match (or numbers) using the Interpro
website
(http:www.ebi.ac.uk/interpro~.
DOMAIN results for GPCR10 were collected from the Conserved Domain Database
(CDD) with Reverse Position Specific BLAST. This BLAST samples domains found
in the
Smart and Pfam collections. The results are listed in Table lOK with the
statistics and domain
description. The results indicate that this protein contains the following
protein domains (as
defined by Interpro) at the indicated positions: domain name 7tm 1 (InterPro)
7
transmembrane receptor (rhodopsin family) at amino acid positions 39 to 213.
This indicates
that the sequence of GPCR10 has properties similar to those of other proteins
known to
contain this domain and similar to the properties of this domain.
Table 10K. DOMAIN results for GPCR10
gnllPfam~pfam00001, 7tm 1, 7 transmembrane receptor (rhodopsin
family) Length = 377 (SEQ ID N0:45).
Score = 93.6 bits (231), Expect = 1e-20
I.. 1....1....I ...I.. I . .~....I....I....I....I....I
GPCR10 E~'V~TITS ~ S ~PMY F~T ~ L~'. VDTSKSTT~LK~LTQ
TM7 C SRE ° ° TTN L 5 ' 'YLE VGEWK RIH DIF
~....I....I....(....I....1....1....1....1....1....(....1
GPCR10 F~HFFGGV L TV v Y~' C H TIIM- P CC GG G H IQ
TM7 TVMMCTAS~~~N~C = I~ T ' YL~NTRYS~K'~RVT~'~~~AI~L~ICPM
.I....I....I....I....I....1....1....(....1....1....1....1
GPCR10 LF-MYQIPFCG--------PNIIDHFICDLFQL-- L LAC DTHILGLLVTNSG C
TM7 ~FGLNNTDQNE--------CIIANP--------AFVVYSIVFYV----PFITLL~YI
GPCR10 VAIF'=LI-___________________________________________________
TM7 KIYI RRRRKRVNTKRSSRAFRANLKAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRV
The similarity information for the GPCR10 protein and nucleic acid disclosed
herein
suggest that GPCR10 may have important structural and/or physiological
functions
characteristic of the Olfactory Receptor family and the GPCR family.
Therefore, the nucleic
acids and proteins of the invention are useful in potential diagnostic and
therapeutic
applications and as a research tool. These include serving as a specific or
selective nucleic
acid or protein diagnostic and/or prognostic marker, wherein the presence or
amount of the
nucleic acid or the protein are to be assessed, as well as potential
therapeutic applications such
as the following: (i) a protein therapeutic, (ii) a small molecule drug
target, (iii) an antibody
target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a
nucleic acid useful in
gene therapy (gene delivery/gene ablation), and (v) a composition promoting
tissue
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regeneration in vitro and in vivo (vi) biological defense weapon. The novel
nucleic acid
encoding GPCR10, and the GPCR10 protein of the invention, or fragments
thereof, may
further be useful in diagnostic applications, wherein the presence or amount
of the nucleic acid
or the protein are to be assessed.
The nucleic acids and proteins of the invention are useful in potential
therapeutic
applications implicated in used in the treatment of infections such as
bacterial, fungal,
protozoal and viral infections (particularly infections caused by HIV-1 or HIV-
2), pain, cancer
(including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate
cancer; uterus
cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure,
hypotension,
hypertension, urinary retention, osteoporosis, Crohn's disease; multiple
sclerosis; and
treatment of Albright hereditary ostoeodystrophy, angina pectoris, myocardial
infarction,
ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and
neurological
disorders, including anxiety, schizophrenia, manic depression, delirium,
dementia, severe
mental retardation and dyskinesias, such as Huntington's disease or Gilles de
la Tourette
syndrome and/or other pathologies and disorders. The polypeptides can be used
as
immunogens to produce antibodies specific for the invention, and as vaccines.
They can also
be used to screen for potential agonist and antagonist compounds. For example,
a cDNA
encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-
like protein
may be useful when administered to a subject in need thereof. By way of
nonlimiting
example, the compositions of the present invention will have efficacy for
treatment of patients
suffering from bacterial, fungal, protozoal and viral infections (particularly
infections caused
by HIV-1 or HIV-2), pain, cancer (including but not limited to neoplasm;
adenocarcinoma;
lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma,
Parkinson's disease,
acute heart failure, hypotension, hypertension, urinary retention,
osteoporosis, Crohn's disease;
multiple sclerosis; and treatment of Albright hereditary ostoeodystrophy,
angina pectoris,
myocardial infarction, ulcers, asthma, allergies, benign prostatic
hypertrophy, and psychotic
and neurological disorders, including anxiety, schizophrenia, manic
depression, delirium,
dementia, severe mental retardation and dyskinesias, such as Huntington's
disease or Gilles de
la Tourette syndrome andlor other pathologies and disorders. These materials
are further
useful in the generation of antibodies that bind immuno-specifically to the
novel GPCR10
substances for use in therapeutic or diagnostic methods. These antibodies may
be generated
according to methods known in the art, using prediction from hydrophobicity
charts, as
described in the "Anti-GPCRX Antibodies" section below.
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A summary of the GPCRX nucleic acids and proteins of the invention is provided
in
Table 11.
TABLE 11: Summary Of Nucleic Acids And Proteins Of The Invention
Name Tables Clone; Description of Homolog NucleicAmino
Acid Acid
SEQ SEQ
ID ID
NO NO
GPCR1 1A, GPCRla: ba113a10_B, olfactory 1 2
1B, receptor
1D, GPCRlb: ba32713_A, olfactory 3 4
1E, receptor
1G, GpCRlc: ba113a10_C, olfactory 5 6
1H receptor
GPCR2 2A, GPCR2: 11612531-1, olfactory 7 8
2B receptor
GPCR3 3A, GPCR3: ba145L22_B, olfactory 9 10
3B receptor
GPCR4 4A, GPCR4a1: dj408b20 C, olfactory11 12
4B, receptor
4C, GPCR4a2: dj408b20 C dal, olfactory13 17
receptor
4G, GPCR4a3: CG55358 03, olfactory16
4H receptor
GPCRS 5A, GPCRSal: 115-a-12-A, olfactory18 19
5B, receptor
5C, GPCRSa2: 115-a-12-B, olfactory20 21
SD receptor
SG, GPCR5a3: 115-a-12-A dal, olfactory22 23
5H receptor
GPCR6 6A, GPCR6: 6-L-19-C, olfactory 24 25
6B receptor
GPCR7 7A, GPCR7: dj313i6_D olfactory 28 29
7B receptor
GPCR8 8A, GPCRB: dj408b20_A, olfactory 30 31
8B receptor
GPCR9 9A, GPCR9: 6-L-19-B, olfactory 32 33
9B receptor
GPCR10 10A, GPCRIOa: 6-L-19-A, olfactory 34 35
10B, receptor
IOC, GPCRIOb: 6-L-19-A1, olfactory 36 37
IOD, receptor
IOF, GPCRIOc: 6-L-19-A dal, olfactory38 83
lOG receptor
GPCRX Nucleic Acids and Polypeptides
One aspect of the invention pertains to isolated nucleic acid molecules that
encode
GPCRX polypeptides or biologically active portions thereof. Also included in
the invention
are nucleic acid fragments sufficient for use as hybridization probes to
identify GPCRX-
encoding nucleic acids (e.g., GPCRX mRNAs) and fragments for use as PCR
primers for the
amplification and/or mutation of GPCRX nucleic acid molecules. As used herein,
the term
"nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic
DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using
nucleotide analogs, and derivatives, fragments and homologs thereof. The
nucleic acid
molecule may be single-stranded or double-stranded, but preferably is
comprised double-
stranded DNA.
An GPCRX nucleic acid can encode a mature GPCRX polypeptide. As used herein, a
"mature" form of a polypeptide or protein disclosed in the present invention
is the product of a
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naturally occurnng polypeptide or precursor form or proprotein. The naturally
occurnng
polypeptide, precursor or proprotein includes, by way of nonlimiting example,
the full-length
gene product, encoded by the corresponding gene. Alternatively, it may be
defined as the
polypeptide, precursor or proprotein encoded by an ORF described herein. The
product
"mature" form arises, again by way of nonlimiting example, as a result of one
or more
naturally occurring processing steps as they may take place within the cell,
or host cell, in
which the gene product arises. Examples of such processing steps leading to a
"mature" form
of a polypeptide or protein include the cleavage of the N-terminal methionine
residue encoded
by the initiation codon of an ORF, or the proteolytic cleavage of a signal
peptide or leader
sequence. Thus a mature form arising from a precursor polypeptide or protein
that has
residues 1 to N, where residue 1 is the N-terminal methionine, would have
residues 2 through
N remaining after removal of the N-terminal methionine. Alternatively, a
mature form arising
from a precursor polypeptide or protein having residues 1 to N, in which an N-
terminal signal
sequence from residue 1 to residue M is cleaved, would have the residues from
residue M+1 to
residue N remaining. Further as used herein, a "mature" form of a polypeptide
or protein may
arise from a step of post-translational modification other than a proteolytic
cleavage event.
Such additional processes include, by way of non-limiting example,
glycosylation,
myristoylation or phosphorylation. In general, a mature polypeptide or protein
may result
from the operation of only one of these processes, or a combination of any of
them.
The term "probes", as utilized herein, refers to nucleic acid sequences of
variable
length, preferably between at least about 10 nucleotides (nt), 100 nt, or as
many as
approximately, e.g., 6,000 nt, depending upon the specific use. Probes are
used in the
detection of identical, similar, or complementary nucleic acid sequences.
Longer length
probes are generally obtained from a natural or recombinant source, are highly
specific, and
much slower to hybridize than shorter-length oligomer probes. Probes may be
single- or
double-stranded and designed to have specificity in PCR, membrane-based
hybridization
technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as utilized herein, is one, which
is separated
from other nucleic acid molecules which are present in the natural source of
the nucleic acid.
Preferably, an "isolated" nucleic acid is free of sequences which naturally
flank the nucleic
acid (a.e., sequences located at the 5'- and 3'-termini of the nucleic acid)
in the genomic DNA
of the organism from which the nucleic acid is derived. For example, in
various embodiments,
the isolated GPCRX nucleic acid molecules can contain less than about 5 kb, 4
kb, 3 kb, 2 kb,
1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the
nucleic acid molecule
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in genomic DNA of the cell/tissue from which the nucleic acid is derived
(e.g., brain, heart,
liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a
cDNA molecule,
can be substantially free of other cellular material or culture medium when
produced by
recombinant techniques, or of chemical precursors or other chemicals when
chemically
synthesized.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having
the
nucleotide sequence of SEQ >D NOS:1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24,
28, 30, 32, 34, 36,
and 38, or a complement of this aforementioned nucleotide sequence, can be
isolated using
standard molecular biology techniques and the sequence information provided
herein. Using
all or a portion of the nucleic acid sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11,
13, 16, 18, 20,
22, 24, 28, 30, 32, 34, 36, and 38 as a hybridization probe, GPCRX molecules
can be isolated
using standard hybridization and cloning techniques (e.g., as described in
Sambrook, et al.,
(eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2°a Ed., Cold Spring
Harbor
Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.),
CURRENT
PROTOCOLS rN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively,
genomic DNA, as a template and appropriate oligonucleotide primers according
to standard
PCR amplification techniques. The nucleic acid so amplified can be cloned into
an
appropriate vector and characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to GPCRX nucleotide sequences can be prepared
by standard
synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked
nucleotide
residues, which oligonucleotide has a sufficient number of nucleotide bases to
be used in a
PCR reaction. A short oligonucleotide sequence may be based on, or designed
from, a
genomic or cDNA sequence and is used to amplify, confirm, or reveal the
presence of an
identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having about 10
nt, 50 nt, or
100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment
of the
invention, an oligonucleotide comprising a nucleic acid molecule less than 100
nt in length
would further comprise at least 6 contiguous nucleotides of SEQ ID NOS:1, 3,
5, 7, 9, 11, 13,
16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38, or a complement thereof.
Oligonucleotides may
be chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention
comprises a
nucleic acid molecule that is a complement of the nucleotide sequence shown in
SEQ ID
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NOS:1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38, or
a portion of this
nucleotide sequence (e.g., a fragment that can be used as a probe or primer or
a fragment
encoding a biologically-active portion of an GPCRX polypeptide). A nucleic
acid molecule
that is complementary to the nucleotide sequence shown in SEQ ID NOS:1, 3, 5,
7, 9, 11, 13,
16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38, is one that is sufficiently
complementary to the
nucleotide sequence shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22,
24, 28, 30, 32,
34, 36, and 38, that it can hydrogen bond with little or no mismatches to the
nucleotide
sequence shown SEQ ID NOS:1, 3, 5, 7, 9, I 1, 13, 16, 18, 20, 22, 24, 28, 30,
32, 34, 36, and
38, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen
base
pairing between nucleotides units of a nucleic acid molecule, and the teen
"binding" means
the physical or chemical interaction between two polypeptides or compounds or
associated
polypeptides or compounds or combinations thereof. Binding includes ionic, non-
ionic, van
der Waals, hydrophobic interactions, and the like. A physical interaction can
be either direct
or indirect. Indirect interactions may be through or due to the effects of
another polypeptide or
compound. Direct binding refers to interactions that do not take place
through, or due to, the
effect of another polypeptide or compound, but instead are without other
substantial chemical
intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous)
nucleic
acids or at least 4 (contiguous) amino acids, a length sufficient to allow for
specific
hybridization in the case of nucleic acids or for specific recognition of an
epitope in the case of
amino acids, respectively, and are at most some portion less than a full
length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or
amino acid
sequence of choice. Derivatives are nucleic acid sequences or amino acid
sequences formed
from the native compounds either directly or by modification or partial
substitution. Analogs
are nucleic acid sequences or amino acid sequences that have a structure
similar to, but not
identical to, the native compound but differs from it in respect to certain
components or side
chains. Analogs may be synthetic or from a different evolutionary origin and
may have a
similar or opposite metabolic activity compared to wild type. Homologs are
nucleic acid
sequences or amino acid sequences of a particular gene that are derived from
different species.
Derivatives and analogs may be full length or other than full length, if the
derivative or
analog contains a modified nucleic acid or amino acid, as described below.
Derivatives or
analogs of the nucleic acids or proteins of the invention include, but are not
limited to,
molecules comprising regions that are substantially homologous to the nucleic
acids or
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proteins of the invention, in various embodiments, by at least about 70%, 80%,
or 95%
identity (with a preferred identity of 80-95%) over a nucleic acid or amino
acid sequence of
identical size or when compared to an aligned sequence in which the alignment
is done by a
computer homology program known in the art, or whose encoding nucleic acid is
capable of
hybridizing to the complement of a sequence encoding the aforementioned
proteins under
stringent, moderately stringent, or low stringent conditions. See e.g.
Ausubel, et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and
below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or
variations thereof, refer to sequences characterized by a homology at the
nucleotide level or
amino acid level as discussed above. Homologous nucleotide sequences encode
those
sequences coding for isoforms of GPCRX polypeptides. Isoforms can be expressed
in
different tissues of the same organism as a result of, for example,
alternative splicing of RNA.
Alternatively, isoforms can be encoded by different genes. In the invention,
homologous
nucleotide sequences include nucleotide sequences encoding for an GPCRX
polypeptide of
species other than humans, including, but not limited to: vertebrates, and
thus can include, e.g.,
frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous
nucleotide
sequences also include, but are not limited to, naturally occurring allelic
variations and
mutations of the nucleotide sequences set forth herein. A homologous
nucleotide sequence
does not, however, include the exact nucleotide sequence encoding human GPCRX
protein.
Homologous nucleic acid sequences include those nucleic acid sequences that
encode
conservative amino acid substitutions (see below) in SEQ ID NOS:1, 3, 5, 7, 9,
11, 13, 16, 18,
20, 22, 24, 28, 30, 32, 34, 36, and 38, as well as a polypeptide possessing
GPCRX biological
activity. Various biological activities of the GPCRX proteins are described
below.
