CA2216835A1 - Human g-protein coupled receptors - Google Patents

Abstract

Human G-protein coupled receptor polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptides for identifying antagonists and agonists to such polypeptides and methods of using the agonists and antagonists therapeutically to treat conditions related to the underexpression and overexpression of the G-protein coupled receptor polypeptides, respectively. Also disclosed are diagnostic methods for detecting a mutation in the G-protein coupled receptor nucleic acid sequences and an altered level of the soluble form of the receptors.

Description

CA 0221683~ 1997-09-29 W O 961304~6 PCTnUS95104079 EI~UN G-PROTEIN CO~PLED RE~r~KS

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are human 7-tr~n~m~mhrane receptors. The tr~n~m~mhrane receptors are G-protein coupled receptors sometimes hereina~ter referred to individually as GPR1, GPR2, GPR3 and GPR4. The invention also relates to inhibiting the action of such polypeptides.
It is well established that many medically signi~icant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Le~kowitz, Nature, 351:353-354 (1991)). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG
proteins. Some examples of these proteins include the GPC
receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., PNAS, 84:46-50 (1987); Kobilka, B.K., et al., Science, 238:650-656 (1987); Bunzow, J.R., et al., Nature, 336:783-787 (1988)), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclaser and phosphodiesterase, and actuator proteins, e.g., protein ~ --1--CA 0221683~ 1997-09-29 W 096/30406 PCTrUS9~01~

kinase A and protein kinase C (Simon, M.I., et al., Science, 252:802-8 (1991)).
For example, in one form of signal transduction, the effect of hormone binding is activation of an enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP, and GTP also influences hormone binding. A G-protein connects the hormone receptors to adenylate cyclase. G-protein was shown to exchange GTP for bound GDP when activated by hormone receptors. The GTP-carrying form then binds to an activated adenylate cyclase. Hydrolysis of GTP
to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
The membrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative tr~ncme~rane ~om~in~. The ~or~;n~ are believed to represent tr~n~m~mhrane ~-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
G-protein coupled receptors have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of mer.~bers of this family include calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, hist~mi ne, thrombin, kinin, follicle stimulating hormone, opsins and rhodopsins, odorant, cytomegalovirus receptors, etc.

CA 0221683~ 1997-09-29 W O 96/30406 PCTnUS~5~4~79 Most G-protein coupled receptors have sinyle conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. The 7 tr~n-cm~hrane regions are designated as TMl, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 is also implicated in signal transduction.
Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors. Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several G-protein coupled receptors, such as the ~-adrenoreceptor, phosphorylation by protein kinase A
and/or specific receptor kinases mediates receptor desensitization.
The ligand binding sites of G-protein coupled receptors are believed to comprise a hydrophilic socket formed by several G-protein coupled receptors tr~n~m~mhrane ~ ;n~, which socket is surrounded by hydrophobic residues of t:he G-protein coupled receptors. The hydrophilic side of each G-protein coupled receptor tr~n~m~mhrane helix is postulated to ~ace inward and form the polar ligand binding site. I~I3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as including the TM3 aspartate residue. Additionally, TM5 serines, a TM6 asparagine and TM6 or TM7 phenyl~l~nines or tyrosines are also implicated in ligand binding.
G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intrace].lular enzymes, ion channels and transporters (see, Johnson e t al ., Endoc., Rev., 10:317-331 (1989)). Different G-protein ~-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell.
Phosphorylation of cytoplasmic residues of G-protein coupled receptors have been identified as an important me~h~nism for .

CA 0221683~ 1997-09-29 W 096/30406 PCTrUS95/04079 the regulation of G-protein coupling of some G-protein coupled receptors.
G-protein coupled receptors are found in numerous sites within a m~ l ian host, for example, dopamine is a critical neurotransmitter in the central nervous system and is a G-protein coupled receptor ligand.
In accordance with one aspect of the present invention, there are provided novel polypeptides which have been putatively identified as G-protein coupled receptors and biologically active and diagnostically or therapeutically useful fragments and derivatives thereof. The polypeptides of the present invention are of human origin.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding human G-protein coupled receptors, including mRNAs, DNAs, cDNAs, genomic DNA as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques which comprises culturing recombinant prokaryotic and/or eukaryotic host cells, containing a human G-protein coupled receptor nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery of said protein.
In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.
In accordance with another embodiment, there is provided a process for using the receptors to screen for receptor antagonists and/or agonists and/or receptor ligands.
In accordance with still another embodiment of the present invention there is provided a process of using such agonists to stimulate the G-protein coupled receptors for the CA 0221683~ 1997-09-29 W 096/30406 PCTnUS9~D4079 treatment of conditions related to the under-expression of the G-protein coupled receptors.
In accordance with another aspect of the present invention there is provided a process of using such antagonists for inhibiting the action of the G-protein coupled receptors for treating conditions associated with over-expression of the G-protein coupled receptors.
In accordance with yet another aspect of the present invention there is provided non-naturally occurring synthetic, isolated and/or recombinant G-protein coupled receptor polypeptides which are fra~_ snt.s, consensus fragments and/or sequences having conservative amino acid substitutions, of at least one tr~n~ -.nhrane ~ n of the G-protein coupled receptor, such that G-protein coupled receptor polypeptides of the present invention may bind G-protein coupled receptor ligands, or which may also modulate, quantitatively or qualitatively, G-protein coupled receptor ligand binding.
In accordance with still another aspect of the present invention there are provided synthetic or recombinant G-protein coupled receptor polypeptides, conser~rative substitution and derivatives thereof, antibodies, anti-idiotype antibodies, compositions and methods that can be useful as potential modulators of G-protein coupled receptor function, by hin~ing to ligands or modulating ligand bi~sding, due to their expected biological properties, which may be used in diagnostic, therapeutic and/or research applications.
It is still another object of the present invention to provide synthetic, isolated or recombinant polypeptides which are designed to inhibit or mimic various G-protein coupled receptors or fragments thereof, as receptor type.s and subtypes.
In accordance with yet a further aspect of the p~esent invention, there is also provided diagnostic probes comprising nucleic acid molecules of sufficient length to CA 0221683~ 1997-09-29 W 096/30406 PCTrUS95/04079 specifically hybridize to the G-protein coupled receptor nucleic acid sequences.
In accordance with yet another object of the present invention, there is provided a diagnostic assay for detecting a disease or susceptibility to a disease related to a mutation in a G-protein coupled receptor nucleic acid sequence.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as ~ncomrassed by the claims.
Figures 1-4 show the cDNA sequences and the corresponding deduced amino acid sequences of the four G-protein coupled receptors of the present invention, respectively. The stAn~Ard one-letter abbreviation for amino acids are used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).
Se~l~nc~ng accuracy is predicted to be greater than 97%
accurate.
Figure 5 is an illustration of the amino acid homology between GPR1 (top line) and odorant receptor-like protein (bottom line).
Figure 6 illustrates the amino acid homology between GPR2 (top line) and the human Endothelial Differentiation Gene-1 (EDG-1) (bottom line).
Figure 7 illustrates the amino acid homology between GPR3 (top line) and a human G-protein coupled receptor open reading frame (ORF) (bottom line).
Figure 8 illustrates the amino acid homology between GPR4 and the chick orphan G-protein coupled receptor (bottom line).
In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) CA 0221683~ 1997-09-29 W O 96/30406 PcT/U~ 79 which encode for the mature polypeptides having the deduced amino acid sequence~ of Figures 1-4 (SEQ ID No. 2, 4, 6 and 8) or for the mature polypeptides encoded by the cDNAs of the clones deposited as ATCC Deposit No. 75981 (GPRl), 75983 (GPR2), 75976 (GPR3), 75979 (GPR4) on December 16, 1994.
A polynucleotide encoding the GPR1 polypeptide of the present invention may be isolated from the human breast. The polynucleotide encoding GPR1 was discovered in a cDNA library derived from human eight-week-old embryo. It is structurally related to the G protein-coupled receptor family. It contains an open reading frame encoding a protein of 296 amino acid residues. The protein exhibits the highest degree of homology to an odorant receptor-like protein with 66 %
identity and 83 % similarity over a 216 amino acid stretch.
A polynucleotide encoding the GPR2 polypeptide of the present invention may be isolated from human liver, heart and leukocytes. The polynucleotide encoding GPR2 was discovered in a cDNA library derived from human adrenal gland tumor. It is structurally related to the G protein-coupled receptor family. It contains an open reading frame encoding a protein of 393 amino acid residues. The protein exhibits the highest degree of homology to human EDG-l with 30 ~ identity and 52 % similarity over a 383 amino acid stretch. Potential ligands to GPR2 include but are not limited to ~n~n~m~ de, serotonin, adrènalin and noradrenalin.
A polynucleotide ~nco~ng the GPR3 polypeptide of the present invention may be isolated from human liver, kidney and pancreas. The polynucleotide encoding GPR3 was discovered in a cDNA library derived from human neutrophil.
It is structurally related to the G protein-coupled receptor family. It contains an open reading frame encoding a protein of 293 amino acid residues. The protein exhibits the highest degree of homology to a human G-Protein Coupled Receptor open reading frame with 39 % identity and 61 ~ similarity over the entire amino acid sequence. Potential CA 0221683~ 1997-09-29 W 096~0406 PCTrUS95/04079 ligands to GPR3 include but are not limited to platelet activating factor, thrombin, C5a and bradykinin.
A polynucleotide encoding the GPR4 polypeptide of the present invention may be found in human heart, spleen and leukocytes. The polynucleotide encoding GPR4 was discovered in a cDNA library derived from hum.an twelve-week-old embryo.
It is structurally related to the G-protein coupled receptor family. It contains an open re~' ng frame encoding a protein of 344 amino acid residues. The protein exhibits the highest degree of homology to a chick orphan G-protein coupled receptor with 82 % identity and 91 ~ similarity over a 291 amino acid stretch. Potential ligands to GPR4 include but are not limited to thrombin, ~hemokine~ and platelet activating factor.
The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptides may be identical to the coding sequence shown in Figures 1-4 (SEQ ID
No. 1, 3, 5 and 7) or that of the deposited clones or may be a different coding sequence which coding sequence, as a result of the re~nn~ncy or degeneracy of the genetic code, encodes the same mature polypeptides as the DNA of Figures 1-4 (SEQ ID No. 1, 3, 5 and 7) or the deposited cDNAs.
The polynucleotides which encode for the mature polypeptides of Figures 1-4 (SEQ ID No. 2, 4, 6 and 8) or for the mature polypeptides encoded by the deposited cDNAs may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3 of the coding sequence for the mature polypeptide.

