CA2221706A1 - G-protein receptor htnad29 - Google Patents

Abstract

Human G-protein PAF 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 PAF receptor polypeptides. Also disclosed are diagnostic methods for detecting a mutation in the PAF receptor nucleic acid sequences and detecting a level of the soluble form of the receptors in a sample derived from a host.

Description

W O 96/39442 PCT~US95/07288 This invention relates to newly identi~ied polynucleotides, polypeptides encoded by such polynucleotides, the use o~ such polynucleotides and polypeptides, as well as the production o~ such polynucleotides and polypeptides. More particularly, the polypeptide o~ the present invention is a human 7-transmembrane receptor which has been putatively identi~ied as a platelet activating ~actor receptor, sometimes hereina~ter re~erred to as "G-protein PAF Receptor". The invention also relates to inhibiting the action o~ 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 o~ these proteins include the GPC
receptors, such as those ~or adrenergic agents and dor~m; n~
(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 themsel~es, e~ector proteins, e.g., phospholipase C, adenyl cyclase, and O 96/39442 PCTrUS95/07288 phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 252:802-8 (1991)).
For example, in one ~orm o~ signa] transduction, the e~ect o~ hormone binding is activation o~ an enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence o~ the nucleotide GTP, and GTP also in~luences hormone b;n~ing A G-protein connects the hormone receptors to adenylate cyclase. G-protein was shown to ~x~h~nge GTP ~or bound GDP when activated by hormone receptors. The GTP-carrying ~orm then binds to an activated adenylate cyclase. Hydrolysis o~ GTP
to GDP, catalyzed by the G-protein itsel~, returns the G-protein to its basal, inactive ~orm. l'hus, the G-protein serves a dual role, as an intermediate that relays the signal ~rom receptor to e~ector, and as a clock that controls the duration o~ the signal.
The membrane protein gene super~amily o~ G-protein coupled receptors has been characterize~d as having seven putative transmembrane ~om~; n~ . The domains are believed to represent transmembrane ~-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range o~ biologically active receptors, such as hormone, viral, growth ~actor and neuroreceptors.
G-protein coupled receptors have been characterized as including these seven conserved hydrophobic stretches o~
about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein ~amily o~ coupled receptors includes dopamine receptors which bind to neuroleptic drugs used ~or treating psychotic and neurological disorders. Other examples of members o~ this ~amily include calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, hist~m; n~, thrombin, kinin, ~ollicle stimulating hormone, opsins, O 96/39442 PCT~US95/07288 endothelial differentiation gene-l receptor and rhodopsins, odorant, cytomegalovirus receptors, etc.
Most G-protein coupled receptors have single conserved cysteine residues in each of the ~irst two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.
Phosphorylation and lipidation (palmitylation or ~arnesylation) 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 transmembrane ~om~in~, which socket is surrounded by hydrophobic residues of the G-protein coupled receptors. The hydrophilic side o~ each G-protein coupled receptor transmembrane helix is postulated to face inward and form the polar ligand binding site. TM3 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 phenylalanines or tyrosines are also implicated in ligand binding.
G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al ., Endoc., Rev., 10:317-331 (1989)). Different G-protein ~-subunits preferentially stimulate particular effectors to modulate ~arious biological functions in a cell.

W O 96/39442 PCT~US95/07288 Phosphorylation of cytoplasmic residues of G-protein coupled receptors have been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a m~mm~l ian host.
In accordance with one aspect o~ the present invention, there are provided novel mature receptor polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The receptor 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 the receptor polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as antisense analogs thereof and biologi.cally active and diagnostically or therapeutically useful fragments thereof.
In accordance with a further aspect o~ the present invention, there are provided processes for producing such receptor polypeptides by recombinant techniques comprising culturing reco~m--binant prokaryotic and/or eukaryotic host cells, containing nucleic acid sequences encoding the receptor polypeptides of the present invention, under conditions promoting expression of said polypeptides and subsequent recovery of said polypeptides.
In accordance with yet a further aspect of the present invention, there are provided antibodies against such receptor polypeptides.
In accordance with another aspect: of the present invention there are provided methods of screening for compounds which bind to and activate or inhibit activation of the receptor polypeptides of the present invention.
In accordance with still another embodiment of the present invention there are provided processes of ~m;n; ~tering compounds to a host which bind to and activate -W O 96/39442 PCT~US95/07288 the receptor polypeptide of the present invention which are use~ul in the prevention and/or treatment o~ hemophilia by inducing platelet aggregation, and to promote wound healing.
In accordance with still another embodiment o~ the present invention there are provided processes o~
~m; n ~ stering compounds to a host which bind to and inhibit activation o~ the receptor polypeptides of the present invention which are use~ul in the prevention and/or treatment o~ allergy, in~lammation, restenosis a~ter angioplasty, unstable angina, myocardial in~arction and thrombotic or thromboembolytic stroke.
In accordance with yet another aspect o~ the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of su~icient length to specifically hybridize to the polynucleotide sequences o~ the present invention.
In accordance with still another aspect o~ the present invention, there are provided diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences encoding such polypeptides and ~or detecting an altered level o~ the soluble ~orm o~ the receptor polypeptides.
In accordance with yet a ~urther aspect o~ the present invention, there are provided processes ~or utilizing such receptor polypeptides, or polynucleotides encoding such polypeptides, ~or in vitro purposes related to scienti~ic research, synthesis o~ DNA and manu~acture o~ DNA vectors.
These and other aspects o~ the present invention should be apparent to those skilled in the art ~rom the teachings herein.
The following drawings are illustrative o~ embodiments o~ the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figure 1 shows the cDNA sequence and the correspon~;ng deduced amino acid sequence o~ the G-protein receptor o~ the present invention which has been putatively identi~ied as a 2 PCT~US95/07288 platelet-activating factor receptor. The stAn~rd one-letter abbreviation for amino acids is used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).
Figure 3 illustrates an amino acid alignment of the G-protein receptor of the present invention (top line) and the human PAF receptor (bottom line).
In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. on June 1, 1995.
A polynucleotide encoding a polypep~ide of the present invention may be found in leukocytes, lung and kidney. The polynucleotide of this invention was discovered in a cDNA
library derived from a human thyroid. It is structurally related to the G protein-PAF receptor fami.ly. It contains an open re~A; ng frame encoding a protein of 337 amino acid residues. The protein exhibits the highest degree of homology to a human PAF receptor with 29.375 % identity and 53.438 % similarity over a 334 amino acid stretch.
The polynucleotide 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-csA; ng (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID
NO:1) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the rPAllnA~ncy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 (SEQ ID NO:1) or the deposited cDNA.