An GPCRX polypeptide is encoded by the open reading frame ("ORF") of an GPCRX
nucleic acid. An ORF corresponds to a nucleotide sequence that could
potentially be translated
into a polypeptide. A stretch of nucleic acids comprising an ORF is
uninterrupted by a stop
codon. An ORF that represents the coding sequence for a full protein begins
with an ATG
"start" codon and terminates with one of the three "stop" codons, namely, TAA,
TAG, or
TGA. For the purposes of this invention, an ORF may be any part of a coding
sequence, with
or without a start codon, a stop codon, or both. For an ORF to be considered
as a good
candidate for coding for a bona fide cellular protein, a minimum size
requirement is often set,
e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human GPCRX genes
allows for the generation of probes and primers designed for use in
identifying and/or cloning
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GPCRX homologues in other cell types, e.g. from other tissues, as well as
GPC1RX
homologues from other vertebrates. The probe/primer typically comprises
substantially
purified oligonucleotide. The oligonucleotide typically comprises a region of
nucleotide
sequence that hybridizes under stringent conditions to at least about 12, 25,
50, 100, 150, 200,
250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID
NOS:1, 3, 5, 7,
9, 11, 13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38; or an anti-sense
strand nucleotide
sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 28, 30, 32,
34, 36, and 38; or
of a naturally occurring mutant of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 16, 18,
20, 22, 24, 28, 30,
32, 34, 36, and 38.
Probes based on the human GPCRX nucleotide sequences can be used to detect
transcripts or genomic sequences encoding the same or homologous proteins. In
various
embodiments, the probe further comprises a label group attached thereto, e.g.
the label group
can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-
factor. Such
probes can be used as a part of a diagnostic test kit for identifying cells or
tissues which mis-
express an GPCRX protein, such as by measuring a level of an GPCRX-encoding
nucleic acid
in a sample of cells from a subject e.g., detecting GPCRX mRNA levels or
determining
whether a genomic GPCRX gene has been mutated or deleted.
"A polypeptide having a biologically-active portion of an GPCRX polypeptide"
refers
to polypeptides exhibiting activity similar, but not necessarily identical to,
an activity of a
polypeptide of the invention, including mature forms, as measured in a
particular biological
assay, with or without dose dependency. A nucleic acid fragment encoding a
"biologically-
active portion of GPCIRX" can be prepared by isolating a portion SEQ ID NOS:1,
3, 5, 7, 9,
11, 13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38 that encodes a
polypeptide having an
GPCRX biological activity (the biological activities of the GPCRX proteins are
described
below), expressing the encoded portion of GPCRX protein (e.g., by recombinant
expression in
vitro) and assessing the activity of the encoded portion of GPCRX.
GPCRX Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the
nucleotide sequences shown SEQ ID NOS:1, 3, 5, 7, 9, 1.1, 13, 16, 18, 20, 22,
24, 28, 30, 32,
34, 36, and 38 due to degeneracy of the genetic code and thus encode the same
GPCRX
proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:1, 3,
5, 7, 9, 11,
13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38. In another embodiment, an
isolated nucleic
acid molecule of the invention has a nucleotide sequence encoding a protein
having an amino
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acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23, 25, 29,
31, 33, 35, 37,
and 83.
In addition to the human GPCRX nucleotide sequences shown in SEQ ID NOS:1, 3,
5,
7, 9, 11, 13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38 it will be
appreciated by those skilled
in the art that DNA sequence polymorphisms that lead to changes in the amino
acid sequences
of the GPCRX polypeptides may exist within a population (e.g., the human
population). Such
genetic polymorphism in the GPCRX genes may exist among individuals within a
population
due to natural allelic variation. As used herein, the terms "gene" and
"recombinant gene" refer
to nucleic acid molecules comprising an open reading frame (ORF) encoding an
GPCRX
protein, preferably a vertebrate GPCRX protein. Such natural allelic
variations can typically
result in 1-5% variance in the nucleotide sequence of the GPCRX genes. Any and
all such
nucleotide variations and resulting amino acid polymorphisms in the GPCRX
polypeptides,
which are the result of natural allelic variation and that do not alter the
functional activity of
the GPCRX polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding GPCRX proteins from other species,
and
thus that have a nucleotide sequence that differs from the human sequence SEQ
ID NOS:1, 3,
5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38 are intended
to be within the scope
of the invention. Nucleic acid molecules corresponding to natural allelic
variants and
homologues of the GPCRX cDNAs of the invention can be isolated based on their
homology
to the human GPCRX nucleic acids disclosed herein using the human cDNAs, or a
portion
thereof, as a hybridization probe according to standard hybridization
techniques under
stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the
invention is at least 6 nucleotides in length and hybridizes under stringent
conditions to the
nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3,
5, 7, 9, 11,
13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38. In another embodiment, the
nucleic acid is at
least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides
in length. In yet
another embodiment, an isolated nucleic acid molecule of the invention
hybridizes to the
coding region. As used herein, the term "hybridizes under stringent
conditions" is intended to
describe conditions for hybridization and washing under which nucleotide
sequences at least
60% homologous to each other typically remain hybridized to each other.
Homologs (i.e., nucleic acids encoding GPCRX proteins derived from species
other
than human) or other related sequences (e.g., paralogs) can be obtained by
low, moderate or
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high stringency hybridization with all or a portion of the particular human
sequence as a probe
using methods well known in the art for nucleic acid hybridization and
cloning.
As used herein, the phrase "stringent hybridization conditions" refers to
conditions
under which a probe, primer or oligonucleotide will hybridize to its target
sequence, but to no
other sequences. Stringent conditions are sequence-dependent and will be
different in
different circumstances. Longer sequences hybridize specifically at higher
temperatures than
shorter sequences. Generally, stringent conditions are selected to be about 5
°C lower than the
thermal melting point (Tm) for the specific sequence at a defined ionic
strength and pH. The
Tm is the temperature (under defined ionic strength, pH and nucleic acid
concentration) at
which SO% of the probes complementary to the target sequence hybridize to the
target
sequence at equilibrium. Since the target sequences are generally present at
excess, at Tm,
50% of the probes are occupied at equilibrium. Typically, stringent conditions
will be those in
which the salt concentration is less than about 1.0 M sodium ion, typically
about 0.01 to 1.0 M
sodium ion (or other salts) at
pH 7.0 to 8.3 and the temperature is at least about 30°C for short
probes, primers or
oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for
longer probes, primers and
oligonucleotides. Stringent conditions may also be achieved with the addition
of destabilizing
agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in
Ausubel,
et al., (eds.), CURRENT PROTOCOLS ~ MOLECULAR BIOLOGY, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at
least about 65%,
70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain
hybridized to each other. A non-limiting example of stringent hybridization
conditions are
hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH
7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm
DNA
at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at
50°C. An isolated
nucleic acid molecule of the invention that hybridizes under stringent
conditions to the
sequences of SEQ ID NOS:I, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 28, 30, 32,
34, 36, and 38
corresponds to a naturally-occurring nucleic acid molecule. As used herein, a
"naturally-occurnng" nucleic acid molecule refers to an RNA or DNA molecule
having a
nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the
nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9,
11, 13, 16, 18,
20, 22, 24, 28, 30, 32, 34, 36, and 38 or fragments, analogs or derivatives
thereof, under
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conditions of moderate stringency is provided. A non-limiting example of
moderate
stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's
solution,
0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by
one or more
washes in 1X SSC, 0.1% SDS at 37°C. Other conditions of moderate
stringency that may be
used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993,
CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990;
GENE
TRANSFER AND EXPRESSION, A LABORATORY MANUAL, StOCktOn PreSS, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid
molecule
comprising the nucleotide sequences of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 16,
18, 20, 22, 24,
28, 30, 32, 34, 36, and 38 or fragments, analogs or derivatives thereof, under
conditions of low
stringency, is provided. A non-limiting example of low stringency
hybridization conditions
are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM
EDTA,
0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X
SSC, 25 mM Tris-HCl
(pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low
stringency that may
be used are well known in the art (e.g., as employed for cross-species
hybridizations). See,
e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
John Wiley
& Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY
MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA
78:
6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of GPCRX sequences that
may exist
in the population, the skilled artisan will further appreciate that changes
can be introduced by
mutation into the nucleotide sequences of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13,
16, 18, 20, 22, 24,
28, 30, 32, 34, 36, and 38 thereby leading to changes in the amino acid
sequences of the
encoded GPCRX proteins, without altering the functional ability of said GPCRX
proteins. For
example, nucleotide substitutions leading to amino acid substitutions at "non-
essential" amino
acid residues can be made in the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12,
17, 19, 21, 23,
25, 29, 31, 33, 35, 37, and 83. A "non-essential" amino acid residue is a
residue that can be
altered from the wild-type sequences of the GPCRX proteins without altering
their biological
activity, whereas an "essential" amino acid residue is required for such
biological activity. For
example, amino acid residues that are conserved among the GPCRX proteins of
the invention
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are predicted to be particularly non-amenable to alteration. Amino acids for
which
conservative substitutions can be made are well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding
GPCRX
proteins that contain changes in amino acid residues that are not essential
for activity. Such
GPCRX proteins differ in amino acid sequence from SEQ ID NOS:2, 4, 6, 8, 10,
12, 17, 19,
21, 23, 25, 29, 31, 33, 35, 37, and 83 yet retain biological activity. In one
embodiment, the
isolated nucleic acid molecule comprises a nucleotide sequence encoding a
protein, wherein
the protein comprises an amino acid sequence at least about 45% homologous to
the amino
acid sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23, 25, 29, 31,
33, 35, 37, and 83.
Preferably, the protein encoded by the nucleic acid molecule is at least about
60% homologous
to SEQ ID NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and
83; more
preferably at least about 70% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 17,
19, 21, 23,
25, 29, 31, 33, 35, 37, and 83; still more preferably at least about 80%
homologous to SEQ ID
NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and 83; even
more preferably at
least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23,
25, 29, 31, 33,
35, 37, and 83; and most preferably at least about 95% homologous to SEQ ID
NOS:2, 4, 6, 8,
10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and 83.
An isolated nucleic acid molecule encoding an GPCRX protein homologous to the
protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35,
37, and 83 can be
created by introducing one or more nucleotide substitutions, additions or
deletions into the
nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24,
28, 30, 32, 34, 36,
and 38 such that one or more amino acid substitutions, additions or deletions
are introduced
into the encoded protein.
Mutations can be introduced into SEQ ID NOS:2, 4, 6, 8, 10, 12, 17, 19, 21,
23, 25, 29,
31, 33, 35, 37, and 83 by standard techniques, such as site-directed
mutagenesis and
PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions
axe made at
one or more predicted, non-essential amino acid residues. A "conservative
amino acid
substitution" is one in which the amino acid residue is replaced with an amino
acid residue
having a similar side chain. Families of amino acid residues having similar
side chains have
been defined within the art. These families include amino acids with basic
side chains (e.g.,
lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged
polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and
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aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Thus, a predicted
non-essential amino acid residue in the GPC1RX protein is replaced with
another amino acid
residue from the same side chain family. Alternatively, in another embodiment,
mutations can
be introduced randomly along all or part of an GPCItX coding sequence, such as
by saturation
mutagenesis, and the resultant mutants can be screened for GPCRX biological
activity to
identify mutants that retain activity. Following mutagenesis of SEQ ID NOS:1,
3, 5, 7, 9, 1 l,
13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38, the encoded protein can be
expressed by any
recombinant technology known in the art and the activity of the protein can be
determined.
The relatedness of amino acid families may also be determined based on side
chain
interactions. Substituted amino acids may be fully conserved "strong" residues
or fully
conserved "weak" residues. The "strong" group of conserved amino acid residues
may be any
one of the following groups: STA, NEQK, NHQK, NDEQ, QH1RK, MILV, MILF, HY,
FYW,
wherein the single letter amino acid codes are grouped by those amino acids
that may be
substituted for each other. Likewise, the "weak" group of conserved residues
may be any one
of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQH1ZK,
VLIM, HFY, wherein the letters within each group represent the single letter
amino acid code.
In one embodiment, a mutant GPCRX protein can be assayed for (i) the ability
to form
protein:protein interactions with other GPCRX proteins, other cell-surface
proteins, or
biologically-active portions thereof, (ii) complex formation between a mutant
GPC1RX protein
and an GPCRX ligand; or (iii) the ability of a mutant GPC1RX protein to bind
to an
intracellular target protein or biologically-active portion thereof; (e.g.
avidin proteins).
In yet another embodiment, a mutant GPCRX protein can be assayed for the
ability to
regulate a specific biological function (e.g., regulation of insulin release).
Antisense Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid
molecules
that are hybridizable to or complementary to the nucleic acid molecule
comprising the
nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24,
28, 30, 32, 34, 36,
and 38, or fragments, analogs or derivatives thereof. An "antisense" nucleic
acid comprises a
nucleotide sequence that is complementary to a "sense" nucleic acid encoding a
protein (e.g.,
complementary to the coding strand of a double-stranded cDNA molecule or
complementary
to an mltNA sequence). In specific aspects, antisense nucleic acid molecules
are provided that
comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or
500 nucleotides
or an entire GPCRX coding strand, or to only a portion thereof. Nucleic acid
molecules
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encoding fragments, homologs, derivatives and analogs of an GPCRX protein of
SEQ ID
NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and 83, or
antisense nucleic acids
complementary to an GPCRX nucleic acid sequence of SEQ ID NOS:1, 3, 5, 7, 9,
11, 13, 16,
18, 20, 22, 24, 28, 30, 32, 34, 36, and 38, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding
region" of the coding strand of a nucleotide sequence encoding an GPCRX
protein. The term
"coding region" refers to the region of the nucleotide sequence comprising
codons which are
translated into amino acid residues. In another embodiment, the antisense
nucleic acid
molecule is antisense to a "noncoding region" of the coding strand of a
nucleotide sequence
encoding the GPCRX protein. The term "noncoding region" refers to 5' and 3'
sequences
which flank the coding region that are not translated into amino acids (i.e.,
also referred to as
5' and 3' untranslated regions).
Given the coding strand sequences encoding the GPCRX protein disclosed herein,
antisense nucleic acids of the invention can be designed according to the
rules of Watson and
Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be
complementary
to the entire coding region of GPCRX mRNA, but more preferably is an
oligonucleotide that
is antisense to only a portion of the coding or noncoding region of GPCRX
mRNA. For
example, the antisense oligonucleotide can be complementary to the region
surrounding the
translation start site of GPCRX mRNA. An antisense oligonucleotide can be, for
example,
about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An
antisense nucleic acid of
the invention can be constructed using chemical synthesis or enzymatic
ligation reactions
using procedures known in the art. For example, an antisense nucleic acid
(e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally-occurnng
nucleotides or
variously modified nucleotides designed to increase the biological stability
of the molecules or
to increase the physical stability of the duplex formed between the antisense
and sense nucleic
acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides
can be used).
Examples of modified nucleotides that can be used to generate the antisense
nucleic
acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethyl-
2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine,
inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-
dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil,
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2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil,
queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil,
3-(3-amino-3-N-2-carboxypiopyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the
antisense nucleic acid can be produced biologically using an expression vector
into which a
nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the
inserted nucleic acid will be of an antisense orientation to a target nucleic
acid of interest,
described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated in situ such that they hybridize with or bind to cellular
mRNA andlor
genomic DNA encoding an GPCRX protein to thereby inhibit expression of the
protein (e.g.,
by inhibiting transcription and/or translation). The hybridization can be by
conventional
nucleotide complementarity to form a stable duplex, or, for example, in the
case of an
antisense nucleic acid molecule that binds to DNA duplexes, through specific
interactions in
the major groove of the double helix. An example of a route of administration
of antisense
nucleic acid molecules of the invention includes direct injection at a tissue
site. Alternatively,
antisense nucleic acid molecules can be modified to target selected cells and
then administered
systemically. For example, for systemic administration, antisense molecules
can be modified
such that they specifically bind to receptors or antigens expressed on a
selected cell surface
(e.g., by linking the antisense nucleic acid molecules to peptides or
antibodies that bind to cell
surface receptors or antigens). The antisense nucleic acid molecules can also
be delivered to
cells using the vectors described herein. To achieve sufficient nucleic acid
molecules, vector
constructs in which the antisense nucleic acid molecule is placed under the
control of a strong
pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is an
a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms
specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
[3-units, the
strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl.
