AU760468B2 - G-protein receptor HTNAD29 - Google Patents

Description

G-PROTEIN RECEPTOR HTNAD29 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 polypeptide of the present invention is a human 7transmembrane receptor which has been putatively identified as a platelet activating factor receptor, sometimes hereinafter referred to as "G-protein PAF Receptor". The :i invention also relates to inhibiting the action of such polypeptides.

It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, CAMP (Lefkowitz, 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, et al., PNAS, 84:46-50 (1987); Kobilka, B.K., et al., Science, 238:650-656 (1987); Bunzow, et al., Nature, 336:783-787 (1988)), G-proteins themselves, effector proteins, phospholipase C, adenyl cyclase, and 1A

I;

phosphodiesterase, and actuator proteins, protein kinase A and protein kinase C (Simon, 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. Gprotein 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 Gprotein 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 transmembrane domains. The domains are believed to represent transmembrane a-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 members or this family include calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, -2endothelial differentiation gene-i receptor and rhodopsins, odorant, cytomegalovirus receptors, etc.

Most G-protein coupled receptors have single 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 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 farnesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors. Most Gprotein 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 0-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 domains, which socket is surrounded by hydrophobic residues of the Gprotein coupled receptors. The hydrophilic side of each Gprotein 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 asubunits preferentially stimulate particular effectors to modulate various biological functions in a cell.

-3- 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 mammalian host.

In accordance with one aspect of 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 biologically active and diagnostically or therapeutically useful fragments thereof.

In accordance with a further aspect of the present "invention, there are provided processes for producing such receptor polypeptides by recombinant techniques comprising culturing recombinant 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 •oC* 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 administering compounds to a host which bind to and activate the receptor polypeptide of the present invention which are useful in the prevention and/or treatment of hemophilia by inducing platelet aggregation, and to promote wound healing.

In accordance with still another embodiment of the present invention there are provided processes of administering compounds to a host which bind to and inhibit activation of the receptor polypeptides of the present invention which are useful in the prevention and/or treatment of allergy, inflammation, restenosis after angioplasty, unstable angina, myocardial infarction and thrombotic or thromboembolytic stroke.

In accordance with yet another aspect of the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the polynucleotide sequences of the present invention.

In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences encoding such polypeptides and for detecting an altered level of the soluble form of the receptor polypeptides.

In accordance with yet a further aspect. of the present invention, there are provided processes for utilizing such receptor polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.

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 encompassed by the claims.

Figure 1 shows the cDNA sequence and the corresponding deduced amino acid sequence of the G-protein receptor of the present invention which has been putatively identified as a platelet-activating factor receptor. The standard one-letter abbreviation for amino acids is used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).

Figure 2 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. 97184 on June 1, 1995.

A polynucleotide encoding a polypeptide 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 family. It contains an open reading frame encoding a protein of 337 oo. 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-coding (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 redundancy 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.

The polynucleotide which encodes for the mature polypeptide of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence; 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.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone.

The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a nonnaturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides -00 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 of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of 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 of the coding sequence shown in Figure 1 (SEQ ID NO:1) or of the coding sequence of the deposited clone. 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 function of the encoded polypeptide.

The polynucleotides may also encode for a soluble form 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 full-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 hexahistidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide 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 mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, 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 70%, preferably at least and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-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 Figure 1 (SEQ ID NO:1) or the deposited cDNA(s), i.e. function as a -8soluble 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 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 activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO: 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 NO: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 -9- 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 of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 1 (SEQ ID NO:2) or that encoded by the deposited cDNA, means a polypeptide which either retains substantially the same biological function or activity as such polypeptide, i.e. functions as a PAF receptor, or retains the ability to bind the ligand for the receptor even though the polypeptide does not function as a G-protein PAF receptor, for example, a soluble form of the receptor.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2) or that encoded by the deposited cDNA may be 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 are employed for purification of the mature polypeptide or a proprotein sequence or one in which a fragment of the polypeDtide 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 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 polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and includes portions of *I such polypeptides 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 of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following 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 from its original environment the natural environment if it is naturally occurring). For example, a naturallyoccurring polynucleotide or polypeptide present in a living animal 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 polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more prefe-ably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO: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 of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present 000. invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the -12invention and the production of polypeptides of the invention .by recombinant techniques.