W O 96/39442 PCT~US95/07288 The polynucleotide which encodes ~or the mature polypeptide o~ Figure 1 (SEQ ID NO:2) or ~or the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence ~or the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence; the coding sequence ~or the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' o~ the coding sequence for the mature polypeptide.
Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding sequence ~or the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
The present invention ~urther relates to variants o~ the hereinabove described polynucleotides which encode ~or ~ragments, analogs and derivatives o~ the polypeptide having the deduced amino acid sequence o~ Figure 1 ( SEQ ID NO:2) or the polypeptide encoded by the cDNA o~ the deposited clone.
The variant of the polynucleotide may be a naturally occurring allelic variant o~ the polynucleotide or a non-naturally occurring variant o~ the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants o~ such polynucleotides which variants encode ~or a ~ragment, derivative or analog o~ the polypeptide o~ Figure 1 (SEQ ID
NO:2) or the polypeptide encoded by the cDNA o~ the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant o~ the coding sequence shown in Figure 1 ( SEQ ID
NO-l) or o~ the coding sequence o~ the deposited clone. As - known in the art, an allelic variant is an alternate ~orm o~

=

O 96/39442 PCT~US95/07288 a polynucleotide sequence which may have a substitution, deletion or addition o~ one or more nucleotides, which does not substantially alter the function o~ the encoded polypeptide.
The polynucleotides may also encode for a soluble ~orm of the PAF receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved from the TM
and intracellular domain of the ~ull-length polypeptide of the present invention.
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 polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vect:or to provide for puri~ication of the mature polypeptide fused to the marker in the case of a bacterial host, or, ~or e~xample, the marker sequence may be a hemagglutinin (HA) tag when a m~mm~l ian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein tWilson, I., et al., Cell, 37:767 (1984)).
The present invention ~urther relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70~, preferably at least 90~, and more preferably at least 95~ identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the here;n~hove-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. I'he 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 Figure 1 (SEQ ID NO:1) or the deposited cDNA(s), i.e. function as a W O 96/39442 PCT~US95/07288 soluble PAF receptor by retaining the ability to bind the ligands for the receptor even though the polypeptide does not function as a membrane bound PAF receptor, for example, by eliciting a second messenger response.
Alternatively, the polynucleotides may have at least 20 bases, preferably at least 30 bases and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which have an identity thereto, as hereinabove described, and which may or may not retain acti~ity. For example, such polynucleotides may be employed as probes for the polynucleotide o~ SEQ ID N0: 1, or for variants thereof, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
Thus, the present invention is directed to polynucleotides having at least a 70~ identity, preferably at least 90~ and more preferably at least a 95~ identity to a polynucleotide which encodes the polypeptide of SEQ ID N0:2 as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.
The deposit(s) referred to herein will be maintained 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 sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence 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 a PAF receptor ~ polypeptide which has the deduced amino acid sequence of O 96/39442 PCT~US95/07288 Figure 1 (SEQ ID NO:2) or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives o~ such polypeptide.
The terms "fragment," "derivative" and "analog" when re~erring to the polypeptide o~ Figure 1 (SEQ D NO:2) or that encoded by the deposited cDNA, means a polypeptide which either retains substantially the same biological ~unction or activity as such polypeptide, i.e. functions as a PAF
receptor, or retains the ability to bind the ligand ~or the receptor even though the polypeptide does not function as a G-protein PAF receptor, for example, a soluble ~orm o~ the receptor.
The polypeptide of the present i3lvention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
The ~ragment, derivative or analog o~ the polypeptide of Figure 1 (SEQ ID NO:2) or that encoded by the deposited cDNA may be (i) one in which one or more! o~ the amino acid residues are substituted with a conserved or non-conserved amino acid residue (pre~erably 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 o~ the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is ~used with another compound, such as a compound to increase the half-life o~ the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide which are employed ~or puri~ication o~ the mature polypeptide or a proprotein sequence or (v) one in which a fragment 03- the polypeptide is soluble, i.e. not membrane bound, yet still binds ligands to the membrane bound receptor. Such fragments, derivatives and analogs are deemed to be within the scope o~ those skilled in the art from the teachings herein.