Acids Res. 15: .
6625-6641. The antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (see, e.g., moue, et al. 1987. Nucl. Acids Res. 15:
6131-6148) or a
chimeric RNA-DNA analogue (see, e.g., moue, et al., 1987. FEBS Lett. 215: 327-
330.
Ribozymes and PNA Moieties
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Nucleic acid modifications include, by way of non-limiting example, modified
bases,
and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These
modifications are carned out at least in part to enhance the chemical
stability of the modified
nucleic acid, such that they may be used, for example, as antisense binding
nucleic acids in
therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are
capable of
cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described
in
Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically
cleave GPCRX
mRNA transcripts to thereby inhibit translation of GPCRX mRNA. A ribozyme
having
specificity for an GPCRX-encoding nucleic acid can be designed based upon the
nucleotide
sequence of an GPCRX cDNA disclosed herein (i.e., SEQ ID NOS:1, 3, 5, 7, 9, 1
l, 13, 16, 18,
20, 22, 24, 28, 30, 32, 34, 36, and 38). For example, a derivative of a
Tetrahymena L-19 IVS
RNA can be constructed in which the nucleotide sequence of the active site is
complementary
to the nucleotide sequence to be cleaved in an GPCRX-encoding mRNA. See, e.g.,
U.S.
Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al.
GPCRX mRNA can
also be used to select a catalytic RNA having a specific ribonuclease activity
from a pool of
RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, GPCRX gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of the GPCRX nucleic acid
(e.g., the
GPCRX promoter and/or enhancers) to form triple helical structures that
prevent transcription
of the GPCRX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug
Des. 6: 569-84;
Helene, et al. 1992. Ann. N. Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays
14: 807-15.
In various embodiments, the GPCRX nucleic acids can be modified at the base
moiety,
sugar moiety or phosphate backbone to improve, e.g., the stability,
hybridization, or solubility
of the molecule. For example, the deoxyribose phosphate backbone of the
nucleic acids can
be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al:, 1996.
Bioorg Med
Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic
acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is
replaced by
a pseudopeptide backbone and only the four natural nucleobases are retained.
The neutral
backbone of PNAs has been shown to allow for specific hybridization to DNA and
RNA under
conditions of low ionic strength. The synthesis of PNA oligomers can be
performed using
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standard solid phase peptide synthesis protocols as described in Hyrup, et
al., 1996. supra;
Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
PNAs of GPCRX can be used in therapeutic and diagnostic applications. For
example,
PNAs can be used as antisense or antigene agents for sequence-specific
modulation of gene
expression by, e.g., inducing transcription or translation arrest or
inhibiting replication. PNAs
of GPCRX can also be used, for example, in the analysis of single base pair
mutations in a
gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when
used in
combination with other enzymes, e.g., S~ nucleases (see, Hyrup, et al.,
1996.supra); or as
probes or primers for DNA sequence and hybridization (see, Hyrup, et al.,
1996, supra;
Perry-O'Keefe, et al., 1996. supra).
In another embodiment, PNAs of GPCRX can be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other helper groups
to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other techniques
of drug
delivery known in the art. For example, PNA-DNA chimeras of GPCRX can be
generated
that may combine the advantageous properties of PNA and DNA. Such chimeras
allow DNA
recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the
DNA portion
while the PNA portion would provide high binding affinity and specificity. PNA-
DNA
chimeras can be linked using linkers of appropriate lengths selected in terms
of base stacking,
number of bonds between the nucleobases; and orientation (see, Hyrup, et al.,
1996. supra).
The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et
al., 1996.
supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA
chain can
be synthesized on a solid support using standard phosphoramidite coupling
chemistry, and
modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-
thymidine
phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g.,
Mag, et al.,
1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise
manner
to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment.
See, e.g.,
Finn, et al., 1996. supra. Alternatively, chimeric molecules can be
synthesized with a 5' DNA
segment and a 3' PNA segment. See, e.g., Petersen, et al., 1975. Bioorg. Med.
Chem. Lett. 5:
1119-11124.
In other embodiments, the oligonucleotide may include other appended groups
such as
peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across
the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl. Acad. Sci.
U.S.A. 86:
6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT
Publication No.
W088/09810) or the blood-brain barner (see, e.g., PCT Publication No.
W0'89/10134). In
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addition, oligonucleotides can be modified with hybridization triggered
cleavage agents (see,
e.g., Krol, et al., 1988. BioTechniques 6:958-976) or intercalating agents
(see, e.g., Zon, 1988.
Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to
another
molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a
transport agent, a
hybridization-triggered cleavage agent, and the like.
GPCRX Polypeptides
A polypeptide according to the invention includes a polypeptide including the
amino
acid sequence of GPCRX polypeptides whose sequences are provided in SEQ ID
NOS:2, 4, 6,
8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and 83. The invention also
includes a mutant
or variant protein any of whose residues may be changed from the corresponding
residues
shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35,
37, and 83 while
still encoding a protein that maintains its GPCIRX activities and
physiological functions, or a
functional fragment thereof.
In general, an GPCRX variant that preserves GPCRX-like function includes any
variant in which residues at a particular position in the sequence have been
substituted by
other amino acids, and further include the possibility of inserting an
additional residue or
residues between two residues of the parent protein as well as the possibility
of deleting one or
more residues from the parent sequence. Any amino acid substitution,
insertion, or deletion is
encompassed by the invention. In favorable circumstances, the substitution is
a conservative
substitution as defined above.
One aspect of the invention pertains to isolated GPCIRX proteins, and
biologically-
active portions thereof, or derivatives, fragments, analogs or homologs
thereof. Also provided
are polypeptide fragments suitable for use as immunogens to raise anti-GPC12X
antibodies. In
one embodiment, native GPCRX proteins can be isolated from cells or tissue
sources by an
appropriate purification scheme using standard protein purification
techniques. In another
embodiment, GPCRX proteins are produced by recombinant DNA techniques.
Alternative to
recombinant expression, an GPC1RX protein or polypeptide can be synthesized
chemically
using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active
portion thereof
is substantially free of cellular material or other contaminating proteins
from the cell or tissue
source from which the GPCItX protein is derived, or substantially free from
chemical
precursors or other chemicals when chemically synthesized. The language
"substantially free
of cellular material" includes preparations of GPCRX proteins in which the
protein is
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separated from cellular components of the cells from which it is isolated or
recombinantly-
produced. In one embodiment, the language "substantially free of cellular
material" includes
preparations of GPCRX proteins having less than about 30% (by dry weight) of
non-GPCRX
proteins (also referred to herein as a "contaminating protein"), more
preferably less than about
20% of non-GPCRX proteins, still more preferably less than about 10% of non-
GPCRX
proteins, and most preferably less than about 5% of non-GPCRX proteins. When
the GPCRX
protein or biologically-active portion thereof is recombinantly-produced, it
is also preferably
substantially free of culture medium, i.e., culture medium represents less
than about 20%,
more preferably less than about 10%, and most preferably less than about 5% of
the volume of
the GPCRX protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes
preparations of GPCRX proteins in which the protein is separated from chemical
precursors or
other chemicals that are involved in the synthesis of the protein. In one
embodiment, the
language "substantially free of chemical precursors or other chemicals"
includes preparations
of GPCRX proteins having less than about 30% (by dry weight) of chemical
precursors or
non-GPCRX chemicals, more preferably less than about 20% chemical precursors
or
non-GPCRX chemicals, still more preferably less than about 10% chemical
precursors or
non-GPCRX chemicals, and most preferably less than about 5% chemical
precursors or
non-GPCRX chemicals.
Biologically-active portions of GPCRX proteins include peptides comprising
amino
acid sequences sufficiently homologous to or derived from the amino acid
sequences of the
GPCRX proteins (e.g., the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8,
10, 12, 17,
19, 21, 23, 25, 29, 31, 33, 35, 37, and 83) that include fewer amino acids
than the full-length
GPCRX proteins, and exhibit at least one activity of an GPCRX protein.
Typically,
biologically-active portions comprise a domain or motif with at least one
activity of the
GPCRX protein. A biologically-active portion of an GPCRX protein can be a
polypeptide
which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the
protein are
deleted, can be prepared by recombinant techniques and evaluated for one or
more of the
functional activities of a native GPCRX protein.
In an embodiment, the GPCRX protein has an amino acid sequence shown in SEQ ID
NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and 83. In
other embodiments,
the GPCRX protein is substantially homologous to SEQ ID NOS:2, 4, 6, 8, 10,
12, 17, 19, 21,
23, 25, 29, 31, 33, 35, 37, and 83, and retains the functional activity of the
protein of SEQ ID
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NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and 83, yet
differs in amino acid
sequence due to natural allelic variation or mutagenesis, as described in
detail, below.
Accordingly, in another embodiment, the GPCRX protein is a protein that
comprises an amino
acid sequence at least about 45% homologous to the amino acid sequence SEQ ID
NOS:2, 4,
6, 8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and 83, and retains the
functional activity of
the GPCRX proteins of SEQ ID NOS:2, 4, 6, 8, 10, 12, 17, 19, 21, 23, 25, 29,
31, 33, 35, 37,
and 83.
Determining Homology Between Two or More Sequences
To determine the percent homology of two amino acid sequences or of two
nucleic
acids, the sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced
in the sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a
second amino or nucleic acid sequence). The amino acid residues or nucleotides
at
corresponding amino acid positions or nucleotide positions are then compared.
When a
position in the first sequence is occupied by the same amino acid residue or
nucleotide as the
corresponding position in the second sequence, then the molecules are
homologous at that
position (i.e., as used herein amino acid or nucleic acid "homology" is
equivalent to amino
acid or nucleic acid "identity")
The nucleic acid sequence homology may be determined as the degree of identity
between two sequences. The homology may be determined using computer programs
known
in the art, such as GAP software provided in the GCG program package. See,
Needleman and
Wunsch, 1970. JMoI Biol 48: 443-453. Using GCG GAP software with the following
settings
for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP
extension
penalty of 0.3, the coding region of the analogous nucleic acid sequences
referred to above
exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%,
95%, 98%, or
99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOS:1,
3, S, 7, 9,
11, 13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38.
The term "sequence identity" refers to the degree to which two polynucleotide
or
polypeptide sequences are identical on a residue-by-residue basis over a
particular region of
comparison. The term "percentage of sequence identity" is calculated by
comparing two
optimally aligned sequences over that region of comparison, determining the
number of
positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I,
in the case of
nucleic acids) occurs in both sequences to yield the number of matched
positions, dividing the
number of matched positions by the total number of positions in the region of
comparison (i. e.,
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the window size), and multiplying the result by 100 to yield the percentage of
sequence
identity. The term "substantial identity" as used herein denotes a
characteristic of a
polynucleotide sequence, wherein the polynucleotide comprises a sequence that
has at least 80
percent sequence identity, preferably at least 85 percent identity and often
90 to 95 percent
sequence identity, more usually at least 99 percent sequence identity as
compared to a
reference sequence over a comparison region.
Chimeric and Fusion Proteins
The invention also provides GPCRX chimeric or fusion proteins. As used herein,
an
GPCRX "chimeric protein" or "fusion protein" comprises an GPCRX polypeptide
operatively
linked to a non-GPCRX polypeptide. An "GPCRX polypeptide" refers to a
polypeptide
having an amino acid sequence corresponding to an GPCRX protein (SEQ ID
NOS:2,.4, 6, 8,
10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and 83), whereas a "non-GPCRX
polypeptide"
refers to a polypeptide having an amino acid sequence corresponding to a
protein that is not
substantially homologous to the GPCRX protein, e.g., a protein that is
different from the
GPCRX protein and that is derived from the same or a different organism.
Within an GPCRX
fusion protein the GPCRX polypeptide can correspond to all or a portion of an
GPCRX
protein. In one embodiment, an GPCRX fusion protein comprises at least one
biologically-
active portion of an GPCRX protein. In another embodiment, an GPCRX fusion
protein
comprises at least two biologically-active portions of an GPCRX protein. In
yet another
embodiment, an GPCRX fusion protein comprises at least three biologically-
active portions of
an GPCRX protein. Within the fusion protein, the term "operatively-linked" is
intended to
indicate that the GPCRX polypeptide and the non-GPCRX polypeptide are fused in-
frame
with one another. The non-GPC1RX polypeptide can be fused to the N-terminus or
C-terminus
of the GPCRX polypeptide.
In one embodiment, the fusion protein is a GST-GPCRX fusion protein in which
the
GPCRX sequences are fused to the C-terminus of the GST (glutathione S-
transferase)
sequences. Such fusion proteins can facilitate the purification of recombinant
GPCRX
polypeptides.
In another embodiment, the fusion protein is an GPCRX protein containing a
heterologous signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host
cells), expression and/or secretion of GPCRX can be increased through use of a
heterologous
signal sequence.
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In yet another embodiment, the fusion protein is an GPCRX-immunoglobulin
fusion
protein in which the GPCRX sequences are fused to sequences derived from a
member of the
immunoglobulin protein family. The GPCRX-immunoglobulin fusion proteins of the
invention can be incorporated into pharmaceutical compositions and
administered to a subject
to inhibit an interaction between an GPCRX ligand and an GPCRX protein on the
surface of a
cell, to thereby suppress GPCRX-mediated signal transduction in vivo. The
GPCRX-
immunoglobulin fusion proteins can be used to affect the bioavailability of an
GPCRX
cognate ligand. Inhibition of the GPCRX ligand/GPCRX interaction may be useful
therapeutically for both the treatment of proliferative and differentiative
disorders, as well as
modulating (e.g. promoting or inhibiting) cell survival. Moreover, the
GPCRX-immunoglobulin fusion proteins of the invention can be used as
immunogens to
produce anti-GPCRX antibodies in a subject, to purify GPCRX ligands, and in
screening
assays to identify molecules that inhibit the interaction of GPCRX with an
GPCRX ligand.
An GPCRX chimeric or fusion protein of the invention can be produced by
standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
polypeptide sequences are ligated together in-frame in accordance with
conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for
ligation, restriction
enzyme digestion to provide for appropriate termini, filling-in of cohesive
ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and enzymatic
ligation. In
another embodiment, the fusion gene can be synthesized by conventional
techniques including
automated DNA synthesizers. Alternatively, PCR amplification of gene fragments
can be
carned out using anchor primers that give rise to complementary overhangs
between two
consecutive gene fragments that can subsequently be annealed and reamplified
to generate a
chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are
commercially
available that already encode a fusion moiety (e.g., a GST polypeptide). An
GPCRX-encoding nucleic acid can be cloned into such an expression vector such
that the
fusion moiety is linked in-frame to the GPCRX protein.
GPCRX Agonists and Antagonists
The invention also pertains to variants of the GPCRX proteins that function as
either
GPCRX agonists (i.e., mimetics) or as GPCRX antagonists. Variants of the GPCRX
protein
can be generated by mutagenesis (e.g., discrete point mutation or truncation
of the GPCRX
protein). An agonist of the GPCRX protein can retain substantially the same,
or a subset of,
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the biological activities of the naturally occurring form of the GPCRX
protein. An antagonist
of the GPCRX protein can inhibit one or more of the activities of the
naturally occurnng form
of the GPCRX protein by, for example, competitively binding to a downstream or
upstream
member of a cellular signaling cascade which includes the GPCRX protein. Thus,
specific
biological effects can be elicited by treatment with a variant of limited
function. In one
embodiment, treatment of a subject with a variant having a subset of the
biological activities
of the naturally occurnng form of the protein has fewer side effects in a subj
ect relative to
treatment with the naturally occurring form of the GPCRX proteins.