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 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 -13examples of such promoters, there may be mentioned: LTR or promoter, the E. coli. lac or tr, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.

The expression vector also contains a ribosome binding 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 containing 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, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenovirus; 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 -14and promoters are known to those of 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 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 containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian 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, Dibner, Battey, 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.

Mature proteins can be expressed in mammalian 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, (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 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 cytomegalovirus early S. 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, 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), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and terrmination sequences, and preferably, a leader sequence 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, -16stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence ehcoding 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 of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for 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 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 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 -17cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian 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. Mammalian 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 terr..ination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The G-protein PAF receptor polypeptides of the present invention can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic 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 mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present -18invention may be glycosylated or may be non-glycosylated.

Polypeptides of the invention may also include an initial methionine amino acid residue.

The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease.

The G-protein PAF receptors of the present invention may be employed in a process for screening for compounds which activate (agonists) or inhibit activation (antagonists) of the receptor polypeptide of the present invention *In general, such screening procedures involve providing appropriate cells which express the receptor polypeptide of the present invention on the surface thereof. Such cells include cells from mammals, yeast, drosophila or E. Coli. In particular, a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby -los* express the G-protein PAF receptor. The expressed receptor is then contacted with a test compound to observe binding, stimulation or inhibition of a functional response.

One such screening procedure involves the use of melanophores which are transfected to express the G-protein PAF 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 compound which inhibits activation of the receptor polypeptide of the present invention by contacting the melanophore cells which encode the 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, inhibits activation of the receptor.

The screen may be employed for determining a compound which activates the receptor by contacting such cells with -19compounds to be screened and determining whether such compound generates a signal, 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 technique 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 detection 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 Gprotein 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 polypeptide 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 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, by radioactivity. The amount of labeled ligand bound to the receptors is measured, 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 mammalian host and are responsible for many biological functions, n* 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 of asthma, Parkinson's disease, acute heart failure, hypotension, urinary retention, and osteoporosis.

In general, compounds which inhibit activation of the Gprotein PAF receptor 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, 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 of the G- -21protein PAF receptors is prevented. Antibodies include antiidiotypic antibodies which recognize unique determinants generally associated with the antigen-binding site of an antibody. Potential antagonist compounds also include proteins which are closely related to the ligand of the Gprotein PAF receptors, i.e. a fragment of the ligand, which have lost biological function and when binding to the Gprotein PAF receptor, elicit no response.

An antisense construct prepared through the use of antisense technology, may 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 Spolynucleotide 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 Spairs 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 of mRNA molecules into G-protein PAF receptor (antisense Okano, J.

Neurochem., 56:560 (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 vi;-o to inhibit production of 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 -22activation of the receptor polypeptide of the present invention.

A soluble form of the G-protein PAF receptor, e.g. a fragment of 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 mammalian 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 for determining agonists and/or antagonists may ~also be employed for determining ligands which bind to the Sreceptor.

This invention also provides a method of detecting expression of a G-protein PAF receptor polypeptide of the present invention on the surface of a cell by detecting the presence of mRNA coding for the receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 10 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the receptor by the cell.

The present invention also provides a method for identifying receptors related to the receptor polypeptides of the present invention. These related receptors may be -23identified 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 Gprotein PAF receptor polypeptides of the present invention.

The agonists identified by the screening method as described above, may be employed to treat 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, as hereinafter described.

The antagonist or agonist compounds 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 administration.

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 administration. In addition, the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.

-24- 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 effective for treating and/or prophylaxis of the specific indication. In general, the pharmaceutical compositions will be administered in an amount of at least about 10 pg/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 pg/kg to about 1 mg/kg body weight daily, *taking into account the routes of administration, 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 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 containing 'NA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, 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 administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering 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 may 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.