W O 96/39442 PCTAUS~ /'88 The polypeptides and polynucleotides o~ the present invention are preferably provided in an isolated ~orm, and pre~erably are puri~ied to homogeneity.
The polypeptides of the present invention include the polypeptide o~ SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70%
similarity (preferably at least 70~ identity) to the polypeptide of SEQ ID NO:2 and more pre~erably at least 90%
similarity (more preferably at least 90% identity) to the polypeptide o~ SEQ ID NO:2 and still more preferably at least 95% similarity (still more pre~erably at least 95~ identity) to the polypeptide o~ SEQ ID NO:2 and includes portions of such polypeptides generally cnnt~;n;ng at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "similarity~ between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes o~ one polypeptide to the sequence of a second polypeptide.
Fragments or portions o~ the polypeptides o~ the present invention may be employed for producing the correspon~; ng ~ull-length polypeptide by peptide synthesis, there~ore, the fragments may be employed as intermediates ~or producing the full-length polypeptides. Fragments or portions o~ the polynucleotides o~ the present invention may be used to synthesize full-length polynucleotides o~ the present invention.
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and ~ollowing the coding region "leader and trailer~ as well as intervening sequences (introns) between individual coding segments (exons).
The term "isolated" means that the material is removed ~rom its original environment (e.g., the natural envi~ el~t i~ it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living W O 96/39442 PCT~US95/07288 ~nim~l iS not isolated, but the same polynucleotide or polypeptide, separated from some or al] o~ 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 t:hat such vector or composition is not part of its natural e~vironment.
The polypeptides of the present invention include the polypeptide of SEQ ID N0:2 (in part:icular the mature polypeptide) as well as polypeptides whic:h have at least 70~
similarity (pre~erably at least 70~ identity) to the polypeptide of SEQ ID N0:2 and more preferably at least 90~
similarity (more pre~erably at least 90~ identity) to the polypeptide o~ SEQ ID N0:2 and still more pre~e-ably at least 95~ similarity (still more pre~erably at least 95~ identity) to the polypeptide of SEQ ID N0:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes o~ one polypeptide to the sequence of a second polypeptide.
Fragments or portions o~ the polypept:ides o~ the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; there~ore, the ~ragments may be employed as intermediates for producing the ~ull-length polypeptides. Fragments or portions of the polynucleotides o~ the present invention may be used to synthesize ~ull-length polynucleotides of the present invention.
The present invention also relates to vectors which include polynucleotides o~ the present invention, host cells which are genetically engineered witll vectors o~ the invention and the production of polypeptides of the invention by recombinant techni~ues.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of 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 modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing 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.
However, 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 ~n~onllclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope o~
those skilled in ~he 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 -~ =~

examples o~ such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli ac or trp, the phage lambda PL
promoter and other promoters known to control expression o~
genes in prokaryotic or eukaryotic cells or their viruses.
The expression vector also contains a r-bosome binding site ~or translation initiation and a transcription terminator.
The vector may also include appropriate sequences ~or ampli~ying expression.
In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait ~or selection o~ transformed host cells such as dihydro~olate reductase or neomycin resisl_ance ~or eukaryotic cell culture, or such as tetracycl:ine 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 trans~orm an appropriate host to permit the host to express the protein.
As representative examples o~ appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomvces, Salmonella t~phimurium; ~ungal cells, such as yeast; insect cells such as Drosophila and SPodoPtera S~9;
~n;m~l cells such as CHO, COS or Bowes melanoma; adenovirus;
plant cells, etc. The selection o~ an appropriate host is deemed to be within the scope o~ those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs comprising one or more o~ the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence o~ the invention has been inserted, in a ~orward or reverse orientation. In a pre~erred aspect o~ this embodiment, the construct ~urther contprises regulatory sequences, including, ~or example, a promoter, operably linked to the sequence. Large numbers o~ suitable vectors W O 96/394~2 PCT~US95/07288 and promoters are known to those o~ skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, 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 (chloramphenicol 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 ;mme~;ate 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 containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a m~mm~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, DEAE-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 encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