Variants of the GPCRX proteins that function as either GPCRX agonists (i.e.,
mimetics) or as GPCRX antagonists can be identified by screening combinatorial
libraries of
mutants (e.g., truncation mutants) of the GPCRX proteins for GPCRX protein
agonist or
antagonist activity. In one embodiment, a variegated library of GPCRX variants
is generated
by combinatorial mutagenesis at the nucleic acid level and is encoded by a
variegated gene
library. A variegated library of GPCRX variants can be produced by, for
example,
enzymatically ligating a mixture of synthetic oligonucleotides into gene
sequences such that a
degenerate set of potential GPCRX sequences is expressible as individual
polypeptides, or
alternatively, as a set of larger fusion proteins (e.g., for phage display)
containing the set of
GPCRX sequences therein. There are a variety of methods which can be used to
produce
libraries of potential GPCRX variants from a degenerate oligonucleotide
sequence. Chemical
synthesis of a degenerate gene sequence can be performed in an automatic DNA
synthesizer,
and the synthetic gene then ligated into an appropriate expression vector. Use
of a degenerate
set of genes allows for the provision, in one mixture, of all of the sequences
encoding the
desired set of potential GPCRX sequences. Methods for synthesizing degenerate
oligonucleotides are well-known within the art. See, e.g., Narang, 1983.
Tetrahedron 39: 3;
Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al., 1984.
Science 198: 1056;
Ike, et al., 1983. Nucl. Acids Res. 11: 477.
Polypeptide Libraries
In addition, libraries of fragments of the GPCRX protein coding sequences can
be used
to generate a variegated population of GPCRX fragments for screening and
subsequent
selection of variants of an GPCRX protein. In one embodiment, a library of
coding sequence
fragments can be generated by treating a double stranded PCR fragment of an
GPCRX coding
sequence with a nuclease under conditions wherein nicking occurs only about
once per
molecule, denaturing the double stranded DNA, renaturing the DNA to form
double-stranded
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DNA that can include sense/antisense pairs from different nicked products,
removing single
stranded portions from reformed duplexes by treatment with S ~ nuclease, and
ligating the
resulting fragment library into an expression vector. By this method,
expression libraries can
be derived which encodes N-terminal and internal fragments of various sizes of
the GPCRX
proteins.
Various techniques are known in the art for screening gene products of
combinatorial
libraries made by point mutations or truncation, and for screening cDNA
libraries for gene
products having a selected property. Such techniques are adaptable for rapid
screening of the
gene libraries generated by the combinatorial mutagenesis of GPCRX proteins.
The most
widely used techniques, which are amenable to high throughput analysis, for
screening large
gene libraries typically include cloning the gene library into replicable
expression vectors,
transforming appropriate cells with the resulting library of vectors, and
expressing the
combinatorial genes under conditions in which detection of a desired activity
facilitates
isolation of the vector encoding the gene whose product was detected.
Recursive ensemble
mutagenesis (REM), a new technique that enhances the frequency of functional
mutants in the
libraries, can be used in combination with the screening assays to identify
GPCRX variants.
See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815;
Delgrave, et
al., 1993. Protein Engineering 6:327-331.
Anti-GPCRX Antibodies
The invention encompasses antibodies and antibody fragments, such as Fab or
(Fab)2,
that bind immunospecifically to any of the GPCRX polypeptides of said
invention.
An isolated GPCRX protein, or a portion or fragment thereof, can be used as an
immunogen to generate antibodies that bind to GPCRX polypeptides using
standard
techniques for polyclonal and monoclonal antibody preparation. The full-length
GPCRX
proteins can be used or, alternatively, the invention provides antigenic
peptide fragments of
GPCRX proteins for use as immunogens. The antigenic GPCRX peptides comprises
at least 4
amino acid residues of the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8,
10, 12, 17,
19, 21, 23, 25, 29, 31, 33, 35, 37, and 83 and encompasses an epitope of
GPCItX such that an
antibody raised against the peptide forms a specific immune complex with
GPCItX.
Preferably, the antigenic peptide comprises at least 6, 8, 10, 15, 20, or 30
amino acid residues.
Longer antigenic peptides are sometimes preferable over shorter antigenic
peptides, depending
on use and according to methods well known to someone skilled in the art.
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In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a region of GPCRX that is located on the surface of the
protein (e.g., a
hydrophilic region). As a means for targeting antibody production, hydropathy
plots showing
regions of hydrophilicity and hydrophobicity may be generated by any method
well known in
the art, including, for example, the Kyte Doolittle or the Hopp Woods methods,
either with or
without Fourier transformation (see, e.g., Hopp and Woods, 1981. Proc. Nat.
Acad. Sci. USA
78: 3824-3828; Kyte and Doolittle, 1982. f Mol. Biol. 157: 105-142, each
incorporated herein
by reference in their entirety).
As disclosed herein, GPCRX protein sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12,
17,
19, 21, 23, 25, 29, 31, 33, 35, 37, and 83, or derivatives, fragments, analogs
or homologs
thereof, may be utilized as immunogens in the generation of antibodies that
immunospecifically-bind these protein components. The term "antibody" as used
herein refers
to immunoglobulin molecules and immunologically-active portions of
immunoglobulin
molecules, i. e., molecules that contain an antigen binding site that
specifically-binds
(immunoreacts with) an antigen, such as GPCRX. Such antibodies include, but
are not limited
to, polyclonal, monoclonal, chimeric, single chain, Fab and F~ab~~z fragments,
and an Fab
expression library. In a specific embodiment, antibodies to human GPCRX
proteins are
disclosed. Various procedures known within the art may be used for the
production of
polyclonal or monoclonal antibodies to an GPCRX protein sequence of SEQ ID
NOS:2, 4, 6,
8, 10, 12, 17, 19, 21, 23, 25, 29, 31, 33, 35, 37, and 83, or a derivative,
fragment, analog or
homolog thereof. Some of these proteins are discussed below.
For the production of polyclonal antibodies, various suitable host animals
(e.g., rabbit,
goat, mouse or other mammal) may be immunized by injection with the native
protein, or a
synthetic variant thereof, or a derivative of the foregoing. An appropriate
immunogenic
preparation can contain, for example, recombinantly-expressed GPCRX protein or
a
chemically-synthesized GPCRX polypeptide. The preparation can further include
an adjuvant.
Various adjuvants used to increase the immunological response include, but are
not limited to,
Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide),
surface active
substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions,
dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and
Corynebacterium
parvum, or similar immunostimulatory agents. If desired, the antibody
molecules directed
against GPCRX can be isolated from the mammal (e.g., from the blood) and
further purified
by well known techniques, such as protein A chromatography to obtain the IgG
fraction.
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The term "monoclonal antibody" or "monoclonal antibody composition", as used
herein, refers to a population of antibody molecules that contain only one
species of an antigen
binding site capable of immunoreacting with a particular epitope of GPCRX. A
monoclonal
antibody composition thus typically displays a single binding affinity for a
particular GPCRX
protein with which it immunoreacts. For preparation of monoclonal antibodies
directed
towards a particular GPCRX protein, or derivatives, fragments, analogs or
homologs thereof,
any technique that provides for the production of antibody molecules by
continuous cell line
culture may be utilized. Such techniques include, but are not limited to, the
hybridoma
technique (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the
trioma technique; the
human B-cell hybridoma technique (see, e.g., Kozbor, et al., 1983. Immunol.
Today 4: 72) and
the EBV hybridoma technique to produce human monoclonal antibodies (see, e.g.,
Cole, et al.,
1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-
96).
Human monoclonal antibodies may be utilized in the practice of the invention
and may be
produced by using human hybridomas (see, e.g., Cote, et al., 1983. Proc Natl
Acad Sci USA
80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in
vitro (see, e.g.,
Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.
Liss, Inc.,
pp. 77-96). Each of the above citations is incorporated herein by reference in
their entirety.
According to the invention, techniques can be adapted for the production of
single-chain antibodies specific to an GPCRX protein (see, e.g., U.S. Patent
No. 4,946,778).
In addition, methods can be adapted for the construction of Fab expression
libraries (see, e.g.,
Huse, et al., 1989. Science 246: 1275-1281) to allow rapid and effective
identification of
monoclonal Fab fragments with the desired specificity for an GPCRX protein or
derivatives,
fragments, analogs or homologs thereof. Non-human antibodies can be
"humanized" by
techniques well known in the art. See, e.g., U.S. Patent No. 5,225,539.
Antibody fragments
that contain the idiotypes to an GPCRX protein may be produced by techniques
known in the
art including, but not limited to: (i) an F~ab')z fragment produced by pepsin
digestion of an
antibody molecule; (ii) an Fab fragment generated by reducing the disulfide
bridges of an F~ab')z
fragment; (iii) an Fab fragment generated by the treatment of the antibody
molecule with
papain and a reducing agent; and (iv) F,, fragments.
Additionally, recombinant anti-GPCRX antibodies, such as chimeric and
humanized
monoclonal antibodies, comprising both human and non-human portions, which can
be made
using standard recombinant DNA techniques, are within the scope of the
invention. Such
chimeric and humanized monoclonal antibodies can be produced by recombinant
DNA
techniques known in the art, for example using methods described in
International Application
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No. PCT/US86/02269; European Patent Application No. 184,187; European Patent
Application No. 171,496; European Patent Application No. 173,494; PCT
International
Publication No. WO 86/01533; U.S. Patent No. 4,816,567; U.S. Pat. No.
5,225,539; European
Patent Application No. 125,023; Better, et al., 1988. Science 240: 1041-1043;
Liu, et al., 1987.
Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139:
3521-3526; Sun,
et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987.
Cancer Res. 47:
999-1005; Wood, et al., 1985. Nature 314 :446-449; Shaw, et al., 1988. J.
Natl. Cancer Inst.
80: 1553-1559); Mornson(1985) Science 229:1202-1207; Oi, et al. (1986)
BioTechniques
4:214; Jones, et al., 1986. Nature 321: 552-525; Verhoeyan, et al., 1988.
Science 239: 1534;
and Beidler, et al., 1988. J. Immunol. 141: 4053-4060. Each of the above
citations are
incorporated herein by reference in their entirety.
In one embodiment, methods for the screening of antibodies that possess the
desired
specificity include, but are not limited to, enzyme-linked immunosorbent assay
(ELISA) and
other immunologically-mediated techniques known within the art. In a specific
embodiment,
selection of antibodies that are specific to a particular domain of an GPCRX
protein is
facilitated by generation of hybridomas that bind to the fragment of an GPCRX
protein
possessing such a domain. Thus, antibodies that are specific for a desired
domain within an
GPCRX protein, or derivatives, fragments, analogs or homologs thereof, are
also provided
herein.
Anti-GPCRX antibodies may be used in methods known within the art relating to
the
localization and/or quantitation of an GPCRX protein (e.g., for use in
measuring levels of the
GPCRX protein within appropriate physiological samples, for use in diagnostic
methods, for
use in imaging the protein, and the like). In a given embodiment, antibodies
for GPCRX
proteins, or derivatives, fragments, analogs or homologs thereof, that contain
the antibody
derived binding domain, are utilized as pharmacologically-active compounds
(hereinafter
"Therapeutics").
An anti-GPCRX antibody (e.g., monoclonal antibody) can be used to isolate an
GPCRX polypeptide by standard techniques, such as affinity chromatography or
immunoprecipitation. An anti-GPCRX antibody can facilitate the purification of
natural
GPCRX polypeptide from cells and of recombinantly-produced GPCRX polypeptide
expressed in host cells. Moreover, an anti-GPCRX antibody can be used to
detect GPCRX
protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate
the abundance and
pattern of expression of the GPCRX protein. Anti-GPCRX antibodies can be used
diagnostically to monitor protein levels in tissue as part of a clinical
testing procedure, e.g., to,
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for example, determine the efficacy of a given treatment regimen. Detection
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
streptavidin/biotin and avidin/biotin; 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 l2sh 13~I, 3sS or 3H.
GPCRX Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors,
containing a nucleic acid encoding an GPCRX protein, or derivatives,
fragments, analogs or
homologs thereof. As used herein, the term "vector" refers to a nucleic acid
molecule capable
of transporting another nucleic acid to which it has been linked. One type of
vector is a
"plasmid", which refers to a circular double stranded DNA loop into which
additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein
additional DNA
segments can be ligated into the viral genome. Certain vectors are capable of
autonomous
replication in a host cell into which they are introduced (e.g., bacterial
vectors having a
bacterial origin of replication and episomal mammalian vectors). Other vectors
(e.g.,
non-episomal mammalian vectors) are integrated into the genome of a host cell
upon
introduction into the host cell, and thereby are replicated along with the
host genome.
Moreover, certain vectors are capable of directing the expression of genes to
which they are
operatively-linked. Such vectors are referred to herein as "expression
vectors". In general,
expression vectors of utility in recombinant DNA techniques are often in the
form of plasmids.
In the present specification, "plasmid" and "vector" can be used
interchangeably as the
plasmid is the most commonly used form of vector. However, the invention is
intended to
include such other forms of expression vectors, such as viral vectors (e.g.,
replication defective
retroviruses, adenoviruses and adeno-associated viruses), which serve
equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the
invention in a form suitable for expression of the nucleic acid in a host
cell, which means that
the recombinant expression vectors include one or more regulatory sequences,
selected on the
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basis of the host cells to be used for expression, that is operatively-linked
to the nucleic acid
sequence to be expressed. Within a recombinant expression vector, "operably-
linked" is
intended to mean that the nucleotide sequence of interest is linked to the
regulatory
sequences) in a manner that allows for expression of the nucleotide sequence
(e.g., in an in
S vitro transcription/translation system or in a host cell when the vector is
introduced into the
host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers
and other
expression control elements (e.g., polyadenylation signals). Such regulatory
sequences are
described, for example, iri Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
1O ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory
sequences include
those that direct constitutive expression of a nucleotide sequence in many
types of host cell
and those that direct expression of the nucleotide sequence only in certain
host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by those skilled
in the art that the
design of the expression vector can.depend on such factors as the choice of
the host cell to be
15 transformed, the level of expression of protein desired, etc. The
expression vectors of the
invention can be introduced into host cells to thereby produce proteins or
peptides, including
fusion proteins or peptides, encoded by nucleic acids as described herein
(e.g., GPCRX
proteins, mutant forms of GPCRX proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression of
20 GPCRX proteins in prokaryotic or eukaryotic cells. For example, GPCRX
proteins can be
expressed in bacterial cells such as Escherichia coli, insect cells (using
baculovirus expression
vectors) yeast cells or mammalian cells. Suitable host cells are discussed
further in Goeddel,
GENE EXPRESSTON TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San
Diego, Calif. (1990). Alternatively, the recombinant expression vector can be
transcribed and
25 translated in vitro, for example using T7 promoter regulatory sequences and
T7 polymerise.
Expression of proteins in prokaryotes is most often carried out in Escherichia
coli with
vectors containing constitutive or inducible promoters directing the
expression of either fusion
or non-fusion proteins. Fusion vectors add a number of amino acids to a
protein encoded
therein, usually to the amino terminus of the recombinant protein. Such fusion
vectors
30 typically serve three purposes: (i) to increase expression of recombinant
protein; (ii) to
increase the solubility of the recombinant protein; and (iii) to aid in the
purification of the
recombinant protein by acting as a ligand in affinity purification. Often, in
fusion expression
vectors, a proteolytic cleavage site is introduced at the junction of the
fusion moiety and the
recombinant protein to enable separation of the recombinant protein from the
fusion moiety
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subsequent to purification of the fusion protein. Such enzymes, and their
cognate recognition
sequences, include Factor Xa, thrombin and enterokinase. Typical fusion
expression vectors
include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40),
pMAL
(New England Biolabs, Beverly, Mass.) and pRIT$ (Pharmacia, Piscataway, N.J.)
that fuse
$ glutathione S-transferase (GST), maltose E binding protein, or protein A,
respectively, to the
target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc
(Amrann et al., (1988) Gene 69:301-31$) and pET l 1d (Studier et al., GENE
EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 18$, Academic Press, Sari Diego, Calif.