Retroviruses from which the retroviral 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, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

~In one embodiment, the retroviral plasmid vector is derived from 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 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other ~promoter cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and 0-actin promoters).. Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of 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 -26promoters; 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 modified retroviral LTRs hereinabove described); the 3-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 form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, V-2, i-AM, PAl2, T19-14X, VT-19-17-H2, OCRE, 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 reference 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 CaPO 4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or PAF to a lipid, and then administered 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 vitro or in vivo. The transduced eukaryotic cells will 000 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 of the present invention as a diagnostic, for example, some diseases result from inherited defective genes. These genes can be detected by comparing the sequences of the -27defective gene with that of a normal one. Subsequently, 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 for expression in a functional assay system colorimetric assay, expression on MacConkey plates, complementation experiments, in a receptor deficient strain of HEK293 cells) as yet another means to verify or identify mutations. Once "mutant" genes have been identified, one can then screen population for 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 of techniques. 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 I 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 identified by hybridizing amplified DNA to radio labeled RNA of the invention or alternatively, radio labeled antisense DNA sequences of the invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Such a diagnostic would be particularly useful for prenatal or even neonatal testing.

Sequence differences between the reference gene and "mutants" may be revealed by the direct DNA sequencing method. In addition, cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of -28this 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 specific locations may also be revealed by nucleus protection assays, such RNase and Sl .protection or the chemical cleavage method 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 reference 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 specific to antigens of the PAF receptor polypeptides, preferably a monoclonal antibody. :n 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 -29binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a nonspecific 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 is a measurement of the amount of PAF receptor proteins present in a given volume of patient sample when compared against a standard curve.

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 9 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 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 containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure

I.

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 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 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 -31of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per kb).

The polypeptides, their fragments or other derivatives, or analogs thereof, 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 chimeric, single chain, and humanized 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 animal or by administering the polypeptides to an animal, preferably a nonh-.an. The antibody so obtained will then bind the polypeptides itself. In this manner, 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 hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBVhybridoma 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 Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to -32express humanized 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 examplzs. 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 h 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 ug of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 Al of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 ig 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 -33electrophoresed 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 synthesized.

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, 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 mg 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, Virology, 52:456-457 (1973).

Example 1 Bacterial Expression and Purification of G-protein coupled receptor HTNAD29 (PAF receptor) The DNA sequence encoding for the PAF receptor, ATCC No. 97184 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 corresponding to the PAF receptor were added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5' CGAATTCCTCCATGAACAGCACATGTATT 3' 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 a bacterial origin of replication (ori), an IPTGregulatable promoter operator a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 was 4@S. then digested with EcoRI and HindIII. The amplified 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 Manual, 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 see* selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired constructs were grown overnight in liquid culture in ~LB media supplemented with both Amp (100 ug/ml) and Kan 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.

6 of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") was 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 were grown an extra 3 to 4 hours.

Cells were then harvested by centrifugation. The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HC1. After clarification, solubilized PAF receptor was 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).

PAF receptor was eluted from the column in 6 molar guanidine HCI pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HC1, 100mM sodium phosphate, mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.

Example 2 Expression of Recombinant PAF receptor in COS cells The expression of plasmid, PAF receptor 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 PAF receptor precursor and a HA tag fused in frame to its 3' end was 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 Wilson, H. Niman, R.

Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to our target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

000 The plasmid construction strategy is described as follows: The DNA sequence encoding for PAF receptor, ATCC No. 97184, was constructed by PCR using two primers: the 5' primer GTCCAAGCTTGCCACCATGAACAGCACATGTATT 3' contains a HindIII ite followed by 18 nucleotides of the PAF receptor coding sequence starting from the initiation codon; the 3' sequence

CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCAAGGACCTCTAATT

CCATA 3' contains complementary sequences to an XhoI site, translation stop codon, HA tag and the last 18 nucleotides of the PAF receptor coding sequence (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 transformed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and examined 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. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the PAF receptor HA protein was detected by radiolabelling and immunoprecipitation method.

Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with "S-cysteine two days post transfection.

Culture media were then collected and cells were lysed with detergent (RIPA buffer (150 mM NaC1, 1% NP-40, 0.1% SDS, 1% 0.5% DOC, 50mM Tris, pH (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.

Example 3 -37- Clonin2 and expression of G-Protein coupled receptor (PAF receptor) using the baculovirus expression system The DNA sequence encoding the full length PAF receptor protein, ATCC No.