W O 96/39442 PCTAUS9~/07288 Mature proteins can be expressed ln m~mm~l ian cells, yeast, bacteria, or other cells under the control o~
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 ~nll~l, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
Transcription of the DNA encoding ~he polypeptides of the present invention by higher eukaryot:es is increased by inserting an enhancer 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 cy1_omegalovirus 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 operon~ encoding glycolytic enzymes such as 3-phosphoglycerate kina~e (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, and preferably, a leader ~2quence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification o~ expressed recombinant product.
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 phase with a functional promoter. The vector will comprise one r 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, Salmonella tyPhimurium and various species within the genera Pseudomonas, 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 o~ replication derived from commercially available plasmids comprising genetiG
elements of the well known cloning vector pBR322 (ATCC
37017). Such commercial vectors include, ~or example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone"
sections are combined with an appropriate promoter and the structural sequence to be expressed.
Following trans~ormation of a suitable host strain and growth o~ 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 centri~ugation, 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 ~reeze-thaw W O 96/39442 PCT~US95/07~88 cycling, sonication, mechanical disrupti.on, or use o~ cell lysing agents, such methods are well know to those skilled in the art.
Various m~mm~l ian cell culture systems can also be employed to express recombinant protein. Examples of m~mm~l ian expression systems include the COS-7 lines o~
monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, ~or example, the C127, 3T3, CHO, He~a and BHK cell lines. ~mm~l ian expression vectors will comprise an origin o~ replication, a suitable promoter and enhancer, and also any necessary ribosome b; n~; n~ sites, polyadenylation site, splice donor and acceptor sites, transcriptional terr.;ination sequences, and 5' ~lanking nontranscribed sequences. DNA sequence~ derived ~rom the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The G-protein PAF receptor polypept:ides o~ the present invention can be recovered and puri~ied ~rom recombinant cell cultures by methods including ~mmo~; um sulfate or ethanol precipitation, acid extraction, anion or cation ~h~nge chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, af~inity chromatography, hydroxylapatite chromatography and lectin chromatography.
Protein refolding steps can be used, as necessary, in completing con~iguration o~ the mature protein. Finally, high per~ormance liquid chromatography (HPLC) can be employed ~or ~inal puri~ication steps.
The polypeptides o~ the present invention may be a naturally puri~ied product, or a product o~ chemical synthetic procedures, or produced by recombinant techniques ~rom a prokaryotic or eukaryotic host (~or example, by bacterial, yeast, higher plant, insect and m-mm~lian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides o~ the present invention may be glycosylated or may be non-glycosylated.
Polypeptides o~ the invention may also include an initial methionine amino acid residue.
The polynucleotides and polypeptides o~ the present invention may be employed as research reagents and materials ~or discovery of treatments and diagnostics to human disease.
The G-protein PAF receptors o~ the present invention may be employed in a process for screening ~or compounds which activate (agonists) or inhibit activation (antagonists) o~
the receptor polypeptide of the present invention .
In general, such screening procedures involve providing appropriate cells which express the receptor polypeptide o~
the present invention on the sur~ace thereo~. Such cells include cells from m~mm~ls, yeast, drosophila or E. Coli. In particular, a polynucleotide encoding the receptor o~ the present invention is employed to transfect cells to thereby express the G-protein PAF receptor. The expressed receptor is then contacted with a test compound to obgerve b; n~; ng, stimulation or inhibition o~ a ~unctional response.
One such screening procedure involves the use o~
melanophores which are trans~ected to express the G-protein PAF receptor o~ 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 ~or screening ~or a compound which inhibits activation o~ the receptor polypeptide o~ the present invention by contacting the melanophore cells which encode the receptor with both the receptor ligand and a compound to be screened. Inhibition o~
the signal generated by the ligand indicates that a compound is a potential antagonist ~or the receptor, i.e., inhibits activation o~ the receptor.
The screen may be employed ~or determining a compound which activates the receptor by contacting such cells with W O 96/39442 PCT~US95/07288 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 PAF receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, volume 246, pages 181-296 (October 1989). For example, compounds may be contacted with a cell which expresses the receptor polypeptide of the present invention and a second messenger response, e.g.
signal transduction or pH changes, may be measured to determine whether the potential compound activates or inhibits the receptor.
Another such screening technic~e involves introducing RNA encoding the G-protein PAF receptor into Xenopus oocytes to transiently express the receptor. The receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detectioll of inhibition or activation of a calcium signal in the case of screening for compounds which are thought to inhibit: activation of the receptor.
Another screening technique involves expressing the G-protein PAF receptor in which the receptor is linked to a phospholipase C or D. As representative examples of such cells, there may be mentioned endothelial cells, smooth muscle cells, embryonic kidney cells, etc-. The screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal.
Another method involves screening for compounds which inhibit activation of the receptor polype~ptide of the present invention 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 PAF receptor W O 96/39442 PCTrUS95/07288 such that the cell expresses the receptor on its surface and contacting the cell with a compound 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 compound 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 PAF receptors are ubiquitous in the m~mm~ 1 ian host and are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate the G-protein PAF
receptor on the one hand and which can inhibit the function of a G-protein PAF receptor on the other hand.
For example, compounds which activate the G-protein PAF
receptor may be employed for therapeutic purposes, such as the treatment o~ a6thma, Par~inson's disease, acute heart failure, hypotension, urinary retention, and osteoporosis.
In general, compounds which inhibit activation of the G-protein PAF receptor may be employed for a variety of therapeutic purposes, for example, for the treatment of hypertension, angina pectoris, myocardial in~arction, ulcers, asthma, allergies, benign prostatic hypertrophy and psychotic and neurological disorders, including schizophrenia, manic excitement, depression, delirium, dementia or severe mental retardation, dyskinesias, such as Huntington's disease or Gilles dila Tourett's syndrome, among others. Compounds which inhibit G-protein PAF receptors have also been useful in reversing endogenous anorexia and in the control of bulimia.
An antibody may antagonize a G-protein PAF receptor of ~ the present invention, or in some cases an oligopeptide, which bind to the G-protein PAF receptor but does not elicit a second messenger response such that the activity o~ the G-protein PAF receptors is prevented. Antibodies include anti-idiotypic antibodies which recognize unique determinants generally associated with the antigen-binding site of an antibody. Potential antagonist compc~unds also include proteins which are closely related to the ligand of the G-protein PAF receptors, i.e. a fragment of the ligand, which have lost biological ~unction and when binding to the G-protein PAF receptor, elicit no response.
An antisense construct prepared t:hrough the use of antisense technology, may be used to cont:rol 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' co~i ng portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of ~rom 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 Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988);
and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of G-protein PAF
receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation o~ mRNA molecules into G-protein PAF receptor (antisense - Okano, J.
Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors o~ 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 o~ G-protein PAF
receptor.
A small molecule which binds to the G-protein PAF
receptor, making it inaccessible to ligands such that normal biological activity is prevented, for example small peptides or peptide-like molecules, may also be used to inhibit W O 96/39442 PCT~US95/07288 activation o~ the receptor polypeptide of the present nvent lon .
A soluble form o~ the G-protein PAF receptor, e.g. a fragment o~ the receptors, may be employed to inhibit activation of the receptor by binding to the ligand to a polypeptide of the present invention and preventing the ligand from interacting with membrane bound G-protein PAF
receptors.
The present invention also provides a method for determining whether a ligand not known to be capable of binding to a G-protein PAF receptor can bind to such receptor which comprises contacting a m~mm~ 1 ian cell which expresses a G-protein PAF receptor with the ligand under conditions permitting binding of ligands to the G-protein PAF receptor, detecting the presence of a ligand which binds to the receptor and thereby determining whether the ligand binds to the G-protein PAF receptor. The systems hereinabove described ~or determining agonists and/or antagonists may also be employed for determining ligands which bind to the receptor.
This invention also provides a method of detecting expression o~ a G-protein PAF receptor polypeptide o~ the present invention on the sur~ace o~ a cell by detecting the presence o~ mRNA coding ~or the receptor which comprises obtaining total mRNA ~rom the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule o~ at least 10 nucleotides capable of specifically hybridizing with a sequence included within the sequence o~
a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence of mRNA
hybridized to the probe, and thereby detecting the expression o~ the receptor by the cell.
- The present invention also provides a method ~or identifying receptors related to the receptor polypeptides of the present invention. These related receptors may be W O 96/39442 PCT~US95/07288 identified by homology to a G-protein PAF receptor polypeptide of the present invention, by low stringency cross hybridization, or by identifying receptors that interact with related natural or synthetic ligands and or elicit similar behaviors after genetic or pharmacological blockade of the G-protein PAF receptor polypeptides of the present invention.
The agonists identified by the screening method as described above, may be employed to t:reat hemophilia by inducing platelet aggregation and to stimulate wound healing.
The antagonist compounds to the G-protein PAF receptor may be employed to prevent and/or treat restenosis after angioplasty, unstable angina, thrombotic or thromboembolytic stroke, allergy, inflammation and myocardial infarction.
The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e g., as hereinafter described.
The antagonist or agonist compound~ may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of ~m;n~stration.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental 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 ~m;n;~tration. In addition, the polypeptides of the present invention may be - employed in conjunction with other therapeutic compounds.