(1990)
60-89).
One strategy to maximize recombinant protein expression in E. coli is to
express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the recombinant
protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY
18$, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to
alter the
1$ nucleic acid sequence of the nucleic acid to be inserted into an expression
vector so that the
individual codons for each amino acid are those preferentially utilized in E.
coli (see, e.g.,
Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of
nucleic acid
sequences of the invention can be carned out by standard DNA synthesis
techniques.
In another embodiment, the GPCRX expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast Saccharomyces cerivisae include
pYepSecl
(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz,
1982. Cell 30:
933-943), pJRY88 (Schultz et al., 1987. Gene $4: 113-123), pYES2 (Invitrogen
Corporation,
San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, GPCRX can be expressed in insect cells using baculovirus
expression
2$ vectors. Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g.,
SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:
21$6-216$) and the
pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian
cells using a mammalian expression vector. Examples of mammalian expression
vectors
include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987.
EMBO
J. 6: 187-19$). When used in mammalian cells, the expression vector's control
functions are
often provided by viral regulatory elements. For example, commonly used
promoters are
derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For
other suitable
expression systems for both prokaryotic and eukaryotic cells see, e.g.,
Chapters 16 and 17 of
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Sambrook, et al., MOLECULAR CLONING: A LABORATORY MArrUAt,. 2nd ed., Cold
Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable
of
directing expression of the nucleic acid preferentially in a particular cell
type (e.g.,
tissue-specific regulatory elements are used to express the nucleic acid).
Tissue-specific
regulatory elements are known in the art. Non-limiting examples of suitable
tissue-specific
promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987.
Genes Dev. 1:
268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol.
43:
235-275), in particular promoters of T cell receptors (Winoto and Baltimore,
1989. EMBO J.
8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740;
Queen and
Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the
neurofilament
promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477),
pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and
mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316
and European
Application Publication No. 264,166). Developmentally-regulated promoters are
also
encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science
249: 374-379)
and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-
546).
The invention further provides a recombinant expression vector comprising a
DNA
molecule of the invention cloned into the expression vector in an antisense
orientation. That
is, the DNA molecule is operatively-linked to a regulatory sequence in a
manner that allows
for expression (by transcription of the DNA molecule) of an RNA molecule that
is antisense to
GPCRX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned
in the
antisense orientation can be chosen that direct the continuous expression of
the antisense RNA
molecule in a variety of cell types, for instance viral promoters and/or
enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific or cell type
specific expression
of antisense RNA. The antisense expression vector can be in the form of a
recombinant
plasmid, phagemid or attenuated virus in which antisense nucleic acids are
produced under the
control of a high efficiency regulatory region, the activity of which can be
determined by the
cell type into which the vector is introduced. For a discussion of the
regulation of gene
expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA
as a molecular
tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms "host cell"
and
"recombinant host cell" are used interchangeably herein. It is understood that
such terms refer
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not only to the particular subject cell but also to the progeny or potential
progeny of such a
cell. Because certain modifications may occur in succeeding generations due to
either
mutation or environmental influences, such progeny may not, in fact, be
identical to the parent
cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, GPCRX
protein
can be expressed in bacterial cells such as E. coli, insect cells, yeast or
mammalian cells (such
as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to
those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional
transformation or transfection techniques. As used herein, the terms
"transformation" and
"transfection" are intended to refer to a variety of art-recognized techniques
for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate
or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting host cells
can be found in
Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989),
and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may integrate
the foreign DNA into their genome. In order to identify and select these
integrants, a gene that
encodes a selectable marker (e.g., resistance to antibiotics) is generally
introduced into the
host cells along with the gene of interest. Various selectable markers include
those that confer
resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acid
encoding a
selectable marker can be introduced into a host cell on the same vector as
that encoding
GPCRX or can be introduced on a separate vector. Cells stably transfected with
the
introduced nucleic acid can be identified by drug selection (e.g., cells that
have incorporated
the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can
be used to produce (i.e., express) GPCRX protein. Accordingly, the invention
further provides
methods for producing GPCRX protein using the host cells of the invention. In
one
embodiment, the method comprises culturing the host cell of invention (into
which a
recombinant expression vector encoding GPCRX protein has been introduced) in a
suitable
medium such that GPCRX protein is produced. In another embodiment, the method
further
comprises isolating GPCRX protein from the medium or the host cell.
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Transgenic GPCRX Animals
The host cells of the invention can also be used to produce non-human
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized oocyte or
an embryonic stem cell into which GPCItX protein-coding sequences have been
introduced.
Such host cells can then be used to create non-human transgenic animals in
which exogenous
GPCRX sequences have been introduced into their genome or homologous
recombinant
animals in which endogenous GPCRX sequences have been altered. Such animals
are useful
for studying the function and/or activity of GPCRX protein and for identifying
and/or
evaluating modulators of GPCRX protein activity. As used herein, a "transgenic
animal" is a
non-human animal, preferably a mammal, more preferably a rodent such as a rat
or mouse, in
which one or more of the cells of the animal includes a transgene. Other
examples of
transgenic animals include non-human primates, sheep, dogs, cows, goats,
chickens,
amphibians, etc. A transgene is exogenous DNA that is integrated into the
genome of a cell
from which a transgenic animal develops and that remains in the genome of the
mature
animal, thereby directing the expression of an encoded gene product in one or
more cell types
or tissues of the transgenic animal. As used herein, a "homologous recombinant
animal" is a
non-human animal, preferably a mammal, more preferably a mouse, in which an
endogenous
GPCItX gene has been altered by homologous recombination between the
endogenous gene
and an exogenous DNA molecule introduced into a cell of the animal, e.g., an
embryonic cell
of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing GPCRX-
encoding
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. The
human GPCRX cDNA sequences of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 16, 18, 20,
22, 24, 28,
30, 32, 34, 36, and 38 can be introduced as a transgene into the genome of a
non-human
animal. Alternatively, a non-human homologue of the human GPCRX gene, such as
a mouse
GPC1RX gene, can be isolated based on hybridization to the human GPC1RX cDNA
(described
further supra) and used as a transgene. Intronic sequences and polyadenylation
signals can
also be included in the transgene to increase the efficiency of expression of
the transgene. A
tissue-specific regulatory sequences) can be operably-linked to the GPCRX
transgene to
direct expression of GPCRX 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
Nos. 4,736,866;
4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING T~ MotlsE EMBRYO,
Cold
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Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are
used for
production of other transgenic animals. A transgenic founder animal can be
identified based
upon the presence of the GPCRX transgene in its genome and/or expression of
GPCRX
mRNA in tissues or cells of the animals. A transgenic founder animal can then
be used to
breed additional animals carrying the transgene. Moreover, transgenic animals
carrying a
transgene-encoding GPCRX protein can further be bred to other transgenic
animals carrying
other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains
at
least a portion of an GPCRX gene into which a deletion, addition or
substitution has been
introduced to thereby alter, e.g., functionally disrupt, the GPCRX gene. The
GPCRX gene can
be a human gene (e.g., the cDNA of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 16, 18,
20, 22, 24, 28,
30, 32, 34, 36, and 38), but more preferably, is a non-human homologue of a
human GPCRX
gene. For example, a mouse homologue of human GPCRX gene of SEQ >D NOS:1, 3,
5, 7, 9,
11, 13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38 can be used to
construct a homologous
recombination vector suitable for altering an endogenous GPCRX gene in the
mouse genome.
In one embodiment, the vector is designed such that, upon homologous
recombination, the
endogenous GPCRX gene is functionally disrupted (i. e., no longer encodes a
functional
protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous
recombination,
the endogenous GPCRX gene is mutated or otherwise altered but still encodes
functional
protein (e.g., the upstream regulatory region can be altered to thereby alter
the expression of
the endogenous GPCRX protein). In the homologous recombination vector, the
altered
portion of the GPCRX gene is flanked at its S'- and 3'-termini by additional
nucleic acid of the
GPCRX gene to allow for homologous recombination to occur between the
exogenous
GPCRX gene carried by the vector and an endogenous GPCRX gene in an embryonic
stem
cell. The additional flanking GPCRX nucleic acid is of sufficient length for
successful
homologous recombination with the endogenous gene. Typically, several
kilobases of
flanking DNA (both at the 5'- and 3'-termini) are included in the vector. See,
e.g., Thomas, et
al., 1987. Cell 51: 503 for a description of homologous recombination vectors.
The vector is
ten introduced into an embryonic stem cell line (e.g., by electroporation) and
cells in which the
introduced GPCRX gene has homologously-recombined with the endogenous GPCRX
gene
are selected. See, e.g., Li, et al., 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a
mouse) to
form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND
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EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152.
A chimeric embryo can then be implanted into a suitable pseudopregnant female
foster animal
and the embryo brought to term. Progeny harboring the homologously-recombined
DNA in
their germ cells can be used to breed animals in which all cells of the animal
contain the
homologously-recombined DNA by germline transmission of the transgene. Methods
for
constructing homologous recombination vectors and homologous recombinant
animals are
described further in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCT
International
Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that
contain
selected systems that allow for regulated expression of the transgene. One
example of such a
system is the cre/loxP recombinase system ofbacteriophage P1. For a
description of the
cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Proc. Natl. Acad.
Sci. USA 89:
6232-6236. Another example of a recombinase system is the FLP recombinase
system of
Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science 251:1351-1355.
If a cre/loxP
recombinase system is used to regulate expression of the transgene, animals
containing
transgenes encoding both the Cre recombinase and a selected protein are
required. Such
animals can be provided through the construction 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, et al., 1997. Nature 385: 810-
813. 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 Go phase. The quiescent cell can then be fused, e.g.,
through 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 blastocyte and then transferred to pseudopregnant female foster
animal. The
offspring borne of this female foster animal will be a clone of the animal
from which the cell
(e.g., the somatic cell) is isolated.
Pharmaceutical Compositions
The GPCRX nucleic acid molecules, GPCRX proteins, and anti-GPCRX antibodies
(also referred to herein as "active compounds") of the invention, and
derivatives, fragments,
analogs and homologs thereof, can be incorporated into pharmaceutical
compositions suitable
for administration. Such compositions typically comprise the nucleic acid
molecule, protein,
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or antibody and a pharmaceutically acceptable Garner. As used herein,
"pharmaceutically
acceptable Garner" is intended to include any and all solvents, dispersion
media, coatings,
antibacterial and antifungal agents, isotonic and absorption delaying agents,
and the like,
compatible with pharmaceutical administration. Suitable carriers are described
in the most
recent edition of Remington's Pharmaceutical Sciences, a standard reference
text in the field,
which is incorporated herein by reference. Preferred examples of such carriers
or diluents
include, but are not limited to, water, saline, finger's solutions, dextrose
solution, and S%
human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may
also be
used. The use of such media and agents for pharmaceutically active substances
is well known
in the art. Except insofar as any conventional media or agent is incompatible
with the active
compound, use thereof in the compositions is contemplated. Supplementary
active
compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with its
intended route of administration. Examples of routes of administration include
parenteral,
e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical),
transmucosal, and rectal administration. Solutions or suspensions used for
parenteral,
intradermal, or subcutaneous application can include the following components:
a sterile
diluent such as water for injection, saline solution, fixed oils, polyethylene
glycols, glycerine,
propylene glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;
chelating agents such
as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates
or phosphates,
and agents for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral
preparation can be enclosed in ampoules, disposable syringes or multiple dose
vials made of
glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. For intravenous
administration,
suitable carriers include physiological saline, bacteriostatic water,
Cremophor ELTM (BASF,
Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the
composition must be
sterile and should be fluid to the extent that easy syringeability exists. It
must be stable under
the conditions of manufacture and storage and must be preserved against the
contaminating
action of microorganisms such as bacteria and fungi. The carrier can be a
solvent or
dispersion medium containing, for example, water, ethanol, polyol (for
example, glycerol,
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propylene glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating
such as lecithin, by
the maintenance of the required particle size in the case of dispersion and by
the use of
surfactants. Prevention of the action of microorganisms can be achieved by
various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol, ascorbic
acid, thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents,
for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride
in the
composition. Prolonged absorption of the injectable compositions can be
brought about by
including in the composition an agent which delays absorption, for example,
aluminum
monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound (e.g.,
an GPCRX protein or anti-GPCRX antibody) in the required amount in an
appropriate solvent
with one or a combination of ingredients enumerated above, as required,
followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the active
compound into a
sterile vehicle that contains a basic dispersion medium and the required other
ingredients from
those enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, methods of preparation are vacuum drying and freeze-drying that
yields a powder of
the active ingredient plus any additional desired ingredient from a previously
sterile-filtered
solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can be
enclosed in gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic
administration, the active compound can be incorporated with excipients and
used in the form
of tablets, troches, or capsules. Oral compositions can also be prepared using
a fluid Garner
for use as a mouthwash, wherein the compound in the fluid Garner is applied
orally and
swished and expectorated or swallowed. Pharmaceutically compatible binding
agents, and/or
adjuvant materials can be included as part of the composition. The tablets,
pills, capsules,
troches and the like can contain any of the following ingredients, or
compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient
such as starch or lactose, a disintegrating agent such as alginic acid,
Primogel, or corn starch; a
lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as
peppermint,
methyl salicylate, or orange flavoring.
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For administration by inhalation, the compounds are delivered in the form of
an
aerosol spray from pressured container or dispenser which contains a suitable
propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art, and
include, for example, for transmucosal administration, detergents, bile salts,
and fusidic acid
derivatives. Transmucosal administration can be accomplished through the use
of nasal sprays
or suppositories. For transdermal administration, the active compounds are
formulated into
ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention enemas
for rectal delivery.
In one embodiment, the active compounds are prepared with Garners that will
protect
the compound against rapid elimination from the body, such as a controlled
release
formulation, including implants and microencapsulated delivery systems.
Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for
preparation of
such formulations will be apparent to those skilled in the art. The materials
can also be
obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal
suspensions (including liposomes targeted to infected cells with monoclonal
antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers. These can
be prepared
according to methods known to those skilled in the art, for example, as
described in U.S.
Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in
dosage
unit form for ease of administration and uniformity of dosage. Dosage unit
form as used
herein refers to physically discrete units suited as unitary dosages for the
subject to be treated;
each unit containing a predetermined quantity of active compound calculated to
produce the
desired therapeutic effect in association with the required pharmaceutical
Garner. The
specification for the dosage unit forms of the invention are dictated by and
directly dependent
on the unique characteristics of the active compound and the particular
therapeutic effect to be
achieved, and the limitations inherent in the art of compounding such an
active compound for
the treatment of individuals.
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The nucleic acid molecules of the invention can be inserted into vectors and
used as
gene therapy vectors. Gene therapy vectors can be delivered to a subject by,
for example,
intravenous injection, local administration (see, e.g., U.S. Patent No.
5,328,470) or by
stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci.
USA 91: 3054-3057).