CA 0221683~ 1997-09-29 W096l30406 PCT~ ~/O~n79 Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding se~uence for the polypeptide as well as a polynucl~otide which includes additional coding and/or non-coding seq~ence.
The present invention further relates to variants of the hereinabove described polynucleotides which encode ~or fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequence of Figures 1-4 (SEQ ID No. 2, 4, 6 and 8) or the polypeptides encoded by the cDNAs of the deposited clones. The variants of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleot.ides.
Thus, the present invention includes polynucleotides encoding the same mature polypeptides as shown in Figures 1-4 (SEQ ID No. 2, 4, 6 and 8) or the same mature polypeptides encoded by the cDNAs of the depo~ited clones as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptides of Figure 1-4 (SEQ ID No. 2, 4, 6 and 8) or the polypeptides encoded by the cDNAs of the deposited clones. Such nucleotide vaîiants include deletion variants, substitution variants and addition or insertion variants.
As hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequences shown in Figures 1-4 (SEQ ID
No. 1, 3, 5 and 7) or of the coding sequences of the deposited clones. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the f~lction of the encoded polypeptides.
The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention. The marker sequence may be a hexa-_9_ CA 0221683~ 1997-09-29 W 096/30406 PCT/U~S/OIC79 histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a m~mm~ lian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70~
identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the here~n~bove-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95~ and preferably at least 97~ identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figures 1-4 (SEQ
ID No. 1, 3, 5 and 7) or the deposited cDNAs, i.e. function as a G-protein coupled receptor or retain the ability to bind the ligand for the receptor even though the polypeptides do not function as a G-protein coupled receptor, for example, soluble form of the receptors.
Alternatively, the polynucleotide may be a polynucleotide which has at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which does not retain activity. Such polynucleotides may be employed as probes for the polynucleotide of SEQ ID No. 1, ~or exa~ple, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.

CA 0221683~ 1997-09-29 W 096~04~6 PCTIU~95,10IC7Y

The deposit(s) referred to herein will be m~intAined under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. 112.
The ~equence of the polynucleotides contained in the deposited materials, as well as the amino acid seque:nce of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
The present invention further relates to G-protein coupled receptor polypeptides which have the deduced amino acid sequences of Figures 1-4 (SEQ ID No. 2, 4, 6 and 8) or which have the amino acid sequences encoded by the deposited cDNAs, as well as fragments, analogs and deri~atives of such polypeptides.
The terms 'Ifragment," "derivativel' and ~analog" when referring to the polypeptides of Figures 1-4 (SEQ ID No. 2, 4, 6 and 8) or that encoded by the deposited cDNAs, means a polypeptide which either retains substantially the same biological function or activity as such polypeptide, i.e.
functions as a G-protein coupled receptor, or retains the ability to bind the ligand or the receptor even though the polypeptide does not function as a G-protein coupled receptor, for example, a soluble form of the receptor. An analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptides of the present invention may be recombinant polypeptides, a natural polypeptides or synthetic r polypeptides, preferably recombinant polypeptides.

CA 0221683~ 1997-09-29 W O 96/30406 PCTrUS95/04079 The fragment, derivative or analoy of the polypeptides of Figures 1-4 tSEQ ID No. 2, 4, 6 and 8) or that encoded by the deposited cDNAs may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide which is employed for purification of the mature polypeptide.
Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The term "isolated" means that the material is removed from its original environment (e.g., the natural environm~t if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living ~nimAl is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
The present invention also relates to vectors which include polynucleotides o~ the present inventio~n, host cells which are genetically engineered with vectors of the CA 022l683~ l997-09-29 W 096/30406 PCTnUS9Sl04079 invention and the production of polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered (transduced or transfonmed or transfected) with the vectors o~ this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modi~ied as appropriate ~or activating promoters, selecting transformants or amplifying the G-protein coupled receptor genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and wi:ll be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for pro~-lci n~ polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
~owever, any other vector may be used as long as it is replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative CA 0221683~ 1997-09-29 W 096r30406 PCT/U~5~01CIY

examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lamhda PL
promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
The expression vector also c~nt~ng a ribosome h~n~ng site .
for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
The vector cont~n~ng the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, 5treptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodo~tera Sf9;
~n~ m~ 1 cells such as CHO, COS or Bowes melanoma;
adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors CA 0221683~ 1997-09-29 WO9613040G PCT~5101C~

and promoters are known to those of skill in the art, and are commercially available. The following vectors are pro~ided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs, pDl0, phagescript, psiXl74, pbluescript SR, p.~sks, pNH8A, pNHl6a, pNHl8A, pNH46A ~Stratagene); pTRC99a, p~K223-3, pKK233-3, pDR540, pRIT5 ~Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG ~Stratagene) pSVK3, pBPV, ~MSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
Promoter regions can be selected from any desired gene using CAT (chlor~mphPn~col transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR~ PL and trp.
Eukaryotic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
In a further embodiment, the present invention relates to host cells cont~i n~ ng the above-described constructs. The host cell can be a higher eukaryotic cell, such as a m~m~l ian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEA~-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional manner to produce the gene product PnCo~e~ by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
o CA 0221683~ 1997-09-29 W 096130406 PCT~US~5~-79 Mature proteins can be expressed in m~mm~lian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an ~nh~ncer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription.
Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ~-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences.
Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recomh'n~nt product.

CA 0221683~ 1997-09-29 WO ~ ~3n 10~ PCTnUS9~1\4079 Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading ~hase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, S~l~n~lla typhimurium and various species within the genera Pse~ nA.~, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC
37017). Such ~ ~rcial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA). These pBR322 ~bac~bone~
sections are combined with an appropriate promoter and the structural sequence to be expressed.
~ Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of: cell CA 0221683~ 1997-09-29 W 096/30406 PCTrUSgS/OS~79 lysing agents, such methods are well know to those skilled in the art.
Various mAmm~ n cell culture systems can also be employed to express reromhin~nt protein. Examples of m~m~l ian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. ~rm~lian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and S' flanking nontranscribed sequence~. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The G-protein coupled receptor polypeptides can be recovered and purified from recombinant cell cultures by methods including ~mm~ni um sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydropho~ic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and m~mm~lian cells in culture). Depending upon the host employed in ~ recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.

CA 0221683~ 1997-09-29 W O 96/3040~ PCTnUS9S/0407g Polypeptides of the invention may also include an initial methionine amino acid residue.
Fragments of the full length G-protein coupled receptor genes may be employed as a hybridization probe for a cDNA
library to isolate the full length genes and to isolate other genes which have a high sequence similarity to the gene or similar biological activity. Probes of this type generally have at least 20 bases. Preferably, however, the probes have at least 30 bases and may contain, for example, 50 bases or more. In many cases, the pro~e has from 20 to 50 bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete G-protein coupled receptor gene including regulatory and promotor regions, exons, and introns. As an example of a screen comprises isolating the coding region of the G-protein coupled receptor gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members o~ the library the probe hybridizes to.
The G-protein coupled receptors of the present invention may be employed in a process ~or screening ~or antagonists and/or agonists for the receptor.
In general, such screening procedures involve providing appropriate cells which express the receptor on the surface thereof. Such cells include cells from m~mm~1 S, yeast, drosophila or E. Coli. In particular, a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby express the respective G-protein coupled receptor. The expressed receptor is then contacted with a test compound to observe binding, stimulation or inhibition of a functional response.

CA 0221683~ 1997-09-29 W 096130406 PCTr~'~S~ 7~

One such screening procedure involves the use of m~l ~nophores which are transfected to express the respective G-protein coupled receptor of the present invention. Such a screening technique is described in PCT WO 92/01810 published February 6, 1992.
Thus, for example, such assay may be employed for screening for a receptor antagonist by contacting the melanophore cells which encode the G-protein coupled receptor with both the receptor ligand and a compound to be screened.
Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i e., inhibits activation of the receptor.
The screen may be employed for determining an agonist by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the receptor.
Other screening techniques include the use of cells which express the G-protein coupled receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, a6 described in Science, volume 246, pages 181-296 (October 1989). For example, potential agonists or antagonists may be contacted with a cell which expresses the G-protein coupled receptor and a second messenger response, e.g. signal transduction or pH changes, may be measured to determine whether the potential agonist or antagonist is e~ective.
Another such screening technique involves introducing RNA encoding the G-protein coupled receptors into Xenopus oocytes to transiently express the receptor. The receptor oocytes may then be contacted in the case of antagonist screening with the receptor ligand and a compound to be screened, ~ollowed by detection of inhibition of a calcium signal.

