WO1996039438A1

WO1996039438A1 - G-protein receptor hibeb69

Info

Publication number: WO1996039438A1
Application number: PCT/US1995/007180
Authority: WO
Inventors: Yi Li; Jeanine D. Gocayne; Steven M. Ruben
Original assignee: Human Genome Sciences, Inc.
Priority date: 1995-06-06
Filing date: 1995-06-06
Publication date: 1996-12-12
Also published as: AU2698995A

Abstract

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

Description

6-PROTEIN RECEPTOR EZBEB6

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 7- transmembrane receptor which has been putatively identified as a G-protein thrombin receptor. The 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, e.g., cAMP (Lef owitz, Nature, 351:353-354 (1991)) . Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins. Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., PNAS, 84:46-50 (1987); Kobilka, B.K., et al., Science, 238:650-656 (1987); Bunzow, J.R. , et al., Nature, 336:783-787 (1988)), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phoεphodiesterase, and actuator proteins, e.g., protein

-ι- kinase A and protein kinase C (Simon, M.I., et al., Science, 252:802-8 (1991)) .

For example, in one form of signal transduction, the effect of hormone binding is activation of an enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP, and GTP also influences hormone binding. A G-protein connects the hormone receptors to adenylate cyclase. G- protein was shown to exchange GTP for bound GDP when activated by hormone receptors. The GTP-carrying form then binds to an activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G- protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.

Thrombin is a powerful factor in regulating the state of* the cardiovascular system. It is clear that thrombin aids in the formation of blood clots by catalyzing the conversion of fibrinogen to fibrin, which is an integral part of most clots. In addition, thrombin is known to act directly on cells in the blood and in the interior blood vessel wall, and specifically to activate platelets to form clots. Thrombin- induced platelet activation is particularly important for arterial thrombus formation, a process that causes myocardial infarction and some forms of unstable angina and stroke. In addition, thrombin promotes inflammation and other cellular activities. Thrombin is chemotactic for monocytes, mitogenic for lymphocytes, and causes endothelial cells to express the neutrophil adhesive protein GMP-140 on their surfaces and inhibits the growth of these cells. Thrombin elicits platelet-derived growth factor from the endothelium and is a mitogen for mesenchymal cells.

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 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 stimulating platelet aggregation, bone remodeling, and for stimulating fibroblast proliferation to promote wound healing

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

In accordance with yet another aspect of the presen 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 which may detect thrombosis. *

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 coupled receptor of the present invention which has been putatively identified as a thrombin 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 thrombin-like receptor of the present invention and the murine thrombin receptor.

In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. on June 1, 1995.

The polynucleotide of this invention was discovered in a cDNA library derived from human infant brain. It is structurally related to the G-protein thrombin-like receptor family. It contains an open reading frame encoding a protein of 339 amino acid residues. The protein exhibits the highest degree of homology to the murine thrombin receptor with 32.335 % identity and 56.587 % similarity over the entire 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 l (SEQ ID N0:1) 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 N0:1) 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 non- naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure l (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 l (SEQ ID N0:1) 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 G-protein thrombin-like 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 hexa- hiεtidine 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, I., et al., Cell, 37:767 (1984)) .

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90%; and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the 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:l) or the deposited cDNA(s) , i.e. function as a soluble G-protein thrombin-like receptor by retaining the ability to bind the ligands for the receptor even though the polypeptide does not function as a membrane bound G-protien thrombin-like receptor, for example, by eliciting a second messenger response. Alternatively, the polynucleotides may have at least 20 bases, preferably 30 bases and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which 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.

Fragments of the genes may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the genes of the present invention, or which have similar biological activity. Probes of this type are at least 20 bases, preferably at least 30 bases and most preferably at least 50 bases or more. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene of the present invention including-regulatory and promoter regions, exons and introns. An example of a screen of this type comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the genes of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to,.