The pharmaceutical preparation of the gene therapy vector can include the gene
therapy vector
in an acceptable diluent, or can comprise a slow release matrix in which the
gene delivery
vehicle is imbedded. Alternatively, where the complete gene delivery vector
can be produced
intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can
include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser
together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules of the invention can be used to express
GPCRX
protein (e.g., via a recombinant expression vector in a host cell in gene
therapy applications),
1 S to detect GPCRX mRNA (e.g., in a biological sample) or a genetic lesion in
an GPC1ZX gene,
and to modulate GPCItX activity, as described further, below. In addition, the
GPCRX
proteins can be used to screen drugs or compounds that modulate the GPCRX
protein activity
or expression as well as to treat disorders characterized by insufficient or
excessive production
of GPC1RX protein or production of GPCRX protein forms that have decreased or
aberrant
activity compared to GPCRX wild-type protein (e.g.; diabetes (regulates
insulin release);
obesity (binds and transport lipids); metabolic disturbances associated with
obesity, the
metabolic syndrome X as well as anorexia and wasting disorders associated with
chronic
diseases and various cancers, and infectious disease(possesses anti-microbial
activity) and the
various dyslipidemias. In addition, the anti-GPCRX antibodies of the invention
can be used to
detect and isolate GPCRX proteins and modulate GPCRX activity. In yet a
further aspect, the
invention can be used in methods to influence appetite, absorption of
nutrients and the
disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening
assays
described herein and uses thereof for treatments as described, supra.
Screening Assays
The invention provides a method (also referred to herein as a "screening
assay") for
identifying modulators, i.e., candidate or test compounds or agents (e.g.,
peptides,
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peptidomimetics, small molecules or other drugs) that bind to GPCRX proteins
or have a
stimulatory or inhibitory effect on, e.g., GPCRX protein expression or GPCRX
protein
activity. The invention also includes compounds identified in the screening
assays described
herein.
In one embodiment, the invention provides assays for screening candidate or
test
compounds which bind to or modulate the activity of the membrane-bound form of
an
GPCRX protein or polypeptide or biologically-active portion thereof. The test
compounds of
the invention can be obtained using any of the numerous approaches in
combinatorial library
methods known in the art, including: biological libraries; spatially
addressable parallel solid
phase or solution phase libraries; synthetic library methods requiring
deconvolution; the
"one-bead one-compound" library method; and synthetic library methods using
affinity
chromatography selection. The biological library approach is limited to
peptide libraries,
while the other four approaches are applicable to peptide, non-peptide
oligomer or small
molecule libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug Design
12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has
a
molecular weight of less than about S kD and most preferably less than about 4
kD. Small
molecules can be, e.g., nucleic acids, peptides, polypeptides,
peptidomimetics, carbohydrates,
lipids or other organic or inorganic molecules. Libraries of chemical and/or
biological
mixtures, such as fungal, bacterial, or algal extracts, are known in the art
and can be screened
with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in
the art,
for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909;
Erb, et al., 1994.
Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med.
Chem. 37: 2678;
Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem. Int.
Ed. Engl. 33:
2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop,
et al., 1994. .l.
Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, .1992.
Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on
chips (Fodor,
1993. Nature 364: S55-556), bacteria (Ladner, U.S. Patent No. 5,223,409),
spores (Ladner,
U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci.
USA 89:
1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin,
1990. Science
249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-
6382; Felici, 1991.
J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
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In one embodiment, an assay is a cell-based assay in which a cell which
expresses a
membrane-bound form of GPCRX protein, or a biologically-active portion
thereof, on the cell
surface is contacted with a test compound and the ability of the test compound
to bind to an
GPCRX protein determined. The cell, for example, can of mammalian origin or a
yeast cell.
Determining the ability of the test compound to bind to the GPCRX protein can
be
accomplished, for example, by coupling the test compound with a radioisotope
or enzymatic
label such that binding of the test compound to the GPCRX protein or
biologically-active
portion thereof can be determined by detecting the labeled compound in a
complex. For
example, test compounds can be labeled with lzsh 3sS, laC, or 3H, either
directly or indirectly,
and the radioisotope detected by direct counting of radioemission or by
scintillation counting.
Alternatively, test compounds can be enzymatically-labeled with, for example,
horseradish
peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label
detected by
determination of conversion of an appropriate substrate to product. In one
embodiment, the
assay comprises contacting a cell which expresses a membrane-bound form of
GPCRX
protein, or a biologically-active portion thereof, on the cell surface with a
known compound
which binds GPCRX to form an assay mixture, contacting the assay mixture with
a test
compound, and determining the ability of the test compound to interact with an
GPCRX
protein, wherein determining the ability of the test compound to interact with
an GPCRX
protein comprises determining the ability of the test compound to
preferentially bind to
GPCRX protein or a biologically-active portion thereof as compared to the
known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell
expressing a membrane-bound form of GPCRX protein, or a biologically-active
portion
thereof, on the cell surface with a test compound and determining the ability
of the test
compound to modulate (e.g., stimulate or inhibit) the activity of the GPCRX
protein or
biologically-active portion thereof. Determining the ability of the test
compound to modulate
the activity of GPCRX or a biologically-active portion thereof can be
accomplished, for
example, by determining the ability of the GPCRX protein to bind to or
interact with an
GPCRX target molecule. As used herein, a "target molecule" is a molecule with
which an
GPCRX protein binds or interacts in nature, for example, a molecule on the
surface of a cell
which expresses an GPCRX interacting protein, a molecule on the surface of a
second cell, a
molecule in the extracellular milieu, a molecule associated with the internal
surface of a cell
membrane or a cytoplasmic molecule. An GPCRX target molecule can be a non-
GPCRX
molecule or an GPCRX protein or polypeptide of the invention. In one
embodiment, an
GPCRX target molecule is a component of a signal transduction pathway that
facilitates
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transduction of an extracellular signal (e.g. a signal generated by binding of
a compound to a
membrane-bound GPCRX molecule) through the cell membrane and into the cell.
The target,
for example, can be a second intercellular protein that has catalytic activity
or a protein that
facilitates the association of downstream signaling molecules with GPCRX.
S Determining the ability of the GPCRX protein to bind to or interact with an
GPCRX
target molecule can be accomplished by one of the methods described above for
determining
direct binding. In one embodiment, determining the ability of the GPCRX
protein to bind to or
interact with an GPCRX target molecule can be accomplished by determining the
activity of
the target molecule. For example, the activity of the target molecule can be
determined by
detecting induction of a cellular second messenger of the target (i.e.
intracellular Ca2+,
diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the
target an appropriate
substrate, detecting the induction of a reporter gene (comprising an GPCRX-
responsive
regulatory element operatively linked to a nucleic acid encoding a detectable
marker, e.g.,
luciferase), or detecting a cellular response, for example, cell survival,
cellular differentiation,
or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay
comprising
contacting an GPCRX protein or biologically-active portion thereof with a test
compound and
determining the ability of the test compound to bind to the GPCRX protein or
biologically-
active portion thereof. Binding of the test compound to the GPCRX protein can
be determined
either directly or indirectly as described above. In one such embodiment, the
assay comprises
contacting the GPCRX protein or biologically-active portion thereof with a
known compound
which binds GPCRX to form an assay mixture, contacting the assay mixture with
a test
compound, and determining the ability of the test compound to interact with an
GPCRX
protein, wherein determining the ability of the test compound to interact with
an GPCRX
protein comprises determining the ability of the test compound to
preferentially bind to
GPCRX or biologically-active portion thereof as compared to the known
compound.
In still another embodiment, an assay is a cell-free assay comprising
contacting
GPCRX protein or biologically-active portion thereof with a test compound and
determining
the ability of the test compound to modulate (e.g. stimulate or inhibit) the
activity of the
GPCRX protein or biologically-active portion thereof. Determining the ability
of the test
compound to modulate the activity of GPCRX can be accomplished, for example,
by
determining the ability of the GPCRX protein to bind to an GPCRX target
molecule by one of
the methods described above for determining direct binding. In an alternative
embodiment,
determining the ability of the test compound to modulate the activity of GPCRX
protein can
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be accomplished by determining the ability of the GPCRX protein further
modulate an
GPCRX target molecule. For example, the catalytic/enzymatic activity of the
target molecule
on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the GPCRX
protein or biologically-active portion thereof with a known compound which
binds GPCRX
protein to form an assay mixture, contacting the assay mixture with a test
compound, and
determining the ability of the test compound to interact with an GPCRX
protein, wherein
determining the ability of the test compound to interact with an GPCRX protein
comprises
determining the ability of the GPCRX protein to preferentially bind to or
modulate the activity
of an GPCRX target molecule.
The cell-free assays of the invention are amenable to use of both the soluble
form or
the membrane-bound form of GPCRX protein. In the case of cell-free assays
comprising the
membrane-bound form of GPCRX protein, it may be desirable to utilize a
solubilizing agent
such that the membrane-bound form of GPCRX protein is maintained in solution.
Examples
of such solubilizing agents include non-ionic detergents such as n-
octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton~ X-100, Triton~ X-114, Thesit~,
Isotridecypoly(ethylene glycol ether)", N-dodecyl--N,N-dimethyl-3-ammonio-1-
propane
sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS),
or
3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it
may be
desirable to immobilize either GPCRX protein or its target molecule to
facilitate separation of
complexed from uncomplexed forms of one or both of the proteins, as well as to
accommodate
automation of the assay. Binding of a test compound to GPCRX protein, or
interaction of
GPCRX protein with a target molecule in the presence and absence of a
candidate compound,
can be accomplished in any vessel suitable for containing the reactants.
Examples of such
vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In
one embodiment, a
fusion protein can be provided that adds a domain that allows one or both of
the proteins to be
bound to a matrix. For example, GST-GPCRX fusion proteins or GST-target fusion
proteins
can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,
MO) or
glutathione derivatized microtiter plates, that are then combined with the
test compound or the
test compound and either the non-adsorbed target protein or GPCRX protein, and
the mixture
is incubated under conditions conducive to complex formation (e.g., at
physiological
conditions for salt and pH). Following incubation, the beads or microtiter
plate wells are
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washed to remove any unbound components, the matrix immobilized in the case of
beads,
complex determined either directly or indirectly, for example, as described,
supra.
Alternatively, the complexes can be dissociated from the matrix, and the level
of GPCRX
protein binding or activity determined using standard techniques.
S Other techniques for immobilizing proteins on matrices can also be used in
the
screening assays of the invention. For example, either the GPCRX protein or
its target
molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated
GPCRX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g.,
biotinylation kit,
Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well
plates (Pierce Chemical). Alternatively, antibodies reactive with GPCRX
protein or target
molecules, but which do not interfere with binding of the GPCRX protein to its
target
molecule, can be derivatized to the wells of the plate, and unbound target or
GPCRX protein
trapped in the wells by antibody conjugation. Methods for detecting such
complexes, in
addition to those described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the GPCRX protein
or target
molecule, as well as enzyme-linked assays that rely on detecting an enzymatic
activity
associated with the GPCRX protein or target molecule.
In another embodiment, modulators of GPCRX protein expression are identified
in a
method wherein a cell is contacted with a candidate compound and the
expression of GPCRX
mRNA or protein in the cell is determined. The level of expression of GPCRX
mRNA or
protein in the presence of the candidate compound is compared to the level of
expression of
GPCRX mRNA or protein in the absence of the candidate compound. The candidate
compound can then be identified as a modulator of GPCRX mRNA or protein
expression
based upon this comparison. For example, when expression of GPCRX mRNA or
protein is
greater (i.e., statistically significantly greater) in the presence of the
candidate compound than
in its absence, the candidate compound is identified as a stimulator of GPCRX
mRNA or
protein expression. Alternatively, when expression of GPCRX mRNA or protein is
less
(statistically significantly less) in the presence of the candidate compound
than in its absence,
the candidate compound is identified as an inhibitor of GPCRX mRNA or protein
expression.
The level of GPCRX mRNA or protein expression in the cells can be determined
by methods
described herein for detecting GPCRX mRNA or protein.
In yet another aspect of the invention, the GPCRX 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;
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Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem.
268: 12046-12054;
Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.
Oncogene 8:
1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or
interact with
GPC1RX ("GPCRX-binding proteins" or "GPCItX-by") and modulate GPCRX activity.
Such
GPCItX-binding proteins are also likely to be involved in the propagation of
signals by the
GPC1RX proteins as, for example, upstream or downstream elements of the GPCRX
pathway.
The two-hybrid system is based on the modular nature of most transcription
factors,
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 GPCRX
is fused to a
gene encoding the DNA binding domain of a known transcription factor (e.g.,
GAL-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, in vivo, forming an GPCRX-dependent complex, the DNA-binding and
activation
domains of the transcription factor are brought into close proximity. This
proximity allows
transcription of a reporter gene (e.g., LacZ) that is operably linked to a
transcriptional
regulatory site responsive to the transcription factor. Expression 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 that encodes the protein which interacts with
GPCRX.
The invention further pertains to novel agents identified by the
aforementioned
screening assays and uses thereof for treatments as described herein.
Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the
corresponding
complete gene sequences) can be used in numerous ways as polynucleotide
reagents. By way
of example, and not of limitation, these sequences can be used to: (i) map
their respective
genes on a chromosome; and, thus, locate gene regions associated with genetic
disease; (ii)
identify an individual from a minute biological sample (tissue typing); and
(iii) aid in forensic
identification of a biological sample. Some of these applications are
described in the
subsections, below.
Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated,
this
sequence can be used to map the location of the gene on a chromosome. This
process is called
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chromosome mapping. Accordingly, portions or fragments of the GPCRX sequences,
SEQ ID
NOS:1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and 38, or
fragments or
derivatives thereof, can be used to map the location of the GPCRX genes,
respectively, on a
chromosome. The mapping of the GPCRX sequences to chromosomes is an important
first
step in correlating these sequences with genes associated with disease.
Briefly, GPCRX genes can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 by in length) from the GPCRX sequences. Computer analysis of
the
GPCRX, sequences can be used to rapidly select primers that do not span more
than one exon
in the genomic DNA, thus complicating the amplification process. These primers
can then be
used for PCR screening of somatic cell hybrids containing individual human
chromosomes.
Only those hybrids containing the human gene corresponding to the GPCRX
sequences will
yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different
mammals
(e.g., human and mouse cells). As hybrids of human and mouse cells grow and
divide, they
gradually lose human chromosomes in random order, but retain the mouse
chromosomes. By
using media in which mouse cells cannot grow, because they lack a particular
enzyme, but in
which human cells can, the one human chromosome that contains the gene
encoding the
needed enzyme will be retained. By using various media, panels of hybrid cell
lines can be
established. Each cell line in a panel contains either a single human
chromosome or a small
number of human chromosomes, and a full set of mouse chromosomes, allowing
easy
mapping of individual genes to specific human chromosomes. See, e.g.,
D'Eustachio, et al.,
1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of
human
chromosomes can also be produced by using human chromosomes with
translocations and
deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular
sequence to a particular chromosome. Three or more sequences can be assigned
per day using
a single thermal cycler. Using the GPCRX sequences to design oligonucleotide
primers, sub-
localization can be achieved with panels of fragments from specific
chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase
chromosomal spread can further be used to provide a precise chromosomal
location in one
step. Chromosome spreads can be made using cells whose division has been
blocked in
metaphase by a chemical like colcemid that disrupts the mitotic spindle. The
chromosomes
can be treated briefly with trypsin, and then stained with Giemsa. A pattern
of light and dark
bands develops on each chromosome, so that the chromosomes can be identified
individually.
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The FISH technique can be used with a DNA sequence as short as S00 or 600
bases.
However, clones larger than 1,000 bases have a higher likelihood of binding to
a unique
chromosomal location with sufficient signal intensity for simple detection.
Preferably 1,000
bases, and more preferably 2,000 bases, will suffice to get good results at a
reasonable amount
of time. For a review of this technique, see, Verma, et al., HUMAN
CIIROI~tosolvlES: A
MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single
chromosome or a single site on that chromosome, or panels of reagents can be
used for
marking multiple sites and/or multiple chromosomes. Reagents corresponding to
noncoding
regions of the genes actually are preferred for mapping purposes. Coding
sequences are more
likely to be conserved within gene families, thus increasing the chance of
cross hybridizations
during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the
physical
position of the sequence on the chromosome can be correlated with genetic map
data. Such
data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-
line
through Johns Hopkins University Welch Medical Library). The relationship
between genes
and disease, mapped to the same chromosomal region, can then be identified
through linkage
analysis (co-inheritance of physically adjacent genes), described in, e.g.,
Egeland, et al., 1987.
Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and
unaffected with a disease associated with the GPCRX gene, can be determined.
If a mutation
is observed in some or all of the affected individuals but not in any
unaffected individuals,
then the mutation is likely to be the causative agent of the particular
disease. Comparison of
affected and unaffected individuals generally involves first looking for
structural alterations in
the chromosomes, such as deletions or translocations that are visible from
chromosome
spreads or detectable using PCR based on that DNA sequence. Ultimately,
complete
sequencing of genes from several individuals can be performed to confirm the
presence of a
mutation and to distinguish mutations from polymorphisms.
Tissue Typing
The GPCRX sequences of the invention can also be used to identify individuals
from
minute biological samples. In this technique, an individual's genomic DNA is
digested with
one or more restriction enzymes, and probed on a Southern blot to yield unique
bands for
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identification. The sequences of the invention are useful as additional DNA
markers for RFLP
("restriction fragment length polyrnorphisms," described in U.S. Patent No.
5,272,057).
Furthermore, the sequences of the invention can be used to provide an
alternative
technique that determines the actual base-by-base DNA sequence of selected
portions of an
individual's genome. Thus, the GPCRX sequences described herein can be used to
prepare
two PCR primers from the 5'- and 3'-termini of the sequences. These primers
can then be used
to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this
manner,
can provide unique individual identifications, as each individual will have a
unique set of such
DNA sequences due to allelic differences. The sequences of the invention can
be used to
obtain such identification sequences from individuals and from tissue. The
GPCRX sequences
of the invention uniquely represent portions of the human genome. Allelic
variation occurs to
some degree in the coding regions of these sequences, and to a greater degree
in the noncoding
regions. It is estimated that allelic variation between individual humans
occurs with a
frequency of about once per each 500 bases. Much of the allelic variation is
due to single
nucleotide polymorphisms (SNPs), which include restriction fragment length
polymorphisms
(RFLPs).
Each of the sequences described herein can, to some degree, be used as a
standard
against which DNA from an individual can be compared for identification
purposes. Because
greater numbers of polymorphisms occur in the noncoding regions, fewer
sequences are
necessary to differentiate individuals. The noncoding sequences can
comfortably provide
positive individual identification with a panel of perhaps 10 to 1,000 primers
that each yield a
noncoding amplified sequence of 100 bases. If predicted coding sequences, such
as those in
SEQ ID NOS:l, 3, S, 7, 9, 11, 13, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, and
38 are used, a
more appropriate number of primers for positive individual identification
would be 500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which
diagnostic
assays, prognostic assays, pharmacogenomics, and monitoring clinical trials
are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly,
one aspect of the invention relates to diagnostic assays for determining GPCRX
protein and/or
nucleic acid expression as well as GPCRX activity, in the context of a
biological sample (e.g.,
blood, serum, cells, tissue) to thereby determine whether an individual is
afflicted with a
disease or. disorder, or is at risk of developing a disorder, associated with
aberrant GPCRX
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expression or activity. The disorders include metabolic disorders, diabetes,
obesity, infectious
disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative
disorders,
Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic
disorders,
and the various dyslipidemias, metabolic disturbances associated with obesity,
the metabolic
syndrome X and wasting disorders associated with chronic diseases and various
cancers. The
invention also provides for prognostic (or predictive) assays for determining
whether an
individual is at risk of developing a disorder associated with GPCRX protein,
nucleic acid
expression or activity. For example, mutations in an GPC1ZX gene can be
assayed in a
biological sample. Such assays can be used for prognostic or predictive
purpose to thereby
prophylactically treat an individual prior to the onset of a disorder
characterized by or
associated with GPC1RX protein, nucleic acid expression, or biological
activity.
Another aspect of the invention provides methods for determining GPC1RX
protein,
nucleic acid expression or activity in an individual to thereby select
appropriate therapeutic or
prophylactic agents for that individual (referred to herein as
"pharmacogenomics").
Pharmacogenomics allows for the selection of agents (e.g., drugs) for
therapeutic or
prophylactic treatment of an individual based on the genotype of the
individual (e.g., the
genotype of the individual examined to determine the ability of the individual
to respond to a
particular agent.)
Yet another aspect of the invention pertains to monitoring the influence of
agents (e.g.,
drugs, compounds) on the expression or activity of GPC1RX in clinical trials.
These and other agents are described in further detail in the following
sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of GPCRX in a
biological
sample involves obtaining a biological sample from a test subject and
contacting the biological
sample with a compound or an agent capable of detecting GPC1RX protein or
nucleic acid
(e.g., mIRNA, genomic DNA) that encodes GPCRX protein such that the presence
of GPCRX
is detected in the biological sample. An agent for detecting GPC1ZX mltNA or
genomic DNA
is a labeled nucleic acid probe capable of hybridizing to GPC1R.X mRNA or
genomic DNA.
The nucleic acid probe can be, for example, a full-length GPC1RX nucleic acid,
such as the
nucleic acid of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 16, 18, 20, 22, 24, 28, 30,
32, 34, 36, and 38,
or a portion thereof, such as an oligonucleotide of at least 1 S, 30, 50, 100,
250 or 500
nucleotides in length and sufficient to specifically hybridize under stringent
conditions to
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GPCRX mRNA or genomic DNA. Other suitable probes for use in the diagnostic
assays of
the invention are described herein.
An agent for detecting GPCRX protein is an antibody capable of binding to
GPCRX
protein, preferably an antibody with a detectable label. Antibodies can be
polyclonal, or more
preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab
or F(ab')2) can be
used. The term "labeled", with regard to the probe or antibody, is intended to
encompass
direct labeling of the probe or antibody by coupling (i.e., physically
linking) a detectable
substance to the probe or antibody, as well as indirect labeling of the probe
or antibody by
reactivity with another reagent that is directly labeled. Examples of indirect
labeling include
detection of a primary antibody using a fluorescently-labeled secondary
antibody and
end-labeling of a DNA probe with biotin such that it can be detected with
fluorescently-
labeled streptavidin. The term "biological sample" is intended to include
tissues, cells and
biological fluids isolated from a subject, as well as tissues, cells and
fluids present within a
subject. That is, the detection method of the invention can be used to detect
GPCRX mRNA,
protein, or genomic DNA in a biological sample in vitro as well as in vivo.
For example, in
vitro techniques for detection of GPCRX mRNA include Northern hybridizations
and in situ
hybridizations. In vitro techniques for detection of GPCRX protein include
enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and
immunofluorescence. In vitro techniques for detection of GPCRX genomic DNA
include
Southern hybridizations. Furthermore, in vivo techniques for detection of
GPCRX protein
include introducing into a subject a labeled anti-GPCRX antibody. 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.
In one embodiment, the biological sample contains protein molecules from the
test
subject. Alternatively, the biological sample can contain mRNA molecules from
the test
subject or genomic DNA molecules from the test subject. A preferred biological
sample is a
peripheral blood leukocyte sample isolated by conventional means from a
subject.
In another embodiment, the methods further involve obtaining a control
biological
sample from a control subject, contacting the control sample with a compound
or agent
capable of detecting GPCRX protein, mRNA, or genomic DNA, such that the
presence of
GPCRX protein, mRNA or genomic DNA is detected in the biological sample, and
comparing
the presence of GPCRX protein, mRNA or genomic DNA in the control sample with
the
presence of GPCRX protein, mRNA or genomic DNA in the test sample.
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The invention also encompasses kits for detecting the presence of GPC1RX in a
biological sample. For example, the kit can comprise: a labeled compound or
agent capable of
detecting GPCIRX protein or mRNA in a biological sample; means for determining
the amount
of GPCRX in the sample; and means for comparing the amount of GPCRX 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 GPCItX protein or
nucleic acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
identify
subjects having or at risk of developing a disease or disorder associated with
aberrant GPCRX
expression or activity. For example, the assays described herein, such as the
preceding
diagnostic assays or the following assays, can be utilized to identify a
subject having or at risk
of developing a disorder associated with GPCItX protein, nucleic acid
expression or activity.
Alternatively, the prognostic assays can be utilized to identify a subject
having or at risk for
developing a disease or disorder. Thus, the invention provides a method for
identifying a
disease or disorder associated with aberrant GPCRX expression or activity in
which a test
sample is obtained from a subject and GPCRX protein or nucleic acid (e.g.,
mRNA, genomic
DNA) is detected, wherein the presence of GPC1RX protein or nucleic acid is
diagnostic for a
subject having or at risk of developing a disease or disorder associated with
aberrant GPCRX
expression or activity. As used herein, a "test sample" refers to a biological
sample obtained
from a subject of interest. For example, a test sample can be a biological
fluid (e.g., serum),
cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine
whether
a subject can be administered an agent (e.g., an agonist, antagonist,
peptidomimetic, protein,
peptide, nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder
associated with aberrant GPCRX expression or activity. For example, such
methods can be
used to determine whether a subject can be effectively treated with an agent
for a disorder.
Thus, the invention provides methods for determining whether a subject can be
effectively
treated with an agent for a disorder associated with aberrant GPCItX
expression or activity in
which a test sample is obtained and GPCItX protein or nucleic acid is detected
(e.g., wherein
the presence of GPC1RX protein or nucleic acid is diagnostic for a subject
that can be
administered the agent to treat a disorder associated with aberrant GPC1RX
expression or
activity).
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The methods of the invention can also be used to detect genetic lesions in an
GPCRX
gene, thereby determining if a subject with the lesioned gene is at risk for a
disorder
characterized by aberrant cell proliferation and/or differentiation. In
various embodiments, the
methods include detecting, in a sample of cells from the subject, the presence
or absence of a
genetic lesion characterized by at least one of an alteration affecting the
integrity of a gene
encoding an GPCRX-protein, or the misexpression of the GPCRX gene. For
example, such
genetic lesions can be detected by ascertaining the existence of at least one
of: (i) a deletion of
one or more nucleotides from an GPCRX gene; (ii) an addition of one or more
nucleotides to
an GPCRX gene; (iii) a substitution of one or more nucleotides of an GPCRX
gene, (iv) a
chromosomal rearrangement of an GPCRX gene; (v) an alteration in the level of
a messenger
RNA transcript of an GPCRX gene, (vi) aberrant modification of an GPCRX gene,
such as of
the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-
type splicing
pattern of a messenger RNA transcript of an GPCRX gene, (viii) a non-wild-type
level of an
GPCRX protein, (ix) allelic loss of an GPCRX gene, and (x) inappropriate post-
translational
modification of an GPCRX protein. As described herein, there are a large
number of assay
techniques known in the art which can be used for detecting lesions in an
GPCRX gene. A
preferred biological sample is a peripheral blood leukocyte sample isolated by
conventional
means from a subject. However, any biological sample containing nucleated
cells may be
used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a
probe/primer in a
polymerise 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, et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994.
Proc. Natl.
Acid. Sci. USA 91: 360-364), the latter of which can be particularly useful
for detecting point
mutations in the GPCRX-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23:
675-682).
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 more primers that specifically hybridize to an
GPCRX gene
under conditions such that hybridization and amplification of the GPCRX 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. It
is anticipated
that PCR and/or LCR may be desirable to use as a preliminary amplification
step in
conjunction with any of the techniques used for detecting mutations described
herein.
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Alternative amplification methods include: self sustained sequence replication
(see,
Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878),
transcriptional amplification
system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177);
Q[3 Replicase
(see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid
amplification
method, followed by the detection of the amplified molecules using techniques
well known to
those of skill in the art. These detection schemes are especially useful for
the detection of
nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an GPCRX gene from a sample cell
can be
identified by alterations in restriction enzyme cleavage patterns. For
example, sample and
control DNA is isolated, amplified (optionally), digested with one or more
restriction
endonucleases, and fragment length sizes are determined by gel electrophoresis
and compared.
Differences in fragment length sizes between sample and control DNA indicates
mutations in
the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g.,
U.S. Patent
No. 5,493,531) can be used to score for the presence of specific mutations by
development or
loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in GPCRX can be identified by
hybridizing a
sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays
containing
hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al.,
1996. Human
Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example,
genetic
mutations in GPCRX can be identified in two dimensional arrays containing
light-generated
DNA probes as described in Cronin, et al., supra. Briefly, a first
hybridization array of probes
can be used to scan through long stretches of DNA in a sample and control to
identify base
changes between the sequences by making linear arrays of sequential
overlapping probes.
This step allows the identification of point mutations. This is followed by a
second
hybridization array that allows the characterization of specific mutations by
using smaller,
specialized probe arrays complementary to all variants or mutations detected.
Each mutation
array is composed of parallel probe sets, one complementary to the wild-type
gene and the
other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in
the art
can be used to directly sequence the GPCRX gene and detect mutations by
comparing the
sequence of the sample GPCRX with the corresponding wild-type (control)
sequence.
Examples of sequencing reactions include those based on techniques developed
by Maxim and
Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl.
Acad. Sci. USA
74: 5463. It is also contemplated that any of a variety of automated
sequencing procedures
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can be utilized when performing the diagnostic assays (see, e.g., Naeve, et
al., 1995.
Biotechniques 19: 448), including sequencing by mass spectrometry (see, e.g.,
PCT
International Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36:
127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the GPCRX gene include methods in
which
protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or
RNAlDNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In
general, the
art technique of "mismatch cleavage" starts by providing heteroduplexes of
formed by
hybridizing (labeled) RNA or DNA containing the wild-type GPCRX sequence with
potentially mutant RNA or DNA obtained from a tissue sample. The double-
stranded
duplexes are treated with an agent that cleaves single-stranded regions of the
duplex such as
which will exist due to basepair mismatches between the control and sample
strands. For
instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids
treated with
S ~ nuclease to enzymatically digesting the mismatched regions. In other
embodiments, either
DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium
tetroxide
and with piperidine in order to digest mismatched regions. After digestion of
the mismatched
regions, the resulting material is then separated by size on denaturing
polyacrylamide gels to
determine the site of mutation. See, e.g., Cotton, et al., 1988. Proc. Natl.
Acad. Sci. USA 85:
4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an embodiment,
the control
DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or
more
proteins that recognize mismatched base pairs in double-stranded DNA (so
called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point
mutations in
GPCRX cDNAs obtained from samples of cells. For example, the mutt enzyme of E,
coli
cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells
cleaves T
at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 15: 1657-1662.
According to
an exemplary embodiment, a probe based on an GPCRX sequence, e.g., a wild-type
GPCRX
sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is
treated with a DNA mismatch repair enzyme, and the cleavage products, if any,
can be
detected from electrophoresis protocols or the like. See, e.g., U.S. Patent
No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to
identify
mutations in GPCRX genes. For example, single strand conformation polymorphism
(SSCP)
may be used to detect differences in electrophoretic mobility between mutant
and wild type
nucleic acids. See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86:
2766; Cotton,
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1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-
79.
Single-stranded DNA fragments of sample and control GPCRX nucleic acids will
be
denatured and allowed to renature. The secondary structure of single-stranded
nucleic acids
varies according to sequence, the resulting alteration in electrophoretic
mobility enables the
detection of even a single base change. The DNA fragments may be labeled or
detected with
labeled probes. The sensitivity of the assay may be enhanced by using RNA
(rather than
DNA), in which the secondary structure is more sensitive to a change in
sequence. In one
embodiment, the subject method utilizes heteroduplex analysis to separate
double stranded
heteroduplex molecules on the basis of changes in electrophoretic mobility.
See, e.g., Keen, et
al., 1991. Trends Genet. 7: 5.
In yet another embodiment, the movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed using
denaturing gradient
gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495.