CA 0221683~ 1997-09-29 W 096130406 PCTnUS951~1~79 Another screening technique involves expressing t]he G-protein coupled receptors in which the receptor is linked to a phospholipase C or D. As representative examples of such cells, there ,m, y be mentioned endotheliAl cells, smooth muscle cells, em~ryonic kidney cells, etc. The screenin~ for an antagonist or agonist ,m,~y be accomplished as hereinabo~e described by detecting activation o~ the receptor or inhibition of activation of the receptor from the phospholipase second signal.
Another method in~Jolves screening for antagonists by determining inhibition of binding of labeled ligand to cells which have the receptor on the surface thereof. Such a method involves transfecting a eukaryotic cell with DNA
encoding the G-protein coupled receptor such that the cell expresses the receptor on its surface and contacting the cell with a potential antagonist in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the potential antagonist binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.
G-protein coupled receptors are ubiquitous in the m~mm~l ian host and are responsible for many biological functions, including many pathologies. Accordingly, i.t is desirous to find compounds and drugs which stimnl~te the G-protein coupled receptors on the one hand and which can antagonize a G-protein coupled receptor on the other hand, when it is desirable to i nhi hi t the G-protein co~lpled receptor.
For example, agonists for G-protein coupled receptors may be employed for therapeutic purposes, such as the treatment of asthma, Parkinson~s disease, acute heart failure, hypotension, urinary retention, and osteoporosis.

CA 0221683~ 1997-09-29 W 096/30406 PcT/u~5~ 79 In general, antagonists to the G-protein coupled receptors may be employed for a variety of therapeutic purposes, for example, for the treatment of hypertension, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy and psychotic and neurological disorders, including schizophrenia, manic excitement, depression, delirium, ~m~ntia or severe mental retardation, dyskinesias, such as Huntington's disease or Gilles dila Tourett's syndrome, among others. G-protein coupled receptor antagonists have also been useful in reversing endogenous anorexia and in the control of bnl imi ~, Examples of G-protein coupled receptor antagonists include an antibody, or in some cases an oligopeptide, which binds to the G-protein coupled receptors but does not elicit a second messenger response such that the activity of the G-protein coupled receptors is prevented. Antibodies include anti-idiotypic antibodies which recognize unique detenminants generally associated with the antigen-binding site of an antibody. Potential antagonists also include proteins which are closely related to the ligand of the G-protein coupled receptors, i.e. a fragment of the ligand, which have lost biological ~unction and when bin~in~ to the G-protein coupled receptors, elicit no response.
A potential antagonist also includes an antisense construct prepared through the use of antisense technology.
Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids CA 0221683~ 1997-09-29 W 09C/3040~ PCTnUS95~1)4079 Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1~88);
and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production o~ G-protein coupled receptors. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of mRNA
molecules into G-protein coupled receptors (antisense Okano, ~. Neurochem., 56:~60 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA
may be expressed in vivo to inhibit production of G-protein coupled receptors.
Another potential antagonist is a small molecule which binds to the G-protein coupled receptor, making it inacces~ible to ligands such that normal biological activity is prevented. Examples of small molecules include bu~ are not limited to small peptides or peptide-like molecules.
Potential antagonists also include a soluble form o~ a G-protein coupled receptor, e.g. a fragment of the receptors, which binds to the ligand and prevents the ligand from interacting with membrane bound G-protein coupled receptors.
This invention additionally provides a method of treating an abnormal condition related to an excess of G-protein coupled receptor activity which comprises i nt stering to a subject the antagonist as her~in~hove described along with a pharm.aceutically acceptable carrier in an amount effective to block binding of ligands to the G-protein coupled receptors and thereby alleviate the abnormal conditions.
The invention also provides a method of treating abnormal conditions related to an under-expre~ion of G-protein coupled receptor activity which comprises administering to a subject a therapeutically effective amount of the agonist described above in com.~ination with a phanmaceutically acceptable carrier, in an amount effective CA 0221683~ 1997-09-29 W 096/30406 PCT/U~/0~79 to enhance binding of ligands to the G-protein coupled receptor and thereby alleviate the abnormal conditions.
The soluble form of the G-protein coupled receptors, antagonists and agonists m~y be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the antagonist or agonist, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, bu~fered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode o~ ~Ami ni stration.
The invention also provides a pharmaceutical pack or kit comprising one or more cont~iners filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such cont~ine~(s) can be a notice in the form prescribed by a gov~ - tal agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.
The pharmaceutical compositions may be ~mi ni stered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intr~n~l or intradermal routes. The pharmaceutical compositions are ~ministered in an amount which is effective for treating and/or prophylaxis of the specific indication. In yeneral, the pharmaceutical compositions will be ~Ami ni stered in an amount of at least about 10 ~g/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 ~g/kg to about 1 mg/kg body weight daily, taking into account the routes of ~Aministration, symptoms, etc.

CA 0221683~ 1997-09-29 W 0~6130406 PCTnUS9~4079 The G-protein coupled receptor polypeptides, and antagonists or agonists which are polypeptides, may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy."
Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle cont~; n; n~ RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for exa~lple, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA
encoding the polypeptide of the present invention may be ;n;stered to a patient for engineering cells in viv~ and expression of the polypeptide in vivo. These and other method~ for a~m; n; ~tering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells mj~y be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
The present invention also provides a method for determining whether a ligand not known to be capable of binding to a G-protein coupled receptor can bind to such receptor which comprises contacting a m~ n cell which expresses a G-protein coupled receptor with the ligand under conditions permitting binding of ligands to the G-protein coupled receptor, detecting the presence of a ligand which CA 0221683~ 1997-09-29 W 096/30406 PCTrUS3S101-/ 7 binds to the receptor and thereby determining whether the ligand binds to the G-protein coupled receptor.
This invention further provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the human G-protein coupled receptors on the surface of a cell which comprises contacting a mAmm~lian cell comprising an isolated DNA molecule encoding the G-protein coupled receptor with a plurality of drugs, determining those drugs which bind to the ~mm~l~ An cell, and thereby identifying drugs which specifically interact with and bind to a human G-protein coupled receptor of the present invention.
This invention also provides a method of detecting expression of the G-protein coupled receptor on the surface of a ce~l by detecting the presence of mRNA coding for a G-protein coupled receptor which comprises obtA~n~ng total mRNA
from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human G-protein coupled receptor under hybridizing conditions, detecting the presence of mRNA
hybridized to the probe, and thereby detecting the expression of the G-protein coupled receptor by t~e cell.
This invention is also related to the use of the G-protein coupled receptor genes as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the G-protein coupled receptor genes. Such diseases, by way of example, are related to cell transformation, such as tumors and cancers.
Individuals carrying mutations in the human G-protein coupled receptor genes may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA

CA 0221683~ 1997-09-29 W096/304~6 PCT~S95J'04079 may be used directly for detection or may be ampli~ied enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding the G-protein coupled receptor proteins can be used to identify and analyze G-protein coupled receptor ~utations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
Point mutations can be identified by hybridizing amplified DNA to radiolabeled G-protein coupled receptor RNA or alternatively, radiolabeled G-protein coupled receptor antisense DNA sequences. Per$ectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
Sequence differences between the reference gene and genes having mutations may be revealed by the direct DNA
sequencing method. In addition, cloned DNA segments ~ay be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly ~nh~nced when combined with PCR. For example, a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without dena.turing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA
fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial CA 0221683~ 1997-09-29 melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985)).
Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process.
These primers are then used for PCR screening of somatic cell hybrids cont~in~ng individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

CA 0221683~ 1997-09-29 W096/3040~ PCT~S9~4079 PC~ mapping of somatic cell hybrids is a rapid procedure for assiy-ning a particular DNA to a particular chromosome.
Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fra~m~nts from specific chromosomes or pools of large genomic clones in an analogous mAnn~r Other mapping strategies that can similarly be used to map to its chromosome include in si tu hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization ~FISH) of a cDNA
clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This tech~ique can be used with cDNA as short as 500 or 600 bases;
however, clones larger than 2,000 bp have a higher like].ihood of h~n~ng to a unique chromosomal location with suff~cient signal intensity for simple detection. FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. ~or a review of this technique, see Verma et al., Human Chromosomes: A ~nll~l of Basic Techniques, Pe~ydllloll Press, New York (1988).
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, for example, in V. McKusick, M~n~l~l ian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes) Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the CA 0221683~ 1997-09-29 W O 96/30406 PCTIUS~5/0~079 affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immllnogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chi~ric, single chain, and hllm~nized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an ~nim~l or by ~ministering the polypeptides to an ~nim~l, preferably a no~hllm~n. The antibody so obtained will then bind the polypeptides itself. In this m~nnPr, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybrido~ma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies CA 0221683~ 1997-09-29 W ~96/30406 PCTnUS95/~4079 (Cole, et al., 1985, in Monoclonal ~ntih~dies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodie~ to ~mmllnogenic polypeptide products of this invention. Also, transgenic mice may be used to express hnm~nized antibodies to i~nnogenic polypeptide products of this invention.
The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate underst~n~ing o~ the following examples certain frequently occurring methods and/or terms will be described.
~ Plasmids~ are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either co~rcially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with pub]ished procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent t:o the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are com~rcially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 ~g of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20 ~l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~g of DNA are digested with 20 to 250 units of enzyme in a larger CA 0221683~ 1997-09-29 W O9~/301_~ PCTrUS95/04079 volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D.
et al., Nucleic Acids Res., 8:4057 (1980).
~ Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthe~ized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
~ Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase") per 0.5 ~g of approximately equimolar amounts of the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Exam~le 1 Bacterial Expression and Purification of GPR1 The DNA sequence encoding GPR1, ATCC # 75981, is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the processed G-protein coupled receptor nucleotide sequence. Additional CA 0221683~ 1997-09-29 W 096/30406 PCTnUS95~407g nucleotides corresponding to the GPRl nucleotide sequence are added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5' GACTAAAGCTTAATGAGTAGTGAAATGGTG 3' (SEQ ID No. 9) contains a HindIII restriction enzyme site ~ollowed by 19 nucleotides of G-protein coupled receptor coding sequence starting from the presumed terminal amino acid of the processed protein. The 3~ sequence 5' GAA~rTcTAGAcccIc~GG~~ ATcAG 3~ (SEQ ID No.
10) contains complementary sequences to an XbaI site and is ~ollowed by 20 nucleotides of GPRl coding sequence. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc.
Chatsworth, CA). pQE-9 encodes antibiotic resistance (~mpr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 is then digested with HindIII and XbaI. The amplified sequences are ligated into pQE-9 and are inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture is then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory ~nll~ 1, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected.
Plasmid DNA is isolated and confirmed by restriction analysis.
Clones cont~intng the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (2S ug/ml). The O/N
culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.~') of between 0.4 and 0.6. IPTG ("Isopropy]-B-D-CA 022l683~ l997-09-29 W O 96130406 PCT/u~s~o1c7~

thiogalacto pyranoside") is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/0 leading to increased gene expression. Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl.
After clarification, solubilized G-protein coupled receptor is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins contAining the 6-His tag (Hochuli, E. et al., ~.
Chromatography 411:177-184 (1984)). GPR1 is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.