97184, 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 Mol. Biol. 1987, 196, 947-950, Kozak, 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 sequence 5' CGGGATCCCGCTCAAGGACC TCTAATTCCATA 3' and contains the cleavage site for the restriction endonuclease BamHI and 18 nucleotides complementary to the 3' non-translated sequence of the PAF receptor gene. The amplified sequences were isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, The fragment was then digested with the endonucleases BamHI and purified as described above. This fragment is designated F2.

The vector pRGI (modification ofpVL941 vector, discussed below) is used for the expression of the PAF receptor 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 endonuclease BamHI. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant 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 pRGI such as pAc373, pVL941 and pAclMl (Luckow, V.A. and Summers, Virology, 170:31- 39).

The plasmid was digested with the restriction enzymes BamHI 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 HB101 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 jg of the plasmid pBacPAF receptor were co-transfected with 1.0 gg 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)).

lig of BaculoGold virus DNA and 5 jg of the plasmid pBacPAF receptor were mixed in a sterile well of a microtiter ~plate containing 50 pl of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 il Lipofectin plus 90 il 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 CRL 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 -39was then incubated for 5 hours at 27 0 C. After 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 0 C for four days.

After four days the supernatant was 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) was 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., Gaithersburg, page 9o*oe Four days after the serial dilution, the viruses were added to the cells and blue stained plaques were picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing 200 Al of Grace's medium. The agar was removed by a brief centrifugation and the supernatant containing 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 0

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 of 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 "S-methionine and 5 Ci "S cysteine (Amersham) were added. The cells were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.

Example 4 Expression via Gene Therapy Fibroblasts are obtained from a subject by skin biopsy.

The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media Ham F12 media, with 10% FBS, penicillin and streptomycin, is added.

This is then incubated at 37 0 C for approximately one week.

At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked 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 linear vector is fractionated on agarose gel and purified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplified 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 fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is used to transform bacteria HB101, which are then plated onto agarcontaining kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.

-41- 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 MSV vector containing the gene is then aded to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing 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 o S* 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, S.therefore, within the scope of the appended claims, the *invention may be practiced otherwise than as particularly described.

-42- P:,OPER\MRO\2820)495.CLM 23/10/99 -42A- Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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r SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Li, Yi Fuldner, Rebecca A (ii) TITLE OF INVENTION: G-PROTEIN RECEPTOR HTNAD29 (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Carella, Byrne, Bain, Gilt juan, Cecchi,Stewart Olstein STREET: 6 Becker Farm Road CITY: Roseland STATE: NJ COUNTRY: USA ZIP: 07068 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentln Release Version #1.30 CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US95/07288 FILING DATE: 06-JUN-1995

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: MULLINS, J.G.