When DGGE is
used as the method of analysis, DNA will be modified to insure that it does
not completely
denature, for example by adding a GC clamp of approximately 40 by of high-
melting GC-rich
DNA by PCR. In a further embodiment, a temperature gradient is used in place
of a
denaturing gradient to identify differences in the mobility of control and
sample DNA. See,
e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are
not limited
to, selective oligonucleotide hybridization, selective amplification, or
selective primer
extension. For example, oligonucleotide primers may be prepared in which the
known
mutation is placed centrally and then hybridized to target DNA under
conditions that permit
hybridization only if a perfect match is found. See, e.g., Saiki, et al.,
1986. Nature 324: 163;
Saiki, et al., 1989. Proc. Natl. Acid. Sci. USA 86: 6230. Such allele specific
oligonucleotides
are hybridized to PCR amplified target DNA or a number of different mutations
when the
oligonucleotides are attached to the hybridizing membrane and hybridized with
labeled target
DNA.
Alternatively, allele specific amplification technology that depends on
selective PCR
amplification may be used in conjunction with the instant invention.
Oligonucleotides used as
primers for specific amplification may carry the mutation of interest in the
center of the
molecule (so that amplification depends on differential hybridization; see,
e.g., Gibbs, et al.,
1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one
primer where,
under appropriate conditions, mismatch can prevent, or reduce polymerise
extension (see, e.g.,
Prossner, 1993. Tibtech. 11: 238). In addition it may be desirable to
introduce a novel
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restriction site in the region of the mutation to create cleavage-based
detection. See, e.g.,
Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in
certain embodiments
amplification may also be performed using Taq ligase for amplification. See,
e.g., Barany,
1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur
only if there is a
perfect match at the 3'-terminus of the 5' sequence, making it possible to
detect the presence of
a known mutation at a specific site by looking for the presence or absence of
amplification.
The methods described herein may be performed, for example, by utilizing
pre-packaged diagnostic kits comprising at least one probe nucleic acid or
antibody reagent
described herein, which may be conveniently used, e.g., in clinical settings
to diagnose
patients exhibiting symptoms or family history of a disease or illness
involving an GPCRX
gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes,
in which
GPCRX is expressed may be utilized in the prognostic assays described herein.
However, any
biological sample containing nucleated cells may be used, including, for
example, buccal
mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on GPCRX
activity
(e.g., GPCRX gene expression), as identified by a screening assay described
herein can be
administered to individuals to treat (prophylactically or therapeutically)
disorders (The
disorders include metabolic disorders, diabetes, obesity, infectious disease,
anorexia, cancer-
associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease,
Parkinson's
Disorder, immune disorders, and hematopoietic disorders, and the various
dyslipidemias,
metabolic disturbances associated with obesity, the metabolic syndrome X and
wasting
disorders associated with chronic diseases and various cancers.) In
conjunction with such
treatment, the pharmacogenomics (i.e., the study of the relationship between
an individual's
genotype and that individual's response to a foreign compound or drug) of the
individual may
be considered. Differences in metabolism of therapeutics can lead to severe
toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the
pharmacologically active drug. Thus, the pharmacogenomics of the individual
permits the
selection of effective agents (e.g., drugs) for prophylactic or therapeutic
treatments based on a
consideration of the individual's genotype. Such pharmacogenomics can further
be used to
determine appropriate dosages and therapeutic regimens. Accordingly, the
activity of GPCRX
protein, expression of GPCRX nucleic acid, or mutation content of GPCRX genes
in an
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individual can be determined to thereby select appropriate agents) for
therapeutic or
prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in
the
response to drugs due to altered drug disposition and abnormal action in
affected persons. See
e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985; Linder,
1997. Clin.
Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be
differentiated. Genetic conditions transmitted as a single factor altering the
way drugs act on
the body (altered drug action) or genetic conditions transmitted as single
factors altering the
way the body acts on drugs (altered drug metabolism). These pharmacogenetic
conditions can
occur either as rare defects or as polymorphisms. For example, glucose-6-
phosphate
dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the
main
clinical complication is hemolysis after ingestion of oxidant drugs (anti-
malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a
major
1 S determinant of both the intensity and duration of drug action. The
discovery of genetic
polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and
cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to
why
some patients do not obtain the expected drug effects or show exaggerated drug
response and
serious toxicity after taking the standard and safe dose of a drug. These
polymorphisms are
expressed in two phenotypes in the population, the extensive metabolizer (EM)
and poor
metabolizer (PM). The prevalence of PM is different among different
populations. For
example, the gene coding for CYP2D6 is highly polymorphic and several
mutations have been
identified in PM, which all lead to the absence of functional CYP2D6. Poor
metabolizers of
CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and
side
effects when they receive standard doses. If a metabolite is the active
therapeutic moiety, PM
show no therapeutic response, as demonstrated for the analgesic effect of
codeine mediated by
its CYP2D6-formed metabolite morphine. At the other extreme are the so called
ultra-rapid
metabolizers who do not respond to standard doses. Recently, the molecular
basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
Thus, the activity of GPCRX protein, expression of GPCRX nucleic acid, or
mutation
content of GPCRX genes in an individual can be determined to thereby select
appropriate
agents) for therapeutic or prophylactic treatment of the individual. In
addition,
pharmacogenetic studies can be used to apply genotyping of polymorphic alleles
encoding
drug-metabolizing enzymes to the identification of an individual's drug
responsiveness
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phenotype. This knowledge, when applied to dosing or drug selection, can avoid
adverse
reactions or therapeutic failure and thus enhance therapeutic or prophylactic
efficiency when
treating a subject with an GPCRX modulator, such as a modulator identified by
one of the
exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression
or
activity of GPCRX (e.g., the ability to modulate aberrant cell proliferation
and/or
differentiation) can be applied not only in basic drug screening, but also in
clinical trials. For
example, the effectiveness of an agent determined by a screening assay as
described herein to
increase GPCRX gene expression, protein levels, or upregulate GPCRX activity,
can be
monitored in clinical trails of subjects exhibiting decreased GPCRX gene
expression, protein
levels, or downregulated GPCRX activity. Alternatively, the effectiveness of
an agent
determined by a screening assay to decrease GPCRX gene expression, protein
levels, or
downregulate GPCRX activity, can be monitored in clinical trails of subjects
exhibiting
increased GPCRX gene expression, protein levels, or upregulated GPCRX
activity. In such
clinical trials, the expression or activity of GPCRX and, preferably, other
genes that have been
implicated in, for example, a cellular proliferation or immune disorder can be
used as a "read
out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including GPCRX, that are
modulated in cells by treatment with an agent (e.g., compound, drug or small
molecule) that
modulates GPCRX activity (e.g., identified in a screening assay as described
herein) can be
identified. Thus, to study the effect of agents on cellular proliferation
disorders, for example,
in a clinical trial, cells can be isolated and RNA prepared and analyzed for
the levels of
expression of GPCRX and other genes implicated in the disorder. The levels of
gene
expression (i. e., a gene expression pattern) can be quantified by Northern
blot analysis or
RT-PCR, as described herein, or alternatively by measuring the amount of
protein produced,
by one of the methods as described herein, or by measuring the levels of
activity of GPCRX or
other genes. In this manner, the gene expression pattern can serve as a
marker, indicative of
the physiological response of the cells to the agent. Accordingly, this
response state may be
determined before, and at various points during, treatment of the individual
with the agent.
In one embodiment, the invention provides a method for monitoring the
effectiveness
of treatment of a subject with an agent (e.g., an agonist, antagonist,
protein, peptide,
peptidomimetic, nucleic acid, small molecule, or other drug candidate
identified by the
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screening assays described herein) comprising the steps of (i) obtaining a pre-
administration
sample from a subject prior to administration of the agent; (ii) detecting the
level of expression
of an GPCRX protein, mIRNA, or genomic DNA in the preadministration sample;
(iii)
obtaining one or more post-administration samples from the subject; (iv)
detecting the level of
expression or activity of the GPCRX protein, mRNA, or genomic DNA in the
post-administration samples; (v) comparing the level of expression or activity
of the GPCRX
protein, mRNA, or genomic DNA in the pre-administration sample with the GPC1RX
protein,
mRNA, or genomic DNA in the post administration sample or samples; and (vi)
altering the
administration of the agent to the subject accordingly. For example, increased
administration
of the agent may be desirable to increase the expression or activity of GPCRX
to higher levels
than detected, i.e., to increase the effectiveness of the agent.
Alternatively, decreased
administration of the agent may be desirable to decrease expression or
activity of GPCIRX to
lower levels than detected, i.e., to decrease the effectiveness of the agent.
Methods of Treatment
The invention provides for both prophylactic and therapeutic methods of
treating a
subject at risk of (or susceptible to) a disorder or having a disorder
associated with aberrant
GPCRX expression or activity. The disorders include cardiomyopathy,
atherosclerosis,
hypertension, congenital heart defects, aortic stenosis, atrial septal defect
(ASD),
atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis,
subaortic stenosis,
ventricular septal defect (VSD), valve diseases, tuberous sclerosis,
scleroderma, obesity,
transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia,
prostate cancer,
neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia,
hypercoagulation,
idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host
disease, AIDS,
bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright
Hereditary
Ostoeodystrophy, and other diseases, disorders and conditions of the like.
These methods of treatment will be discussed more fully, below.
Disease and Disorders
Diseases and disorders that are characterized by increased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity may be
treated with
Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics
that antagonize
activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that may
be utilized include, but are not limited to: (i) an aforementioned peptide, or
analogs,
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derivatives, fragments or homologs thereof; (ii) antibodies to an
aforementioned peptide; (iii)
nucleic acids encoding an aforementioned peptide; (iv) administration of
antisense nucleic acid
and nucleic acids that are "dysfunctional" (i.e., due to a heterologous
insertion within the
coding sequences of coding sequences to an aforementioned peptide) that are
utilized to
"knockout" endoggenous function of an aforementioned peptide by homologous
recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v)
modulators ( i.e.,
inhibitors, agonists and antagonists, including additional peptide mimetic of
the invention or
antibodies specific to a peptide of the invention) that alter the interaction
between an
aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity may be
treated with
Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that
upregulate activity
may be administered in a therapeutic or prophylactic manner. Therapeutics that
may be
utilized include, but are not limited to, an aforementioned peptide, or
analogs, derivatives,
fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide
and/or
RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and
assaying it in vitro for
RNA or peptide levels, structure and/or activity of the expressed peptides (or
mRNAs of an
aforementioned peptide). Methods that are well-known within the art include,
but are not
limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation
followed by
sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis,
immunocytochemistry, etc.)
and/or hybridization assays to detect expression of mRNAs (e.g., Northern
assays, dot blots, in
situ hybridization, and the like).
Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a
disease or
condition associated with an aberrant GPCRX expression or activity, by
administering to the
subject an agent that modulates GPCRX expression or at least one GPCRX
activity. Subjects
at risk for a disease that is caused or contributed to by aberrant GPCRX
expression or activity
can be identified by, for example, any or a combination of diagnostic or
prognostic assays as
described herein. Administration of a prophylactic agent can occur prior to
the manifestation
of symptoms characteristic of the GPCRX aberrancy, such that a disease or
disorder is
prevented or, alternatively, delayed in its progression. Depending upon the
type of GPCRX
aberrancy, for example, an GPCRX agonist or GPCRX antagonist agent can be used
for
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treating the subject. The appropriate agent can be determined based on
screening assays
described herein. The prophylactic methods of the invention are further
discussed in the
following subsections.
Therapeutic Methods
Another aspect of the invention pertains to methods of modulating GPCRX
expression
or activity for therapeutic purposes. The modulatory method of the invention
involves
contacting a cell with an agent that modulates one or more of the activities
of GPCRX protein
activity associated with the cell. An agent that modulates GPCRX protein
activity can be an
agent as described herein, such as a nucleic acid or a protein, a naturally-
occurnng cognate
ligand of an GPCRX protein, a peptide, an GPCRX peptidomimetic, or other small
molecule.
In one embodiment, the agent stimulates one or more GPCRX protein activity.
Examples of
such stimulatory agents include active GPCRX protein and a nucleic acid
molecule encoding
GPCRX that has been introduced into the cell. In another embodiment, the agent
inhibits one
or more GPCRX protein activity. Examples of such inhibitory agents include
antisense
GPCRX nucleic acid molecules and anti-GPCRX antibodies. These modulatory
methods can
be performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g.,
by administering the agent to a subject). As such, the invention provides
methods of treating
an individual afflicted with a disease or disorder characterized by aberrant
expression or
activity of an GPCRX protein or nucleic acid molecule. In one embodiment, the
method
involves administering an agent (e.g., an agent identified by a screening
assay described
herein), or combination of agents that modulates (e.g., up-regulates or down-
regulates)
GPCRX expression or activity. In another embodiment, the method involves
administering an
GPCRX protein or nucleic acid molecule as therapy to compensate for reduced or
aberrant
GPCRX expression or activity.
Stimulation of GPCRX activity is desirable in situations in which GPCRX is
abnormally downregulated and/or in which increased GPCRX activity is likely to
have a
beneficial effect. One example of such a situation is where a subj ect has a
disorder
characterized by aberrant cell proliferation and/or differentiation (e.g.,
cancer or immune
associated disorders). Another example of such a situation is where the
subject has a
gestational disease (e.g., preclampsia).
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Determination of the Biological Effect of the Therapeutic
In various embodiments of the invention, suitable in vitro or in vivo assays
are
performed to determine the effect of a specific Therapeutic and whether its
administration is
indicated for treatment of the affected tissue.
In various specific embodiments, in vitro assays may be performed with
representative
cells of the types) involved in the patient's disorder, to determine if a
given Therapeutic exerts
the desired effect upon the cell type(s). Compounds for use in therapy may be
tested in
suitable animal model systems including, but not limited to rats, mice,
chicken, cows,
monkeys, rabbits, and the like, prior to testing in human subjects. Similarly,
for in vivo
testing, any of the animal model system known in the art may be used prior to
administration
to human subjects.
Prophylactic and Therapeutic Uses of the Compositions of the Invention
The GPCRX nucleic acids and proteins of the invention are useful in potential
prophylactic and therapeutic applications implicated in a variety of disorders
including, but not
limited to: metabolic disorders, diabetes, obesity, infectious disease,
anorexia, cancer-
associated cancer, neurodegenerative disorders, Alzheimer's Disease,
Parkinson's Disorder,
immune disorders, hematopoietic disorders, and the various dyslipidemias,
metabolic
disturbances associated with obesity, the metabolic syndrome X and wasting
disorders
associated with chronic diseases and various cancers.
As an example, a cDNA encoding the GPCRX protein of the invention may be
useful
in gene therapy, and the protein may be useful when administered to a subject
in need thereof.
By way of non-limiting example, the compositions of the invention will have
efficacy for
treatment of patients suffering from: metabolic disorders, diabetes, obesity,
infectious disease,
anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders,
Alzheimer's
Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and
the various
dyslipidemias.
Both the novel nucleic acid encoding the GPCRX protein, and the GPCItX protein
of
the invention, or fragments thereof, may also be useful in diagnostic
applications, wherein the
presence or amount of the nucleic acid or the protein are to be assessed. A
further use could
be as an anti-bacterial molecule (i.e., some peptides have been found to
possess anti-bacterial
properties). These materials are further useful in the generation of
antibodies which
immunospecifically-bind to the novel substances of the invention for use in
therapeutic or
diagnostic methods.
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EQUIVALENTS
Although particular embodiments have been disclosed herein in detail, this has
been
done by way of example for purposes of illustration only, and is not intended
to be limiting
with respect to the scope of the appended claims, which follow. In particular,
it is
contemplated by the inventors that various substitutions, alterations, and
modifications may be
made to the invention without departing from the spirit and scope of the
invention as defined
by the claims. The choice of nucleic acid starting material, clone of
interest, or library type is
believed to be a matter of routine for a person of ordinary skill in the art
with knowledge of the
embodiments described herein. Other aspects, advantages, and modifications
considered to be
within the scope of the following claims.
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