Exam~le 2 ~3acterial Expression and Purification of GPR2 The DNA sequence encoding GPR2, ATCC # 75983, is initially amplified using PCR oligonucleotide primers corresponding to the 5~ and 3~ end sequences of the processed GPR2 coding sequence. Additional nucleotides corresponding to GPR2 coding sequence are added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5' GACTAAAGCTTAATGAGGCCCACATGGGCA 3~ (SBQ ID No. 11) contains a HindIII restriction enzyme site followed by 19 nucleotides of GPR2 coding sequence starting from the presumed terminal amino acid of the processed protein. The 3' sequence 5' GAACTTCTAGACGAACTAGTGGATCCCCCCGG 3' (SEQ ID No. 12) C~nt~in~
complementary sequences to an XbaI site and is followed by 21 nucleotides of GPR2 coding sequence. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, CA 0221683~ 1997-09-29 W Og6/30406 PCTIU'~ 79 CA). pQE-9 encodes antibiotic resistance (Amp'), a bacterial origin of replication (ori), an IPTG-regulatable pro1~0ter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 is then digested with HindIII and XbaI. The amplified sequences are ligated into pQE-9 and are inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture is then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory ~nllAl, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N
culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.~') o~ between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") is then added to a final concentration of 1 mM. IPTG induces by inacti~ating the lacI
repressor, clearing the P/O leading to increased gene expression. Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl.
After clarification, solubilized GPR2 is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J.
Chromatography 411:177-184 (1984)). GPR2 is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium ..

CA 0221683~ 1997-09-29 W 096/30406 PCTrU~9S~1a79 phosphate, 10 mmolar glutathione ~reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.

ExamPle 3 Bacterial Expression and Purification of GPR3 The DNA sequence encoding GPR3, ATCC # 75976, is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3 ' end sequences of the processed G-protein coupled receptor nucleotide sequence. Additional nucleotides corresponding to the GPR3 coding sequence are added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5' GACTAAAGCTTAATGG~~ ~-l~-l~CT 3' (SEQ ID No. 13) contains a HindIII restriction enzyme site followed by 19 nucleotides of GPR3 coding sequence starting from the presumed terminal amino acid of the processed protein. The 3' sequence 5' GAA~-l-l~-l-AGACTTCACACA~-l-l~lACTAT 3 ' (SEQ ID No. 14) contains complementary sequences to XbaI site and is followed ~y 19 nucleotides of GPR3 coding sequence. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, CA). pQE-9 PncoAP~ antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 is then digested with XbaI and XbaI. The amplified sequences are ligated into pQE-g and are inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture is then used to transform E. coli strain M15/rep 4 (Qiagen Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers CA 0221683~ 1997-09-29 W O 96t30406 PCTnUS9~04079 kanamycin resistance (Kanr). Transformants are identified by their ability to grow on ~B plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and con$irmed by restriction analysis.
Clones cont~n~ng the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N
culture is used to inoculate a large culture at a ratio of 1:100 to 1:250 The cells are grown to an optical density 600 (O.D.~') of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/O leading to increased gene expression. Cells are grown an extra 3 to 4 hours Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl.
After clarification, solubilized GPR3 is purified fronl this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins co~t~ntng the 6-His tag (Hochuli, E. et al., J
Chromatography 411:177-184 (1984~). GPR3 is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 ~molar glutathione (oxidized). After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.

ExamPle 4 Bacterial Ex~ression and Purification of GPR4 The DNA sequence encoding GPR4, ATCC # 75979, is initially amplified using PCR oligonucleotide primers corresponding to the 5~ and 3' end sequences of the processed GPR4 nucleotide sequence. Additional nucleotides corresponding to the GPR4 coding sequence are added to the 5' CA 0221683~ 1997-09-29 W 096130406 PCTnUS9~ 7Y

and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5' GACTAAAGCTTAATGGTAAGCGTTAACAGC 3' (SEQ ID
No. 15) contains a HindIII restriction enzyme site followed by 19 nucleotides of GPR4 coding sequence starting from the presumed terminal amino acid of the processed protein. The 3' sequence 5' GAACTTCTAGACTTCAGGCAGCAGATTCATT 3' (SEQ ID No.
16) contains complementary seguences to XbaI site and is followed by 20 nucleotides of GPR4 coding sequence. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc.
Chatsworth, CA). pQE-9 encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome h;n~;ng site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 is then digested with HindIII and X~aI. The amplified sequences are ligated into pQE-9 and are inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture is then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory ~nll~l, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
Clones cont~i n~ ng the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N
culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.~') of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") is then added to a final concentration o~ 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/O leading to increased gene CA 0221683~ 1997-09-29 W 09'~30~~6 PCTnUS9~ 4079 expression. Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl.
After clarification, solubilized GPR4 is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow ~or tight binding by proteins contAining the 6-His tag (Hochuli, E. et al., J.
Chromatography 411:177-184 (1984)). GPR4 is eluted from the column in 6 molar guanidine HCl pH S.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.

Example 5 Expression of Recombinant GPRl in COS cells The expression of plasmid, GPRl HA is derived from a vector pcDNAI/Amp (Invitrogen) cont~ining: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a Sv40 intron and polyadenylation site. A DNA
fragment encoding the entire GPRl precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The ~A tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H.
Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmld construction strategy is described as follows:

CA 0221683~ 1997-09-29 W O~. 301 ~ PCTrUS95/04079 The DNA sequence encoding GPRl, ATCC # 75981, is constructed by PCR using two primers: the 5' primer 5' GTCCAAGCTTGCCACCATGAGTAGTGAAATGGTG 3' tSEQ ID No 17) contains a HindIII site followed by 18 nucleotides of GPRl coding sequence starting from the initiation codon; the 3' sequence 5' CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCAGG
-l~lAAATCAGG 3' (SEQ ID No. 18) contains complementary sequences to an XhoI site, translation stop codon, HA tag and the last 15 nucleotides of the GPRl coding sequence (not including the stop codon). Therefore, the PCR product contains a HindIII site, GPR1 coding sequence followed by HA
tag fused in frame, a translation termination stop codon next to the HA tag, and an XhoI site. The PCR amplified DNA
fragment and the vector, pcDNAI/Amp, are digested with HindIII and XhoI restriction enzymes and ligated. The ligation mixture is transformed into E. coli strain SURE
(Stratagene Cloning Systems, La Jolla, CA) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment. For expression of the recombinant GPRl, COS cells are transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E.
Fritsch, T. Maniatis, Molecular Cloning: A Laboratory MAn~
Cold Spring haboratory Press, (1989)). The expression of the GPRl HA protein is detected by radiolabeling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hours with 35S-cysteine two days post transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1%
NP-40, 0.1~ SDS, 1% NP-40, 0.5~ DOC, 50mM Tris, pH 7.5).
(Wilson, I. et al , Id. 37:767 (1984)). Both cell lysate and culture media are precipitated with a HA specific monoclonal CA 022l683~ l997-09-29 W096/30406 P~TnUS9S)014079 antibody. Proteins precipitated are analyzed on 15% SDS-PAGE
gels.

Example 6 Expression of Recombinant GPR2 in COS cells The expression of plasmid, GPR2 HA is derived from a vector pcDNAI/Amp (Invitrogen) cont~n~ng: 1) Sv40 origin o~
replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA
fragment encoding the entire GPR2 precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H.
Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as ~ollows:
. The DNA sequence encoding for GPR2, ATCC # 75983, is constructed by PCR using two primers: the 5' primer 5' GTCCAAGCTTGCCACCA~ l w l~CACCTGG 3' (SEQ ID No. 19) contains an HindIII site followed by 18 nucleotides of GPR2 coding sequence starting from the initiation codon; the 3' sequence 5' CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGG~GTG
GATCCCCCGTGC 3' (SEQ ID No. 20) contains complementary sequences to an XhoI site, translation stop codon, HA tag and the last 15 nucleotides of the GPR2 coding sequence (not including the stop codon). Therefore, the PCR product contains a HindIII site, GPR2 coding sequence followed by HA
tag fused in frame, a translation termination stop codon next to the HA tag, and an XhoI site. The PCR amplified DNA

CA 0221683~ 1997-09-29 W 096/30406 PCTrUS9S/04079 ~ragment and the vector, pcDNAI/Amp, are digested with HindIII and XhoI restriction enzymes and ligated. The ligation mixture is transformed into E. coli strain SURE
(Stratagene Cloning Sy~tems, La Jolla, CA) the transformed culture i5 plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and P~mined by restriction analysis for the presence of the correct fragment. For expression of the recombinant GPR2, COS cells are transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E.
Fritsch, T. Maniatis, Molecular Cloning: A Laboratory ~n~
Cold Spring Laboratory Press, (1989)). The expression of the GPR2 HA protein is detected by radiolabelling and immllnoprecipitation method (E. Harlow, D. Lane, Antibodies:
A Laboratory MAnll~l, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hours with 35S-cysteine two days post transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1 NP-40, 0.1~ SDS, 1% NP-40, 0.5~ DOC, 50mM Tris, pH 7.5).
(Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media are precipitated with a HA specific monoclonal antibody. Proteins precipitated are analyzed on 15~ SDS-PAGE
gels.