W O 96/39442 PCT~U59~ / 8 The pharmaceutical compositions may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are administered in an amount which is e~ective ~or treating and/or prophylaxis o~ the specific indication. In general, the pharmaceutical compositions will be ~m; n;stered in an amount o~ 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 ~rom about 10 ~g/kg to about 1 mg/kg body weight daily, taking into account the routes o~ ~min;stration, symptoms, etc.
The G-protein PAF receptor polypeptides and antagonists or agonists which are polypeptides, may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is o~ten re~erred to as "gene therapy."
Thus, ~or example, cells ~rom a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being pro~ided 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 o~ a retroviral particle cont~; n; ng ~NA encoding a polypeptide o~
the present invention.
Similarly, cells may be engineered in vivo ~or expression o~ a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell ~or producing a retroviral particle cont~;n;ng RNA
encoding the polypeptide o~ the present invention may be ~m;n;stered to a patient ~or engineering cells in vivo and expression o~ the polypeptide in vivo. These and other - methods ~or administering a polypeptide o~ the present invention by such method should be apparent to those skilled PCTrUS95/07288 in the art ~rom the teachings o~ the present invention. For example, the expression vehicle ~or engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to ensineer cells in vivo a~ter combination with a suitable delivery vehicle.
Retroviruses ~rom which the retrov:iral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, qibbon ape leukemia virus, human ;mmllnode~iciency virus, adenovirus, Myeloproliferative Sarcoma Virus, and m~mm~y tumor virus.
In one embodiment, the retroviral plasmid vector is derived ~rom Moloney Murine Leukemia Virus.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promc,ter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (~989), or any other promoter ~e.g., cellular promoters such as eukaryotic cellular promoters including, but not: limited to, the histone, pol III, and ~-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and Bl9 parvovirus promoters. The selection o~ a suitable promoter will be apparent to those skil:Led in the art ~rom the teachings contained herein.
The nucleic acid sequence encoding the polypeptide o~
the present invention is under the control o~ a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock W O 96/394~2 PCT~US95/07288 promoters; the albumin promoter; the ApoAI promoter; human globin promoters, viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modi~ied retroviral LTRs hereinabove described); the ~-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the genes encoding the polypeptides.
The retroviral plasmid vector is employed to transduce packaging cell lines to ~orm producer cell lines. Examples o~ packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-17-H2, ~CRE, ~CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene TheraPY, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by re~erence in its entirety. The vector may transduce the packaging cells through any means known in the art. Suc~ means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or PAF to a lipid, and then ~m; n; stered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vi tro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
The present invention also contemplates the use of the genes o~ the present invention as a diagnostic, for example, some diseases result ~rom inherited defective genes. These genes can be detected by comparing the sequences o~ the W O 96/39442 PCTnUS95/07288 def-ective gene with that ofE a normal one. Subsec~uently, one can verify that a "mutant" gene is associated with abnormal receptor activity. In addition, one can insert mutant receptor genes into a suitable vector Eor expression in a functional assay system (e.g., colorimetric assay, expression on MacConkey plates, complementation experiments, in a receptor deficient strain ofE HEK293 ce:Lls) as yet another means to verify or identiEy mutations. Once ~'mutant" genes have been identified, one can then screen population fEor carriers of the "mutant" receptor gene.
Individuals carrying mutations in the gene of the present invention may be detected at the DNA level by a variety ofE technic~ues. Nucleic acids used for diagnosis may be obtained from a patient's cells, including but not limited to such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified 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 complimentary to the nucleic acid of the instant invention can be used to identify and analyze mutations in the gene of the present invention. 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 identifEied by hybridizing amplifEied DNA to radio labeled RNA of the invention or alternatively, radio labeled antisense DNA secluences ofE the invention. PerfEectly matched secluences can be distinguished fErom mismatched duplexes by RNase A digestion or by diffEerences in melting temperatures. Such a diagnostic would be particularly useful fEor prenatal or even neonatal testing.
Sec~uence difEferences between the reference gene and "mutants" may be revealed by the direct DNA secluencing method. In addition, cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of W O 96/39442 PCT/U~55~'~i>8g this method is greatly enhanced when combined with PCR. For example, a sequence primer is used with double stranded PCR
pr~duct or a single stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radio labeled nucleotide or by an automatic sequencing procedure with fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection of alterations in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Sequences changes at speci~ic locations may also be revealed by nucleus protection assays, such RNase and S1 protection or the chemical cleavage method (e.g. Cotton, et al., PNAS, USA, 85:4397-4401 1985).
In addition, some diseases are a result of, or are characterized by changes in gene expression which can be detected by changes in the mRNA. Alternatively, the genes of the present invention can be used as a re~erence to identify individuals expressing a decrease of functions associated with receptors of this type.
The present invention also relates to a diagnostic assay for detecting altered levels of soluble forms of the PAF
receptor polypeptides of the present invention in various tissues. Assays used to detect levels of the soluble receptor polypeptides in a sample derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western blot analysis and preferably as ELISA assay.
An ELISA assay initially comprises preparing an antibody speci~ic to antigens of the PAF receptor polypeptides, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as - radioactivity, fluorescence or in this example a horseradish peroxidase enzyme. A sample is now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that WO 96/39442 PCT~US95/07288 binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any PAF receptor proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to PAF receptor proteins.
Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period i8 a measurement of the amount o~ PAF receptor proteins present in a given volume of patient sample when compared against a st~n~rd curve.
The sec~uences of the present invention are also valuable for chromosome identification. The secluence 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 ac:tual secluence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of I)NAs to chromosomes according to the present invention is an important first step in correlating those secluences with genes associated with disease.
Briefly, secluences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
Computer analysis of the cDNA 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~;n~ng individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