REGISTRATION NUMBER: 33,073 REFERENCE/DOCKET NUMBER: 325800-368 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 201-994-1700 TELEFAX: 201-994-1744 INFORMATION FOR SEQ ID NO:l: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 1753 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DMA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 523. .1533 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CTGCACGAGA GGCACAGATT TATCAAGCTC CTCAGTCAAC AAACACATCA. CCGGAAGAAA CATGGAAGGA AAGGAATTTT AAAAGGAAAT ACCAATCTCT GTGCAAACAA AGCCTTGTAT 120 ATTCATGTTT GCACCAATCT ACTGTGAGAT TTATGAAGAA AAACAAATTG CGGACAACTC 180 TCTATGTACA CTTACAAATG CCTCAGTTGA TGC~TGTGGG CTGTTTGTCA GCGTrCTGTG 240 ATAATGAACA CATGGAC ITC TGTTTATTAA ATrCAGTIGA CCCCTTAGC CAATTGCCAG GAGCCTGGAT TTTTAC=TCC AACTGCTGAT ATCTGTGTAA AAATTGATCT ACATCCACCC TTTAAAAGCA TTGATGAATT AATTAGAACT TrAGACAACA AGAAAAATTG AAAAGAATTC TCAGTAAAAG CGAATTCGAT GTTCAAAACA AACTACAAAG AGACAAGACT TCTCTGTTTA CTTTCTAAGA ACTAATATAA TTGCTACrr AAAAAGGAAA AA ATG AAC AGC ACA Met Asn Ser Thr 1 TGT ATT GAA GAA CAG CAT GAC CTG GAT CAC TAT TTG TTT CCC ATT GTT Cys Ile Giu Glu Gin His Asp Leu Asp His Tyr Leu Phe Pro Ile Val 10 15 TAC ATC TTr GTG ATT ATA GTC AGC ATT CCA GCC AAT ATT GGA TCT CTG 300 360 420 480 534 582 TGT GTG TCT TTC CTG CA.A Cys Val Ser Phe Leu Gin CTC TTC AGT TTG TCA CTA Leu Phe Ser Leu Ser Leu 30 CCC AAG AAG GAA AGT GAA CTA GGA ATT TAC Pro Lys Lys Giu Ser Giu Leu Giy Ile Tyr 45 TCA GAT TTA CTC TAT GCA TTA ACT CTC CCT Ser Asp Leu Leu Tyr Ala Leu Thr Leu Pro 60 TGG AAT AAA GAC AAC TGG ACr TTC TCT CCT Try, Asn Lys Asp Asn Trp Thr Phe Ser Pro 75 so GCT Trr CTC ATG TAC ATG AAG Tr'r TAC AGC 678 726 774 822 TTA TGG ATT GAT Leu Trp Ile Asp GCC TTG TGC AAA Ala Leu Cys Lys AGC ACA GCA TTC Ser Thz Ala Phe GTC TAC CCT TTG Vai Tyr Pro Leu 120 ATG GTC AGC CTG Met Val Ser Leu Giy Ser Ala Phe Leu 90 CTC ACC TGC ATT GCC Leu Thr Cys Ile Ala 105 95 100 GTT GAT CGG TAT TrG GCT OTT Asp Arg Tyr TAT ACT Tyr Thr GGG AGT Leu Ala Val 115 AAG TTT TTT TTC CTA Lys Phe Phe Phe Leu 125 TCC ATC TOG ATA TTG Ser Ile Trp Ile Leu 140 CAT GAA ACA OTT GTT Aso Glu Thr Vai Val AGG ACA AGA AGA ATT GCA CTC Arg Thx Arg Arg Ile Ala Leu 130 GAA ACC ATC TIC AAT OCT GTC Giu Thr Ile Phe Ann Ala Vai 870 918 966 1014 1062 135 ATG TTG TGG GAA Met Leu Trp Giu 150 TCT AAT TTT ACT Ser Asn Phe Thr 165 145 GAA TAT TGC CAT Giu Tyr CYS ASP 160 GCC GAA AAG Ala Giu Lys 155 TIA TGC TAT GAC AAA TAC CCT Leu Cye Tyr Asp Lys Tyr Pro 170 175 TIA GAG AAA TOG CAA Leu Giu Lys Trp Gin 180 ATC AAC CTC MAC Ile Asn Leu An GTC ACC ATC CTG Val Thr Ile Leu 200 TTG TTC AGG ACG TGT ACA GGC TAT GCA ATPA CCT TTG 1110 185 ATC TGT AAC 190 CGG AAA GTC Gly Tyr Ala Ile Pro Leu Ile Cys Ann Arg Lys Val 205 TAC CAA Tyr Gin AGA ATC Arg Ile GCT GTG CGG CAC Ala Val. Arg His 210 ATA AAA CTA C'fl Ilz Lys Leu Leu 225 AAT AAA Asn Lys GTC AGC Vai Ser 230 ATG TTG Met Leu 245 CAC AGC Kin Ser

GCC

Ala 215

ATC

Ile ACG GAA AAC AAG GAA AAG Thr Giu Asn Lys Glu Lys 220 ACA GTT ACT TTT GTC TTA Thr Val Thr Phe Val Leu

MAG

Lys TGC Tfl' ACT CCC TTr CAT GTG Cys Phe Thr Pro Phe His Val 240 CAT GCT GTG MAC TTC GMA GAC His Ala Val Ann Phe Giu Asp 4 CTG ATT CGC TGC Leu Ile Arg CyB 250 TTA GAG Leu Glu 255 260