Exam~le 7 Expression of Recombinant GPR3 in COS cells The expression of plasmid, GPR3 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA
fragment encoding the entire GPR3 precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recomblnant protein expression is directed under the CMV promoter. The HA tag CA 0221683~ 1997-09-29 W 096/3040~ PCTnUS9~1)4079 correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H.
Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The in~usion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as follows:
The DNA sequence encoding for GPR3, ATCC # 75976, is con~tructed by PCR using two primers: the 5' primer 5 GTCCAAGCTTGCCACCATGAACACCACAGTAATG 3' (SEQ ID No. 21) contains a HindIII site followed by 18 nucleotides of GPR3 coding se~uence starting from the initiation codon; the 3' sequence 5' CTAGCTCGAGTCAAGCGTA~r~ w GA~l~lATGGGTAGCAAGG
GATCCATACAAATGT 3' (SEQ ID No. 22) cont~nC complementary sequences to an XhoI site, translation stop codon, HA tag and the last 18 nucleotides of the GPR3 coding sequence (not incl~ding the stop codon). Therefore, the PCR product contains a HindIII site, GPR3 coding sequence followed by HA
tag fused in frame, a translation termination stop codon next to the HA tag, and an XhoI site. The PCR amplified DNA
fragment and the vector, pcDNAI/Amp, are digested with HindIII and XhoI restriction enzymes and ligated. The ligation mixture is transformed into E. coli strain. SURE
(Stratagene Cloning Systems, La Jolla, CA) the trans~ormed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment. For expression of the recombinant GPR3, COS cells are transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E.
Fritsch, T. Maniatis, Molecular Cloning: A Laboratory M~n~
Cold Spring Laboratory Press, (1989)). The expression of the GPR3 HA protein is detected by radiolabellinc~ and oprecipitation method (E. Harlow, D. Lane, Antibodies:

CA 0221683~ 1997-09-29 W 096/30406 PCT/u~lo1n79 A Laboratory M~nll~l, Cold Spring Harbor Laboratory Press, ~1988)). Cells are labelled for 8 hours with 35S-cysteine two days post transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1 NP-40, 0.1~ SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media are precipitated with a HA specific monoclonal antibody. Proteins precipitated are analyzed on 15~ SDS-PAGE
gels.

ExamPle 8 Expression of Recombinant GPR4 in COS cells The expression of plasmid, GPR4 HA is derived from a vector pcDNAI/Amp (Invitrogen) cont~n~ng: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA
fragment encodiny the entire GPR4 precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H.
Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as follows:
The DNA sequence encoding for GPR4, ATCC # 75979, is constructed by PCR using two primers: the 5' primer 5' GTCCAAGCTTGCCACCATGGTAAGCGTTAACAGC 3' (SEQ ID No. 23) contains a HindIII site followed by 18 nucleotides of GPR4 coding sequence starting from the initiation codon; the 3' sequence 5' CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCAGG

CA 0221683~ 1997-09-29 WO 96/30406 PCTrUS95~04079 CAGCAGATTCATTGTC 3' (SEQ ID No. 24) contains complementary sequences to an XhoI site, translation stop codon, HA t~g and the last 18 nucleotides of the GPR4 coding sequence (not including the stop codon). Therefore, the PCR product contains a HindIII site, GPR4 coding sequence followed by EA
tag fused in frame, a translation termination stop codon next to the HA tag, and an XhoI site. The PCR amplified DNA
~ragment and the vector, pcDNAI/Amp, are digested with Hind III and XhoI restriction enzymes and ligated. The ligation mixture is trans~ormed into E. coli strain SURE (Stratagene Cloning Systems, La Jolla, CA) the trans~ormed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment. For expression of the recombinant GPR4, COS cells are transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory ~Anll~l, Cold Spring Laboratory Press, (1989)). The expression of the GPR4 HA
protein is detected by radiolabelling and ~mmllnoprecipitation method (E. Harlow, D. Lane, Antibodies: A ~aboratory ~nl-Al, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hours with 35S-cysteine two days post transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% ~rP-40, 0.1~ SDS, 1% NP-40, 0.5~ DOC, 50mM Tris, pH 7.5) (Wilson, I.
et al., Id. 37:767 (1984)). Both cell lysate and cwlture media are precipitated with a HA specific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE
gels.

Example 9 Cloninq and expression of GPR1 usin~ the baculovirus ex~ression sYstem CA 0221683~ 1997-09-29 W 096/30406 PCTrUS9S/04079 The DNA sequence encoding the full length GPR1 protein, ATCC # 75981, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence 5' CGGGATCCCTCCATGAG
TAGTGAAATGGTG 3' (SEQ ID No. 25) and contains a BamHI
restriction enzyme site (in bold) followed by 4 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (~ozak, M., J. Mol. Biol., 196:947-950 (1987) which is just behind the first 18 nucleotides of the GPR1 gene (the initiation codon for translation "ATG" is underlined).
The 3~ primer has the sequence 5' CGGGATCCCGCT
CAGG~'l-l'~l'AAATCAGG 3' (SEQ ID No. 26) and contains the cleavage site for the BamHI restriction ~n~nnllclease and 18 nucleotides complementary to the 3' non-translated sequence of the GPR1 gene. The amplified sequences are isolated from a 1~ agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La ~olla, Ca.). The fragment is then digested with the Pn~onllclease BamHI and then purified again on a 1% agarose gel. This fragment is designated F2.
The vector pRG1 (modification of pVL941 vector, discussed below) is used for the expression of the GPRl protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555).
This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction ~n~o~l~clease BamHI. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant virus the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The CA 0221683~ 1997-09-29 W O 96130406 PCTnUS95/D4079 polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recom~ination of cotran~fected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRGl such as pAc373, p~VL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
The plasmid is digested with the restriction enzymes BamHI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The DNA i5 then isolated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
This vector DNA is designated V2 Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase. E.coli HB101 cells are then transformed and bacteria identified that cont~;n~ the plasmid (pBacGPR1) with the GPRl gene using the enzymes BamHI The sequence of the cloned fragment is confirmed by DNA sequencing.
5 ~g of the plasmid pBacGPRl is cotransfected with 1.0 ~g of a commercially available linearized baculovirus ("BaculoGold~ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al. Proc. Natl.
Acad. Sci. USA, 84:7413-7417 (1987)).
l~g of BaculoGold~ virus DNA and 5 ~g of the plasmid pBacGPR1 are mixed in a sterile well of a microtiter plate containing 50 ~1 of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~1 Lipofectin plus 90 ~1 Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added dropwise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27~C. After 5 hours the transfection solution is removed from the plate and 1 ml of CA 0221683~ 1997-09-29 W 096/30406 PCTnUS95/04079 Grace's insect medium supplemented with 10~ fetal calf serum is added. The plate is put back into an incubator and cultivation continued at 27~C for four days.
After four days the supernatant is collected and a plaque assay performed similar as described by Summers and Smith (supra). As a modification an agarose gel with ~Blue Gal" (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue stained plaques. (A
detailed description of a "plaque assay" can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithers~urg, page 9-10) .
Four days after the serial dilution, the virus are addedto the cells, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar cnnt~n~ng the recombinant viruses is then resuspended in an Eppendorf tube con~n~ng 200 ~l of Grace's medium. The agar is removed by a brief centrifugation and the supernatant cont~n;ng the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then stored at 4~C.
Sf9 cells are grown in Grace~s medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-GPRl at a multiplicity of infection (MOI) of 2. Six hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 ~Ci of 35S-methionine and 5 ~Ci 35S cysteine (Amersham) are added.
The cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.
Numerous modifications and variations of the present invention are possible in light of the above teachings and therefore, within the scope of the appended claims, the W096/30406 PCT~S9~04~79 invention may be practiced otherwise than as particularly described.

W 096130406 PCTnUS95/04079 SE~N~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: LI, ET AL.
(ii) TITLE OF INVENTION: Human G-Protein Coupled Receptors (iii) NUMBER OF SBQUENCES: 26 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) COUNTRY: USA
(F) ZIP: 07068 (v) CO~ul~ READABLE FORM:
(A) MEDIUM TYPE: 3 5 INCH DISKETTE
(B) CO~ 'U'l~: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: Concurrently (C) CLASSIFICATION:
~vii) PRIOR APPLICATION DATA
(A) APPLIC~TION NUMBER:
(B) FILING DATE:

(~iii) Al-lO~N~Y~AGENT lN~O~ ~TION:
(A) NAME: FERRARO, GREGORY D.
(B) REGISTRATION NUMBER: 36,134 (C) REFERENCE/DOCKET NUMBER: 325800-270 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700 (B) TELEFAX: 201-994-1744 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1713 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR

WO 96/3040~ PCTnU~9S1~79 (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GGCACGAGGT CATTCAACAT TTATTCAACC AAAAATACTA AGTCAGCTCT ATACAAACTA 60ATGGA~GGAT ACAGCTATGC AAATATAGAA CACTAAAGTG TTACATGACA GATGTATGAG 120 TAGTGAAATG GTGAAAAATC AGACAATGGT CACAGAGTTC CTCCTACTGG GA'~ 180 - GGGCCCAAGG ATTCAGATGC lC~i~ l~ G~.l-l~-lCC ~-l~il-lATG TCTTCACCCT 240GCTGGGGAAT GGGACCATCC TGGGGCTCAT CTCACTGGAC TCCAGACTCC ACACCCCCAT 300 GTA~-l-l~-l-lC CTCTCACACC TGGC~l~l CAACATCGCC TATGCCTGCA ACACAGTGCC ~60 CCAGATGCTG GTGAACCTCC TGCATCCAGC CAAGCCCATC l~-l-l-l~CTG GTTGCATGAC 420 ACTAGACTTT ~-l~-l-~-l-l-L~A GTTTTGCACA TACTGAATGC ~-l~-l~ll~G TGCTGATGTC 480 CTACGATCGG TACGTGGCCA TCTGCCACCC l~-lC~ATAT TTCATCATCA TGACCTGGAA 540 AGTCTGCATC ACTCTGGGCA TCA~ GACATGTGGC l~C~ -l~G CTA-l~L~A 600 TGTGAGCCTC ATCCTAAGAC TGCC~-l-l-l-l~ TGGGCCTCGT GAAATCAACC A~-l-l~-l-l~-l~ 660TGAAATCCTG l~-l~l~-l~A GGCTGGCCTG TGCTGATACC TGGCTCAACC AG~l~l~AT 720 CTTTGAAGCC TGCATGTTCA l~-l~L~GG ACCACTCTGC CTGGTGCTGG l--L~--lACTC 780 ACACATCCTG GGGGGCATCC TGAGGATCCA ~L~l~G~AG GGCCGCAGAA AGGC--l-l~-lC 840 CACCTGCTCC TCCCACCTCT GCGTAGTGGG A~-l~-l-l~-ll-l GGSAGCGCCA TCGTCATGTA 900 CATGGCCCCT AAGTCCCGCC ATCCTGAGGA GCAGCAGAAG ~lC~l-l-l-l-l~ TTATTTTACA 960 -lC~-l-l-l~'A ACCCCGATGC TTAAACCCCC TGATTTACAA CCCTGAGGAA TGTAGAGGGT 1020 CAA~GGTGCC CTCCGAGGAG ACCACTGTGC AARGRAAGTC ATTCCTAAGG G~ ACAT 1080 TTGAACTGCC AGCCCCAGTT GCCC~ A ~-l~ ~ATGC CCAATTATTG CCTCAACCCA 1140 GAAAAGTTTA ~-lCCC~-ll lA ACTGTGCTTT ACTGACAGAA GGGCAAGCCT l~lCCC~l-l-l 1200 TTTGCAGATA AAATTTTAGA l~L~~ CAA TCAl-l~G~l-l TCTAGGAGAT ~l~l-ll-lAT 1260 CAGACAATTT l-l-l~-l-l-l-lAT TTCACAATTA CTTTAATATC TGTAAAATAA AGAATTATTT 1320 TAAATCATTT TCCCAGTCCC AAAAGTTAAA TACAGGCCAC TTA~-l-l--l-l-l AACCAAATGA 1380 TATAGTTTGG L-l~-l~l~l~C CCACCCAAAT CTCATGTCAA ATTGTAATCC CCGCATGTCA 1440 GCGGAGGGAC CTGGTGGGAG GTGATTGGAT CATGGGGAGG GAl-l-lCCCCC TTG~-l~l-l--l 1500 GTTGATAGTG AACGAGTTCT CACGAAATCT GAL~l-l-lAA AAGTGCAGCA ~-ll~-lCC~-l-l 1560 TG~~ L~l CTCCTGCTGT GCCATGGTAA GACGTGCCTT G~-l-lSCC--l~ GTGCTTCCGC 1620 CATGATTGTA C'~-1-1-1~-1~A GGC~-l~-L~A GCCATGTGGA ACTGTGAGCC AATTAAACTT 1680 11-1~-1-1-lA G/U~ AAAI~I~ AAA 1713 (2) INFORMATION FOR SEQ ID NO:2:
ti) SEQUENCE CHARACTERISTICS
(A) LENGTH: 296 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAP~
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Ser Glu Met Val Lys Asn Gln Thr Met Val Thr Glu Phe Leu Leu Leu Gly Phe Leu Leu Gly Pro Arg Ile Gln Met Leu Leu Phe Gly Leu Phe Ser Leu Phe Tyr Val Phe Thr Leu Leu Gly Asn Gly Thr Ile Leu Gly Leu Ile Ser Leu Asp Ser Arg Leu His Thr Pro Met Tyr Phe Phe Leu Ser His Leu Ala Val Val Asn Ile Ala Tyr Ala Cys Asn Thr Val Pro Gln Met Leu Val Asn Leu Leu His Pro Ala Lys Pro Ile Ser Phe Ala Gly Cys Met Thr Leu Asp Phe CA 0221683~ 1997-09-29 W 096/30406 PCTrUS95/04079 Leu Phe Leu Ser Phe Ala His Thr Glu Cys Leu Leu Leu Val Leu Met Ser Tyr Asp Arg Tyr Val Ala Ile Cys His Pro Leu Arg Tyr Phe Ile Ile Met Thr Trp Lys Val Cys Ile Thr Leu Gly Ile Thr Ser Trp Thr Cys Gly Ser Leu Leu Ala Met Val His Val Ser Leu Ile Leu Arg Leu Pro Phe Cys Gly Pro Arg Glu Ile Asn His Phe Phe Cys Glu Ile Leu Ser Val Leu Arg Leu Ala Cys Ala Asp Thr Trp Leu Asn Gln Val Val Ile Phe Glu Ala Cys Met Phe Ile Leu Val Gly Pro Leu Cys Leu Val Leu Val Ser Tyr Ser His Ile Leu Gly Gly Ile Leu Arg Ile Gln Ser Gly Glu Gly Arg Arg Lys Ala Phe Ser Thr Cys Ser Ser His Leu Cys Val Val Gly Leu Phe Phe Gly Ser Ala Ile Val Met Tyr Met Ala Pro Lys Ser Arg His Pro Glu Glu Gln Gln Lys Val Leu Phe Leu Ile Leu Gln Phe Leu Ser Thr Pro Met Leu Lys Pro Pro Asp Leu Gln Pro (2) INFORMATION FOR SEQ ID NO : 3:
( i ) S EQUENCE CHARACTERI STI CS
(A) LENGTH: 2185 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
( D ) TOPOLOGY: LINEAR
( ii ) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TCACTATAGG GCGAATTGGG TACGGGCCCC CC~-lC~AGGT CGACGGTATC GATAAGCTTG 60 GGAGAAGGAG CCAGAATTGC l~l~l~L~GA GCCGCCATAG GAGCCAGAGG GGTGGCTAGA 360 GTCTCACTAC AATCTTCACA CGCCTTTATT ATTCACCATG GTTGGTGGCA C~-lG~l-lAGC 780 AGCAAGCGGA AGGCTGAGGC CAGTAGGGGC AGGG~L~l-lA ~laG&G~LcG AAGAAGCCAG 840 AGGCTGGCTG ATGAGATGGT GCTGCCCCCC TGCTGACACG AGGTGCACCA CAl-lC~-l-l-lG 960 CA 022l6835 l997-09-29 W096~0406 PCT~S951~4079 CAGCGGGCGG GCTGCCCCAC AGCAAGCTGG CGCACCTGGG CACCATCCAA AATACAGC-l-l 1020 -l-lCC~-lGG ATTTGGAAGG TGAGAGGTTT G~-11~L-1 CCATTAACCA CTGACGTTGT 1080 GCCAGTGAGA CTAACTCTCC GCGCCAATCT GTCCGCGGCT GAC~-lC-~ ' GCGGGCGTGG 1140 ~l-lC--lGCGG CAGGGCTTGC TGGACACAAA CCTCACTGCG l~G~l~GCCA C'ACTGCTGGC 1260 CATCGCCGTG GAGCGGCACC GCAGTGTGAT GGCCGTGCAG CTGCACAGCC GC~-lGCCC~ 1320 TGGCCGCGTG GTCATGCTCA TTGTGGGCGT ~G~lGGCT GCCCTGGGCC TGGGGCTGCT 1380 GCCTGCCCAC TCCTGGCACT GC~-l~-l~l~C CCTGGACCGC TCCTCACGCA TGGCACCCCT 1440 GCTCAGCCGC TCCTATTTGG C~l~-l~GGC l--l~l~AGC CTG~ l~l~-l TCCTGCTCAT 1500 GGTGGCTGTG TACACCCGCA l-l'l-l~l-l'~l'A CGTGCGGCGG CGAGTGCAGC GCATGGCAGA 1560 GCATGTCAGC TGCCACCCCC GCTACCGAGA GACCACGCTC AGC~-l~l~A AGA~-l~l-l~l 1620 CATCATCCTG GGGGCGTTCG 'l~lL-l~CTG GACACCAGGC CAG~-l~lAC TG~-lC~-L~GA 1680 l~-l-l-lAGGC TGTGAGTCCT GCAATGTCCT GGCGTTAGAA AAGTACTTCC TA~-l~l-lGGC 1740 CGAGCCAACC TCA--lG~l~A ATGCTGCTGT GTACTCTTGC CGAGATGCTG AGATGCGCCG 1800 CAC--l-lC-'GC CGC--l~-lCC TGCTGCGCGT GCCTCCGCCA GTCCACCCGC GA~l~-l~lCC 1860 AAATCCACAG CCCCTGATGA ~-l-l~l~l~ ~-L~'~-l~GCTC AACCCAACCT CGTGCCGAAT 204~
TCCTGCAGCC CGGGGGATCC ACTAGTTCTA GAGCGGCGCC ACCGCGGTGG AGCrCCAGCT 2100 l-i-l~llCCCT TTAGTGAGGG TTAATTTCGA GCTTGGCGTA ATCATGGTCA TAG~-l~l-l-lC 2160 ~-l~l~l~AAA TTGTTATCCG CTCAC 2185 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 393 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Arg Pro Thr Trp Ala Gly Trp Leu Met Arg Trp Cys Cyr, Pro Pro Ala Asp Thr Arg Cys Thr Thr Phe Leu Cys Ser Gly Arg Ala Ala Pro Gln Gln Ala Gly Ala Pro Gly His His Pro Lys Tyr Ser Leu Phe Pro Trp Ile Trp Lys Val Arg Gly Leu Leu Pro Pro Pro Leu Thr Thr Asp Val Val Pro Val Arg Leu Thr Leu Arg Ala Asn Leu Ser Ala Ala Asp Leu Leu Arg Gly Arg Gly Leu Pro Leu Pro His Val Pro His Cys Pro Arg Thr Ala Arg Leu Ser Leu Glu Gly Trp Phe Leu Arg Gln Gly Leu Leu Asp Thr Asn Leu Thr Ala Ser Val Ala Thr Leu Leu Ala Ile Ala Val Glu Arg His Arg Ser Val 12S 130 . 135 Met Ala Val Gln Leu His Ser Arg Leu Pro Arg Gly Arg Val Val Met Leu Ile Val Gly Val Trp Val Ala Ala Leu Gly Leu Gly Leu Leu Pro Ala His Ser Trp His Cys Leu Cy~ Ala Leu Asp Arg Ser CA 02216835 l997-09-29 W 096/30406 PCTrUS95/04079 Ser Arg Met Ala Pro Leu Leu Ser Arg Ser Tyr Leu Ala Val Trp Ala Leu Ser Ser Leu Leu Val Phe heu Leu Met Val Ala Val Tyr Thr Arg I le Phe Phe Tyr Val Arg Arg Arg Val Gln Arg Met Ala Glu His Val Ser Cys His Pro Arg Tyr Arg Glu Thr Thr Leu Ser Leu Val Lys Thr Val Val Ile Ile Leu Gly Ala Phe Val Val Cys Trp Thr Pro Gly Gln Val Val Leu Leu Leu A~p Gly Leu Gly Cys Glu Ser Cys Asn Val Leu Ala Leu Glu Lys Tyr Phe Leu Leu Leu Ala Glu Pro Thr Ser Leu Val Asn Ala Ala Val Tyr Ser Cys Arg Asp Ala Glu Met Arg Arg Thr Phe Arg Arg Leu Leu Leu Leu Arg Val Pro Pro Pro Val His Pro Arg Val Cys Pro Leu Tyr Ile Leu Cys Pro Gly Arg Cys Gln His Ser His His Ala Ser Arg Glu Arg Pro Pro Thr Asp Gly Leu His Pro Leu Ala Thr Leu Asn Tyr Ser Gly Thr Arg Gln Ala Thr Asn Pro Gln Pro Leu Met Thr Cys Gly Cys Ser Trp Leu Asn Pro Thr Ser Cys Arg Ile Pro Ala Ala Arg Gly Ile His (2) INFORMATION FOR SEQ ID NO: 5:
( i ) SEQUENCE CH~RACTERISTICS
(A) LENGTH: 1474 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
( ii ) MOLECULE TYPE: cDNA
(xi) SEQUENOE DESCRIPTION: SEQ ID NO:5:

GATAGTACAG CTGGTATTCC CAGCC~l~lA CACAGTGGTT TTcTTGAccG CAATCCTGCT 180 GAATACTTTG G~l~-l~l~GG l~ll-l'~'ll~'A CATCCCCAGC l~lC~ACCT TCATCATCTA 240 CCTCAAAAAC A~-l-llG~l~G CCGACTTGAT AATGACAGTG ATG~-l-lC~-l-l TCAAAATCCT 300 CTCTGACTCA CACCTGGCAC CCTGGCAGCT CAGAGCTTTT Gl~l~l~C-l-l 'l-l-l.-l-l'~'~'l' 360 Al-lC~-l~AAG ATCATCAGAC CTTTGAGAAA TAl-l-l-l-l~-lA AAAAAACCTG ll-l~GGGAAA 480 AACGGTCTCA ATCTTCATCT G~l-l~-ll-l-lG Gl-lL-l-l~ATC TCCCTGCCAA ATATGATCTT 540 GAGCAACAAG GAAGCAACAC CA'l'C~l'~-l~l GAAAAAGTGT G~-l-lC~-l-lAA AGGGGCCTCT 600 GGGGCTGAAA TGGCATCAAA TGGTAAATAA CATATGCCAG TTTATTTTCT GGA~-l~l-l-l-l 660 TATCCTAATG ~-l-l'~'l'~'l-lll' Al~lG~l-lAT TGCAAAAAAG TATATGATTC TTATAGAAAG 720TCCAAAAGTA AGGACAGAAA A~ACAACAAA AAGCTGGAAG GCAAAGTATT l~l~lC~lG 780 WO 96/30406 PCTnUS~51'01~79 G~-l~l~-l-l~-l 1-l'~'l'~'l~l-l-l' TGCTCCATTT CATTTCGCCA GAGTTCCATA TACTCACAGT 840 A~ l-l-l-l' TGGCAGCAAC TAACATTTGT ATGGATCCCT TAATATACAT ATTCTTATGT 960 AAAAAATTCA CAGAAAAGCT ACCATGTATG CAAGGGAGAA AGACCACAGC ATCAAGCCAA. 1020 GAAAATCATA GCAGTCAGAC AGACAACATA ACCTTAGGCT GACAACTGTA CATAGGGGTA. 1080 ACTTCTATTT ATTGATGAGA ~ lCC~LAGA TAATGTGGAA ATCCAATTTA ACCAAGAAAA. 1140 AAAATTTAAA TCCACATAGA TCTATTCATA AGCTGAATGA ACCATTACTA AEAGAATGC~ 1260 ACAGGATACA AATGGCCACT AGAG&TCATT Al-l-l~l-l-l~-l l-l~-l-l-l'~-l-l-l''l-l-l-l-l-ll-l-l-l' 1320 AATTTCAAGA GCATTTCACT TTAACATTTT GGAAAAGACT AAGGAGAAAC GTATATCCCr 1380 ACAAACCTCC CCTCCAAACA C~-l-l~-l"lACA 'l-l~-l-l-l-lC~A CAATTCACAT AACACTACTE 1440 ~'l-l-l-l'~'l'~CC CCTTAAATGT AGAl-l-l~l-l~ GCTG 1474 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 293 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Asn Thr Thr Val Met Gln Gly Phe Asn Arg Ser Lys Arg Cys Pro Lys Asp Thr Arg Ile Val Gln heu Val Phe Pro Ala Leu Tyr Thr Val Val Phe Leu Thr Gly Ile Leu Leu Asn Thr Leu Ala Leu Trp Val Phe Val His Ile Pro Ser Ser Ser Thr Phe Ile Il.e Tyr Leu Lys Asn Thr Leu Val Ala Asp Leu Ile Met Thr Leu M~t Leu Pro Phe Lys Ile Leu Ser Asp Ser His Leu Ala Pro Trp Gln Leu Arg Ala Phe Val Cys Arg Phe Ser Ser Val Ile Phe Tyr Glu Thr Met Tyr Val Gly Ile Val heu Leu Gly Leu Ile Ala Phe Asp Arg Phe heu Lys Ile Ile Arg Pro Leu Arg Asn Ile Phe Leu hys Lys Pro Val Trp Gly Lys Thr Val Ser Ile Phe Ile Trp Phe Phe Trp Phe Phe Ile Ser Leu Pro A~n Met Ile Leu Ser Asn Lys Glu Ala Thr Pro Ser Ser Val Lys Lys Cy8 Ala Ser Leu Lys Gly Pro Leu Gly heu Lys Trp His Gln Met Val Asn Asn Ile Cys Gln Phe Ile Phe Trp Thr Val Phe Ile Leu Met Leu Val Phe Tyr Val Val Ile Ala hys Lys Tyr Met Ile Leu Ile Glu Ser Pro hys Val Arg Thr Glu hys Thr Thr hys Ser Trp Lys Ala hys Tyr Leu Leu Ser Trp r W 096/30406 PCTrUS~51C1~79 Leu Ser Ser Leu Cys Val Leu Leu His Phe Ile Ser Pro Glu Phe His Ile Leu Thr Val Lys Pro Thr Ile Arg Leu Thr Val Asp Cys Lys Ile Asn Cys Leu Leu Leu Lys Lys Gln Leu Ser Phe Trp Gln Gln Leu Thr Phe Val Trp Ile Pro (2) INFORMATION FOR SBQ ID NO: 7 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1301 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 ll-l-lG~lAT TTCTGAGAAA AAGGAAATAT TTATAAAACC ATCCAAAGAT CCAGATAATT 60 CCACTGCTTC TATAATGACT CCTTTAAGTA CA~-l-l-l~lAT GGGTGCATGT TCAGCATGGT 240 ~ l~lGCTT GGGTTAATAT CCAATTGTGT TGCCATATAC ATTTTCATCT GC~lC--l AA 300 AGTCCGAAAT GAAACTACAA CTTACATGAT TAACTTGGCA ATGTCAGACT TG~-l-ll-l-l~l 360 TTTTACTTTA CC~-l-l~AGGA l-l-l-l-l-lACTT CACAACACGG AATTGGCCAT TTGGAGATTT 420 A~-l-l-l~lAAG Al-l-l--l~lGA TG~-i~l-l-l-lA TACCAACATG TACGGAAGCA 1-l~-l~l-l~-ll 480 AACCTGTATT AGTGTAGATC GAll-l~-lGGC AAl-l~l-lAC CCATTTAAGT CAAAGACTCT 540 AAGAACCAAA AGAAATGCAA AGAl-l~l-l-lG ACATGGCGTG TGGTTAACTG TGATCGGAGG 600 AAGTGCACCC GC~i-l-l-l-lG TTCAGTCTAC CCACTCTCAG GGTAACAATG CCTCAGAAGC 660 CTGCTTTGAA AAl-l-l-lC~AG AAGCCACATG GAAAACATAT CTCTCAAGGA TTGTAATTTT 720 CATCGAAATA GTGGGATTTT TTAl-lC~-l~-l AATTTTAAAT GTAACTTGTT CTAGTATGGT 780 GCTAAAAACT T~AACCAAAC CTGTTACATT AAGTAGAAGC AAAATAAACA AAACTAAGGT 840 TTTAAAAATG Al-~l-l-l~lAC ATTTGATCAT Al-l~-l~l-l-lC '1~1-l-l L~l-lC CTTACAATAT 900 AGCAGTAAGG ACAATGTACC CAATCACTCT CTGTATTGCT ~l-l-lC~AACT ~l~lll-l~A 1020 CCCTATAGTT TACTACTTTA CATCGGACAC AATTCA~AAT TCAATAAAAA TGAAAAACTG 1080 .l~l~AGG AGAAGTGACT TCAGATTCTC TGAAGTTCAT GGTGCAGAGA ATTTTATTCA 1140 GCATAACCTA CAGACCTTAA AAAGTAA~AT ATTTGACAAT GAATCTGCTG CCTGAAATAA 1200 AACCATTAGG ACTCACTGGG ACAGAACTTT CAA~l-lC~-l-l CAACTGTGAA AA~l~l--l-l-l 1260 TTGGACAAAC TAlll-llC~A CCTCCAAAAG AAATTAACAC A 1301 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 344 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Val Ser Val Asn Ser Ser His Cys Phe Tyr Asn Asp Ser Phe CA 022l6835 l997-09-29 W Og6/30406 PCTnUS9~/04079 Lys Tyr Thr Leu Tyr Gly Cys Met Phe Ser Met Val Phe Val Leu Gly Leu Ile Ser Asn Cys Val Ala Ile Tyr Ile Phe Ile Cys Val Leu Lys Val Arg Asn Glu Thr Thr Thr Tyr Met Ile Asn Leu Ala Met Ser Asp Leu Leu Phe Val Phe Thr Leu Pro Phe Arg Ile Phe Tyr Phe Thr Thr Arg Asn Trp Pro Phe Gly Asp Leu Leu Cys Lys Ile Ser Val Met Leu Phe Tyr Thr Asn Met Tyr Gly Ser Ile Leu Phe Leu Thr Cys Ile Ser Val Asp Arg Phe Leu Ala Ile ValL Tyr Pro Phe Lys Ser Lys Thr Leu Arg Thr Lys Arg Asn Ala Lys Ile Val Cys Thr Gly Val Trp Leu Thr Val Ile Gly Gly Ser Ala Pro Ala Val Phe Val Gln Ser Thr His Ser Gln Gly Asn Asn Ala Ser Glu Ala Cys Phe Glu Asn Phe Pro Glu Ala Thr Trp Lys Thr Tyr Leu Ser Arg Ile Val Ile Phe Ile Glu Ile Val Gly Phe Phe Ile Pro Leu Ile Leu Asn Val Thr Cys Ser Ser Met Val Leu Lys Thr Leu Thr Lys Pro Val Thr Leu Ser Arg Ser Lys Ile Asn Lys Thr Lys Val Leu Lys Met Ile Phe Val His Leu Ile Ile Phe Cys Phe Cys Phe Val Pro Tyr Asn Ile Asn Leu Ile Leu Tyr Ser Leu Val Arg Thr Gln Thr Phe Val Asn Cys Ser Val Val Ala Ala Va]. Arg Thr Met Tyr Pro Ile Thr Leu Cys Ile Ala Val Ser Asn Cy~ Cys Phe Asp Pro Ile Val Tyr Tyr Phe Thr Ser Asp Thr Ile Gln Asn Ser Ile Lys Met Lys Asn Trp Ser Val Arg Arg Ser Asp Phe Arg Phe Ser Glu Val His Gly Ala Glu Asn Phe Ile Gln His Asn Leu Gln Thr Leu Lys Ser Lys Ile Phe Asp Asn Glu Ser Ala Ala (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
( C ) STRANDEDNES S: S INGLE
(D) TOPOLOGY: LINEAR