W O 96/39442 PCT~US~J~ 88 PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in si tu hybridization (FISH) of a cDNA
clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases.
For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon 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, Mendelian 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 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 W O 96/39442 PCT~US95/07288 of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
The polypeptides, their fragments Ol- other derivatives, or analogs thereo~, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes ch;meric, single chain, and hllm~n;zed 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 ~n;m~l or by ~m;n;stering the polypeptides to an animal, preferably a nonh nan. The antibody so obtained will then bind the polypeptides itself. In this m~nn~r~ 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 prov-des antibodies produced by continuous cell line cultures can be used. Examples inc].ude the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today ~L:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies 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 antibodies to ;mmllnogenic polypeptide products of this invention. Also, transgenic mice may be used to -express hllm~n;zed antibodies to immunogenic 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 examp =s. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate understanding of 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 commercially available, publicly available on an unrestricted basis, or can ~--constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to 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 commercially 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 ~1 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 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 _ _ _ _ W O 96/39442 PCT~US9J~7~88 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 eitner a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide withou~ 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 acco~nplished 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 o~ Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Example 1 Bacterial Expression and Purification o~ G-protein couPled receptor HTNAD29 (PAF receptor) The ~NA sequence encoding for the PAF receptor, ATCC # _ is initially amplified using PCR oligonucleotide primers corresponding to the 5' and sequences, of the processed protein (minus the signal peptide sequence) and the vector sequences 3' to the PAF receptor gene. Additional nucleotides correspo~; ng to the PAF receptor were added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5' CGAATTCCTCCATGAACAGCACATGTATT 3' W O 96/39442 PCTrUS95/07~88 and contains an EcoRI restriction enzyme site followed by 18 nucleotides of the PAF receptor coding sequence starting from the presumed terminal amino acid of the processed protein codon. The 3' sequence 5' CGGAAGCTTCGTCAAGGACCTCTAATTCC 3' contains complementary sequences to a HindIII site and is followed by 18 nucleotides encoding the PAF receptor. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc.
Chatsworth, CA, 91311). pQE-9 encodes antibiotic resistance (Ampr), a bacterial origin o~ replication (ori), an IPTG-regulatable promoter operator (P/0), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 was then digested with EcoRI and HindIII. The ampli~ied sequences were ligated into pQE-9 and were inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture was 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 M~n~
Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan').
Transformants are identified by their ability to grow on LB
plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and con~irmed by restriction analysis. Clones cont~;n~ng the desired constructs were 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 were grown to an optical density 600 (O.D.~) of between 0.4 and 0.6. IPTG
("Isopropyl-B-D-thiogalacto pyranoside") was then added to a f inal concentration o~ 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression. Cells were grown an extra 3 to 4 hours.
Cells were then harvested by centrifugation. The cell pellet W O 96/39442 PCT~US95/07288 was solubilized in the chaotropic agen~ 6 Molar Guanidine HCl. A~ter clari~ication, solubilized PAF receptor was puri~ied from this solution by chromatography on a Nickel-Chelate column under conditions that allow ~or tight binding by proteins cont~ining the 6-His tag. Hochuli, E. et al., J.
Chromatography 411:177-184 (1984). PAF receptor was eluted ~rom the column in 6 molar guanidine HCl pH 5.0 and ~or the purpose ~~ renaturation adjusted to 3 molar guanidine HCl, lOOmM s~lum phosphate, 10 mmolar glutal:hione (reduced) and 2 mmolar glutathione (oxidized). A~ter incubation in this solution ~or 12 hours the protein was d:ialyzed to 10 mmolar sodium phosphate.