AAT

An GCA TTA ACA Ala Leu Thr GTr ACC GMA Val, Thr Giu 295 ACT GGG AGG Thr Gly Arg 310 GTG TCT ACA Val Ser Thr 325 TCT GGG MAG CGA ACT TAC ACA ATG TAT Scr Gly Lys Arg Thr Tyr Thr Met Tyr 265 270 AGT TTA MAT TGT GTT GCT GAT CCA ATT Ser Leu Ann Cyn Val Ala Asp Pro Ile 280 285 ACA GGA AGA TAT GAT ATG TGG AMT ATA Thr Gly Arg Tyr Asp Met Trp Ann Ile 300 TGT AMT ACA TCA CAA AGA CMA AGA AMA Cys Ann Thr Scr Gin Arg Gin Arg Lys AGA ATC ACG GTT Arg Ile Thr Val 275 Leu

TYA

Lou 305

CGC

Arg Tyr Cyn Phe 290 AAA. rrC TGC Lys Phe Cys ATIL CTT TCT Ile Leu~ Ser

TAG.AACCAMG

1158 1206 1254 1302 1350 1398 1446 1494 1543 1603 1663 1723 1753 AAA GAT ACT ATG GMA TTA Met Glu Leu GAG GTC Glu Val 335; cTr GAG Leu Glu Lys Asp 330 GATGTrTTTGA