W 096/30406 PCTrUS95/04079 (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 31 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) ST~ANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GACTA~AGCT TAATGAGGCC CACATGGGCA 30 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 32 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GACTA~AGCT TAATGGCGTC TTTCTCTGCT 30 W ~96130406 PCTnUS9~104D79 (2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 }3ASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STR~NDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GAACTTCTAG ACTTCACACA ~l-l~lACTAT 30 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 3 0 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 31 BASE PAIRS
(B) TYPE: NUCLE IC ACID
(C) STRANDEDNESS SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GAA~-l-l~-lAG ACTTCAGGCA GCAGATTCAT T 31 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 34 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GTCCAAGCTT GCCACCATGA GTAGTGA~AT GGTG 34 (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS
- (A) LENGTH: 58 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE

W 096130406 PCTnUS95/04079 ~ D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide txi) SEQU~ DESCRIPTION: SEQ ID NO:18:

(2) INPORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 34 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GTCCAAGCTT GCCACCATGG l-l~l~GCAC CTGG 34 (2) INFORMATION FOR SEQ ID NO:20:
(i). SEQUENCE CHARACTERISTICS
(A) LENGTH: 58 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
CTAGCTCGAG TCAAGCGTAG TCTGGGACGT CGTATGGGTA GCAGTGGATC CCC~l~C 58 (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 34 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUEN OE DESCRIPTION: SEQ ID NO:21:

(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 61 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

-CA 022l6835 l997-09-29 W 096/30406 PCTAUS9~4079 CTAGCTCGAG TCAAGCGTAG TCTGGGACGT CGTATGGGTA GC~AGGGATC CATACAAATG 60 ~2) INFORMATION FOR SEQ ID NO:23:
(i~ SEQUENCE CHARACTERISTICS
(A) LENGTH: 34 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 61 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLE IC ACID
(C) STRANDEDNESS SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 29 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Claims (32)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide encoding the polypeptide as set forth in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8 or fragments, analogs or derivatives of said polypeptides;
(b) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a); and (c) a fragment of the polynucleotides of (a) or (b) wherein said fragment has at least 50 nucleotides.

2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA.

4. The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA.

5. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) the polynucleotide of Claim 2 encoding a polypeptide having the amino acid sequence encoded by the DNA contained in ATCC Deposit No. 75981;
(b) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a); and (c) a fragment of the polynucleotides of (a) or (b) wherein said fragment has at least 50 nucleotides.

6. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) the polynucleotide of Claim 2 encoding a polypeptide having the amino acid sequence encoded by the DNA contained in ATCC Deposit No. 75983;

(b) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a); and (c) a fragment of the polynucleotides of (a) or (b) wherein said fragment has at least 50 nucleotides.

7. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) the polynucleotide of Claim 2 encoding a polypeptide having the amino acid sequence encoded by the DNA contained in ATCC Deposit No. 75967;
(b) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a); and (c) a fragment of the polynucleotides of (a) or (b) wherein said fragment has at least 50 nucleotides.

8. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) the polynucleotide of Claim 2 encoding a polypeptide having the amino acid sequence encoded by the DNA contained in ATCC Deposit No. 75979;
(b) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a); and (c) a fragment of the polynucleotides of (a) or (b) wherein said fragment has at least 50 nucleotides.

9. The polynucleotide of Claim 1 encoding the polypeptide having the amino acid sequence as shown in SEQ
ID No. 2.

10. The polynucleotide of claim 9 having the coding sequence as shown in SEQ ID No. 1 from nucleotide 1 to nucleotide 1713.

11. The polynucleotide of Claim 1 encoding the polypeptide having the amino acid sequence as shown in SEQ
ID No. 4.

12. The polynucleotide of claim 11 having the coding sequence as shown in SEQ ID No. 3 from nucleotide 1 to nucleotide 2185.

13. The polynucleotide of Claim 1 encoding the polypeptide having the amino acid sequence as shown in SEQ
ID No. 6.

14. The polynucleotide of claim 13 having the coding sequence as shown in SEQ ID No. 5 from nucleotide 1 to nucleotide 1474.

15. The polynucleotide of Claim 1 encoding the polypeptide having the amino acid sequence as shown in SEQ
ID No. 8.

16. The polynucleotide of claim 15 having the coding sequence as shown in SEQ ID No. 7 from nucleotide 1 to nucleotide 1301.

17. A vector containing the DNA of Claim 2.

18. A host cell genetically engineered with the vector of Claim 17.

19. A process for producing a polypeptide comprising:
expressing from the host cell of Claim 18 the polypeptide encoded by said DNA.

20. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 17.

21. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having G-protein coupled receptor activity.

22. A polypeptide selected from the group consisting of: (i) a polypeptide having the deduced amino acid sequence of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No. 8 and fragments, analogs and derivatives thereof, (ii) a polypeptide encoded by the cDNA of ATCC
Deposit No. 75981, ATCC Deposit No. 75983, ATCC Deposit No.
75976 and ATCC Deposit No. 75979 and fragments, analogs and derivatives of said polypeptide.

23. An antibody against the polypeptide of claim 22.

24. A compound which activates the polypeptide of Claim 22.

25. A compound which inhibits activation of the polypeptide of claim 22.

26. A method for the treatment of a patient having need to activate a G-protein coupled receptor comprising:
administering to the patient a therapeutically effective amount of the compound of Claim 24.

27. A method for the treatment of a patient having need to inhibit activation of a G-protein coupled receptor comprising: administering to the patient a therapeutically effective amount of the compound of Claim 25.

28. The polypeptide of Claim 22 wherein the polypeptide is a soluble fragment of the G-protein coupled receptor and is capable of binding a ligand for the receptor.

29. A process for identifying antagonists and agonists to the polypeptide of claim 22 comprising:
contacting a cell which expresses a G-protein coupled receptor with a known receptor ligand and a compound to be screened; and determining if the compound inhibits or enhances activation of the receptor.

30. A process for determining whether a ligand not known to be capable of binding to the polypeptide of claim 22 can bind thereto comprising:
contacting a mammalian cell which expresses a G-protein coupled receptor with a potential ligand;
detecting the presence of the ligand which binds to the receptor; and determining whether the ligand binds to the G-protein coupled receptor.

31. A method for diagnosing a disease or a susceptibility to a disease comprising:

detecting a mutation in the nucleic acid sequence encoding the polypeptide of claim 22 in a sample derived from a host.

32. A diagnostic process comprising:
analyzing for the presence of the polypeptide of claim 28 in a sample derived from a host.