Example 2 Ex~ression o~ Recombinant PAF receptor in COS cells The expression o~ plasmid, PAF receptor HA is deriveG
~rom a vector pcDNAI/Amp (Invitrogen) cont~;n;ng: 1) SV40 origin o~ replication, 2) ampicillin resistance gene, 3) B.coli replication origin, 4) CMV promoter ~ollowed by a polylinker region, a SV40 intron and polyadenylation site.
A DNA ~ragment encoding the entire PAF receptor precursor and a HA tag ~used in ~rame to its 3' end was cloned into the polylinker region o~ the vector, there~ore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the in~luenza hemagglutinin protein as previously described (I. Wilson, H.
Niman, R. Heighten, A Cherenson, M. Connolly, and R. T~Pr~r~
1984, Cell 37, 767). The in~usion o~ ~A tag to our target protein allows easy detection o~ the recomh;n~nt protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as ~ollows:
The DNA secluence encoding ~or PAF receptor, ATCC #
was constructed by PCR using two primers: the 5' primer 5' GTCCAAGCTTGCCACCATGAACAGCACATGTATT 3' contains a HindIII site W O 96/39442 PCTrUS95/07288 followed by 18 nucleotides of the PAF receptor coding sequence starting from the initiation codon; the 3' sequence 5' CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCAAGGACCTCTAATT
CCATA 3' contains complementary sequences to an XhoI site, translation stop codon, H~ tag and the last 18 nucleotides o~
the PAF receptor coding se~uence (not including the stop codon). Therefore, the PCR product contains a site, PAF
receptor 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, were digested with HindIII and XhoI restriction enzyme and ligated. The ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the trans~ormed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and ~X~m; ned by restriction analysis for the presence of the correct fragment. For expression of the recombinant PAF receptor, COS cells were transfected with the expression vector by DEAE-DEXTRAN
method. (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory M~nll~l, Cold Spring Laboratory Press, (1989)). The expression of the PAF receptor HA protein was detected by radiolabelling and ~mmnnoprecipitation method.
(E. Harlow, D. Lane, Antibodies: A Laboratory M~nll~l, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with 35S-cysteine two days post trans~ection.
Culture media were then collected and cells were 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 were precipitated with a HA specific monoclonal antibody.
Proteins precipitated were analyzed on 15% SDS-PAGE gels.

Exam~le 3 W O 96/39442 PCT~US95/07288 Cloninq and exPression of G-Protein coupled receptor (PAF
receptor) usinq the baculovirus expression system The DNA sequence encoding the full length PAF receptor protein, ATCC # , was amplified using PCR
oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence 5~ CGGGATCCCTCCATG
AACAGCACATGTATT 3' 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 (J. Mol. Biol. 1987, :L96, 947-950, Kozak, M.), and just behind the first 18 nucleotides of the PAF
receptor gene (the initiation codon for translation "ATG" is underlined).
The 3' primer has the secluence 5' ('GGGATCCCGCTCAAGGACC
TCTAATTCCATA 3' and contains the cleavage site for the restriction Pn~onllclease BamHI an~ 18 nucleotides complementary to the 3' non-translated sequence of the PAF
receptor gene. The amplified sec~uences were isolated from a 1% agarose gel using a rommp~cially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment was then digested with the endonucleases BamHI and purified as described above. This fragment is designated F2.
The vector pRG1 (modification of pVL941 vector, discussed below) is used for the expression of the PAF
receptor protein using the baculovirus expression system (~or review see: Summers, M.D. and Smith, G.~3. 1987, A m~nll~l 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 endonuclease BamHI. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant W O 96/39442 PCT~US95/07288 viruses 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 polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRG1 such as pAc373, pVh941 and pAcIM1 (huckow, V.A. and Summers, M.D., Viroloov, 170:31-39)-The plasmid was digested with the restriction enzymesBamHI and dephosphorylated using calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from a 1~ agarose gel as described above. This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E.coli B101 cells were then transformed and bacteria identified that contained the plasmid (pBacPAF receptor ) with the PAF receptor gene using the enzyme BamHI. The sequence of the cloned fragment was confirmed by DNA sequencing.
5 ~g of the plasmid pBacPAF receptor were co-trans~ected 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 S ~g of the plasmid pBacPAF receptor were mixed in a sterile well of a microtiter plate cont~;n~ng 50 ~1 of serum ~ree Grace~s medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~1 hipofectin plus 90 ~l Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture was added drop wise to the Sf9 insect cells (ATCC CRh 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace' medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate .

WO 96/39442 PCT~US95/07288 was then incubated for 5 hours at 27~C. A~ter 5 hours the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum was added. The plate was put back into an incubator and cultivation continued at 27~C for four days.
A~ter ~our days the supernatant ~as collected and a pla~ue assay per~ormed similar as described by Summers and Smith (supra). As a modi~ication an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was used which allows an easy isolation of blue st-~ n~ 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., Gaithersburg, page 9-10) .
Four days after the serial dilution, the viruses were added to the cells and blue st~ne~ pla~les were picked with the tip of an Eppendorf pipette. The agar contA;ning the recombinant viruses was then resuspended in an Eppendorf tube contA~ning 200 ~l of Grace's medium. The agar was removed by a brief centrifugation and the superna~ant contA;n~ng the recombinant baculoviruses was used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes were harvested and then stored at 4~C.
Sf9 cells were grown in Grace's medium supplemented with 10~ heat-inactivated FBS. The cells were infected with the recombinant baculovirus V-PAF receptor at a multiplicity o~
infection (MOI) of 2. Six hours later the medium was 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 I~Ci 35S cysteine (Amersham) were added. The cells were further incubated for 16 hours be~ore they were harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.

Exam~le 4 W O 96/39442 PCT~US95/07288 Ex~ression via Gene Thera~y Fibroblasts are obtained ~rom a subject by skin biopsy.
The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks o~ the tissue are placed on a wet sur~ace o~ a tissue culture ~lask, approximately ten pieces are placed in each ~lask. The ~lask is turned upside down, closed tight and le~t at room temperature over night. A~ter 24 hours at room temperature, the ~lask is inverted and the chunks o~ tissue remain ~ixed to the bottom o~ the flask and ~resh media (e.g., Ham F12 media, with 10% FBS, penicillin and streptomycin, is added.
This is then incubated at 37~C ~or approximately one week.
At this time, fresh media is added and subsequently changed every several days. A~ter an additional two weeks in culture, a monolayer o~ ~ibroblasts emerge. The monolayer is trypsinized and scaled into larger ~lasks.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) ~lanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The 1; ne~r vector is ~ractionated on agarose gel and puri~ied, using glass beads.
The cDNA encoding a polypeptide o~ the present invention is ampli~ied using PCR primers which correspond to the 5' and 3' end sequences respectively. The 5' primer contains an EcoRI site and the 3' primer contains a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the EcoRI and HindIII ~ragment are added together, in the presence o~ T4 DNA ligase. The resulting mixture is maintained under conditions appropriate ~or ligation o~ the two ~ragments. The ligation mixture is used to transform bacteria B 101, which are then plated onto agar-cont~;n~ng kanamycin ~or the purpose o~ con~irming that the vector had the gene o~ interest properly inserted.

W O 96/39442 PCT~US95/07288 The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10~ calf serum (CS), penicillin and streptomycin. The MS~ vector containing the gene is then a~ded to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles cont~in;ng the gene (the packaging cells are now referred to as producer cells).
Fresh media is added to the transd~lced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, cont~in;ng the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.
The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.
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 invention may be practiced otherwise than as particularly described.

Claims (20)

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide encoding the polypeptide as set forth in Figure 1;
(b) a polynucleotide encoding a mature polypeptide encoded by the DNA contained in ATCC Deposit No. ;
(c) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a) or (b); and (d) a polynucleotide fragment of the polynucleotide of (a) or (b).

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

3. The polynucleotide of Claim 1 comprising from nucleotide 523 to nucleotide 1533 as set forth in Figure 1.

4. The polynucleotide of Claim 1 encoding a soluble form of the polypeptide of Figure 1.

5. A vector containing the DNA of Claim 2.

6. A host cell transformed or transfected with the vector of Claim 5.

7. A process for producing a polypeptide comprising:
expressing from the host cell of Claim 8 the polypeptide encoded by said DNA.

8. A process for producing cells capable of expressing a polypeptide comprising transforming or transfecting the cells with the vector of Claim 5.

9. A receptor polypeptide comprising a member selected from the group consisting of:
(i) a polypeptide having the deduced amino acid sequence of Figure 1 and fragments, analogs and derivatives thereof; and (ii) a polypeptide encoded by the cDNA of ATCC
Deposit No. and fragments, analogs and derivatives of said polypeptide.

10. An antibody against the polypeptide of claim 9.

11. A compound which activates the polypeptide of claim 9.

12. A compound which inhibits activation of the polypeptide of claim 9.

13. A method for the treatment of a patient having need to activate a G-protein PAF receptor comprising:
administering to the patient a therapeutically effective amount of the compound of claim 11.

14. A method for the treatment of a patient having need to inhibit a G-protein PAF receptor comprising:
administering to the patient a therapeutically effective amount of the compound of claim 12.

15. The method of claim 13 wherein said compound is a polypeptide and a therapeutically effective amount of the compound is administered by providing to the patient DNA
encoding said agonist and expressing said agonist in vivo.

16. A process for diagnosing a disease or a susceptibility to a disease related to an under-expression of the polypeptide of claim 9 comprising:

determining a mutation in the nucleic acid sequence encoding said polypeptide.

17. The polypeptide of Claim 9 wherein the polypeptide is a soluble fragment of the polypeptide and is capable of binding a ligand for the receptor.

18. A diagnostic process comprising:
analyzing for the presence of the polypeptide of claim 9 in a sample derived from a host.

19. A method for identifying compounds which bind to and activate and which bind to and inhibit the receptor polypeptide of claim 9 comprising:
contacting a cell expressing on the surface thereof the receptor polypeptide, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor polypeptide, with a compound under conditions sufficient to permit binding of the compound to the receptor polypeptide;
and identifying if the compound is an effective agonists or antagonist by detecting the presence or absence of the signal produced by said second component.

20. A process for diagnosing a disease or a susceptibility to a disease related to an under-expression of the polypeptide of claim 9 comprising:
determining a mutation in the nucleic acid sequence encoding said polypeptide.