TGAAAAGGA.A

TCCM.TAAAA

TGTATATTrM AGGGAAGG3GA ACT=rMGTT ATGCATTATT ATCTAGCATG TGPLGGGGACT AAGTGTCTC ATATCTTAAA AcTGCATTGT

ACAGCTCCCT

ACAAAGATCA ATATrrTCTT ATATCATCMA GATTACATTT AGAGTGATGT 'rrrAATCCAG CCCTGCGTTT TATTA'8XTGA, INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 337 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE Met Asn Ser Thr Cys 1 5 Phe Pro Ile Val Tyr Ile Gly Ser Leu Cys Leu Gly Ile Tyr Leu DESCRIPTION: SEQ ID Ile Glu Giu Gin His 10 Ile Phe Val Ile Ile 25 al Ser Phe Leu Gin 40 Phe Ser Leu Ser Leu 55 NO: 2: Asp Leu Asp His Tyr Leu Val Ser le Pro Ala Asn Pro Lye Lye Glu Ser Glu Ser Asp Leu Leu Tyr Ala Leu Thr Lye Tyr Thr Phe Phe Leu Leu Ser Tyr Ala Pro Leu Pro Ala Ser Ser 100 Val Val Trp 70 Leu Thr Tyr Ile Asp Tyr Thr Cys Ala Pro 4 4* 4 4*e* 4 *4*4 4 *e44 115 Lys Phe Leu 120 Leu Glu Thr Gly Ser 90 Leu Thr 105 Lye Phe Ser Ile Asp Glu Leu Cys 170 Trp 75 Ala Cys Phe Trp Thr 155 Tyr An Phe Ile Phe Ile 140 Val Asp Lye Leu Ala Leu 125 Leu Val Lye Asp Met Val 110 Arg Glu Glu Tyr An Tyr Asp Thr Thx Tyr Pro 175 Trp Met Arg Arg Ile Cys 160 Leu Arg Phe 145 Asp Glu Ala Ala Ile 225 Pro Ann Arg Leu Leu 305 Arg Glu Ile Ala Leu Met Val Ser 130 Asn Ala Lye Ile Val 210 Lye Phe Phe Ile Tyri 290 Lye Ala Glu Trp Pro 195 Arg Leu His Glu Thr 275 Cye Phe Val Lye Gin 180 Leu His Leu Val Asp 260 Val Phe Cys Met Ser 165 Ile Val Asn Val Met 245 His Ala Val Thr Val 325 Leu 150 Asn Asn Thr Lye Ser 230 Leu Ser Leu Thr Gly 310 135 Trp Phe Leu Ile Ala 215 Ile Leu An Thr Glu 295 Arg Ann Leu Phe Arg Thr Cys Thr Giy Tyr Leu 200 Thr Thz Ile Ser Ser 280 Thri cys 185 Ile Glu Val Arg Gly 265 Leu Gly Asn An Lye Phe 235 Ile Arg Cys Tyr Ser 315 Arg Lye 205 Glu Lye 220 Val Leu Leu Glu Thr Tyr Vai Ala 285 Asp Met 300 Gin Arg 190 Val Lye Cys His Thr 270 Asp Trp Gin Tyr Gin Arg Ile Phe Thr 240 Ala Vai 255 Met Tyr Pro Ile Asn Ile Arg Lye 320 Ile Leu Ser Ser Thr Lye Asp Thr 330 Met Giu Leu Glu Val Leu 335 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 327 amino acids TYPE: amino acid STRANDEDNESS: single CD) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Asp Ser Ser His Met Asp Ser Glu Phe Arg Tyr Thr Leu Phe Pro Ile 1 5 10 Val Tyr Ser Ile Ile Phe Val Leu Gly Val Ile Ala Asn Gly Tyr Val 25 Leu Trp Val Phe Ala Arg Leu Tyr Pro Cys Lys Lys Phe Ann Glu Ile 35 40 Lys Ile Phe Met Val Asn Leu Thr Met Ala Asp Met Leu Phe Leu Ile 55 Thr Leu Pro Leu Trp Ile Val Tyr Tyr Gin Asn Gin Gly Asn Trp Ile 65 70 75 Leu Pro Lys Phe Leu Cys Asn Val Ala Gly Cys Leu Phe Phe Ile Ann 90 Thr Tyr Cys Ser Val Ala Phe Leu Gly Val Ile Thr Tyr An Arg Phe 100 105 110 Gin Ala Val Thr Arg Pro Ile Lys Thr Ala G1n Ala Asn Thr Arg Lys 115 120 125 !*Arg Gly Ile Ser Leu Ser Leu Val Ile Trp Val Ala Ile Val Gly Ala 130 135 140 Ala Ser Tyr Phe Leu Ile Leu Asp Ser Thr Ann Thr Val Pro Asp Ser 145 150 155 160 Ala Gly Ser Gly Asn Val Thr Arg Cys Phe Glu His Tyr Glu Lys Gly 165 170 175 Ser Val Pro Val Leu Ile Ile His Ile Phe Ile Val Phe Ser Phe Phe 180 185 190 Leu Val Phe Leu Ile Ile Leu Phe Cys Asn Leu Val Ile Ile Arg Thr 195 200 205 Leu Leu Met Gin Pro Val Gin Gin Gin Arg Asn Ala Glu Val Thr Gly 210 215 220 Arg Ala Leu Trp Met Val Cys Thr Val Leu Ala Val Phe Ile Ile Cys 225 230 235 240 Phe Val Pro His His Val Val Gin Leu Pro Trp Thr Leu Ala Glu Leu 245 250 255 Gly Phe Gin Asp Ser Lys Phe His Gin Ala Ile Asn Asp Ala His Gin 260 265 270 Val Thr Leu Cys Leu Leu Ser Thr Ann Cys Val Leu Asp Pro Val Ile 47 275 280 285 Tyr Cys Phe Leu Thr Lys Lys Phe Arg Lys His Leu Thr Glu Lys Phe 290 295 300 Tyr Ser Met Arg Ser Ser Arg Lys Cys Ser Arg Ala Thr Thr Asp Thr 305 310 315 320 Val Thr Glu Val Val Val Pro 325 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CGAATTCCTC CATGAACAGC ACATGTATT 29 INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STIRANDED)NESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (xi) SEQUENCE DESCRIPTION: SEQ ID CGGAAGCTTC GTCAAGGACC TCTAATTCC 29 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: *GTCCAAGCTT GCCPLCCATGA. ACAGCACATG TATT 34 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:?: CTAGCTCGAG TCAAGCGTAG TCTGGGACGT CGTATGGGTA GCAAGGACCT CTAAT~CCAT A 61 INFORMATION FOR SEQ ID NO:8: Wi SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CGGGATCCCT CCATGAACAG CACATGTATT INFORMATION FOR SEQ ID NO: 9: Ci) SEQUENCE CZARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLO)GY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CGGGATCCCG CTCAAGGACC TCTAATI'CCA TA 22

Claims (4)

1. A method of treatment of a haemophilia patient comprising: administering to said patient a therapeutically effective amount of a compound which induces platelet aggregation and or promotes wound healing and which activates a polypeptide comprising a member selected from the group consisting of: a polypeptide comprising a deduced amino acid sequence of SEQ ID No:2; a polypeptide encoded by the cDNA of ATCC Deposit No. 97184; a polypeptide comprising the amino acid sequence of the mature polypeptide portion of SEQ ID NO:2; a polypeptide comprising the amino acid sequence of the mature polypeptide encoded by the cDNA of ATCC Deposit No. 97184; a polypeptide capable of binding a PAF receptor ligand wherein said polypeptide is encoded by a polypeptide that is capable of hybridising to the compliment of SEQ ID NO:1 and wherein said polynucleotide comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO:1 or a complementary nucleotide sequence thereto; a polypeptide capable of binding a PAF receptor ligand wherein said polypeptide is encoded by a polypeptide that is capable of hybridising to the cDNA sequence contained in ATCC Deposit No. 97184 and wherein said *20 polynucleotide comprises a nucleotide sequence that is at least identical to said cDNA sequence or a complementary nucleotide sequence thereto; S(g) an amino acid fragment of any one of to comprising at least 30 amino acids in length; a polypeptide capable of binding a PAF receptor ligand wherein said polypeptide comprises an amino acid sequence that is at least identical to SEQ ID NO:2 or the mature protein region thereof; and a receptor polypeptide capable of binding a PAF receptor ligand wherein r SP said polypeptide comprises an amino acid sequence that is at least 9 identical to the amino acid sequence encoded by the cDNA contained in S ATCC Deposit No. 97184 or the mature protein region thereof; 52/1

2. A method of treatment according to claim 1 wherein the compound is identified by the method of contacting a cell expressing on its surface the polypeptide with a compound being tested, under conditions sufficient to permit binding of said compound to said polypeptide, wherein said polypeptide is associated with a second component capable of providing a detectable signal in response to the binding of a compound that activates said polypeptide; and (ii) detecting the presence or absence of said detectable signal produced by said second component.

3. A method of treatment of a patient suffering from 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 comprising administering to said patient a therapeutically effective amount of a compound wherein the compound inhibits a polypeptide comprising a member selected from the group consisting of: a polypeptide comprising a deduced amino acid sequence of SEQ ID No:2; a polypeptide encoded by the cDNA of ATCC Deposit No. 97184; a polypeptide comprising the amino acid sequence of the mature polypeptide portion of SEQ ID NO:2; a polypeptide comprising the amino acid sequence of the mature 20 polypeptide encoded by the cDNA of ATCC Deposit No. 97184; a polypeptide capable of binding a PAF receptor ligand wherein said Spolypeptide is encoded by a polypeptide that is capable of hybridising to the compliment of SEQ ID NO:1 and wherein said polynucleotide comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO:1 or a complementary nucleotide sequence thereto; S(f) a polypeptide capable of binding a PAF receptor ligand wherein said polypeptide is encoded by a polypeptide that is capable of hybridising to the cDNA sequence contained in ATCC Deposit No. 97184 and wherein said polynucleotide comprises a nucleotide sequence that is at least 30 identical to said cDNA sequence or a complementary nucleotide sequence thereto; 52/2 an amino acid fragment of any one of to comprising at least 30 amino acids in length; -53- a polypeptide capable of binding a PAF receptor ligand wherein said polypeptide comprises an amino acid sequence that is at least identical to SEQ ID NO:2 or the mature protein region thereof; and a receptor polypeptide capable of binding a PAF receptor ligand wherein said polypeptide comprises an amino acid sequence that is at least identical to the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 97184 or the mature protein region thereof;

4. A method according to claim 3 wherein the compound is identified by the method of contacting a cell expressing on its surface the polypeptide with a compound being tested, under conditions sufficient to permit binding of said compound to said polypeptide, wherein said polypeptide is associated with a second component capable of providing a detectable signal in response to the binding of a compound that inhibits said polypeptide; and (ii) detecting the presence or absence of said detectable signal produced by said second component. Dated this THIRTIETH day of MAY 2002. Human Genome Sciences, Inc. Applicant Wray Associates Perth, Western Australia Patent Attorneys for the Applicant *7' 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. 0S O 9 S 0 9* 0 0.-S S 6 S S 0 .RC.