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 G-protein thrombin-like receptor polypeptide which has the deduced amino acid sequence of 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 G-protein thrombin-like receptor, or retains the ability to bind the ligand for the receptor even though the polypeptide does not function as a G-protein thrombin-like 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 (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the mature polypeptide which are employed for purification of the mature polypeptide or a proprotein sequence or (v) one in which a fragment of the polypeptide is soluble, i.e. not membrane bound, yet still binds ligands to the membrane bound receptor. Such fragments, derivatives and analogs are deemed to be within the scope 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 homogene-^'ty.

The polypeptides of the present invention include the polypeptide of SEQ ID NO:I (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least a 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably a 90% similarity (more preferably at least a 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least a 95% similarity (still more preferably at least a 95% identity) to the polypeptide of SEQ ID NO:2 and also includes 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 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 (e.g., the natural environment if it is naturally occurring) . For example, a naturally- occurring 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 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% 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 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.

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 he 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 invention 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 examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P_L 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. Streptomvces. Salmonella typhimurium.- fungal cells, such as yeast; insect cells such, as Drosophila and Spodoptera Sf9r 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 and 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, pDIO, phagescript, psiX174, pbluescript SK, pBSKS,- pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia) . Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, 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 lad, lacZ, T3, T7, gpt, lambda P_R, P_L 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, L., Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)) .

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

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, N.Y., (1989), the disclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryoteε 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 promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl 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) , α-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplas ic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N- erminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding¹ a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella tvphimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcuε, although others may also be employed aε 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 GEMl (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 hoεt strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate meanε (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical meanε, and the resulting crude extract retained for further purification.

Microbial cells employed in expreεεion of proteins can be diszτr~ed by any convenient method, including freeze-thaw cycling, 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 ba employed to express recombinant protein. Examples of mammalian expresεion εyεtemε include the COS-7 lineε of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expresεing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lineε. Mammalian expreεεion vectorε will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The receptor polypeptideε 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 invention may be glycosylated or may be non-glycoεylated. Polypeptideε of the invention may alεo include an initial methionine amino acid residue.

The polynucleotides and polypeptideε of the preεent invention may be employed aε research reagents and materials for discovery of treatments and diagnostics to human disease.

The G-protein thrombin-like receptors of the present invention may be employed in a process for screening for compounds which activate (agonists) or inhibit activation (antagonistε) of the receptor polypeptide of the present invention .

In general, such screening procedures involve providing appropriate cellε which expreεε the receptor polypeptide of the preεent invention on the εurface 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 express the G-protein thrombin-like receptor. The expresεed receptor iε then contacted with a teεt compound to obεerve binding, εtimulation or inhibition of a functional reεponεe.

One εuch εcreening procedure involveε the use of melanophores which are transfected to express the G-protein thrombin-like 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 εignal generated by the ligand indicateε that a compound iε a potential antagoniεt for the receptor, i.e., inhibitε activation of the receptor.

The εcreen may be employed for determining a compound which activates the receptor by contacting such cells with compoundε to be εcreened and determining whether εuch compound generateε a signal, i.e., activates the receptor.

Other screening techniques include the use of cells which express the G-protein thrombin-like 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 expresεeε 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 thrombin-like receptor into .Xeπqpus 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 G- protein thrombin-like 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 antagonistε 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 thrombin-like receptor εuch that the cell expreεses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g. , by measuring radioactivity of the receptors. If the compound bindε to the receptor aε determined by a reduction of labeled ligand which bindε to the receptorε, the binding of labeled ligand to the receptor iε inhibited.

Specific examples include a platelet aggregation aεεay wherein, waεhed human platelets are prepared by the method of Baenzinger, M.G. , Meth. Enzymol., 31:149-155 (1974), or aε described-by Charo, I.F., et al., J. clin. Invest>, 63:866- 873 (1977) . To induce aggregation, approximately 1-20 nM thrombin or EC_JQ of an alternate agonist compound is uεed to εtimulate aggregation in control reactionε; the reεults are followed by lumiaggregometry. Agonist compoundε at variouε concentrationε may be uεed in place of thrombin to εtimulate aggregation. Antagoniεt compoundε are added to the reaction mixture in addition to the thrombin in order tcv-aεεess their ability to prevent aggregation. Washed platelets are suspended in modified Tyrode's buffer, pH 7.4 with 2 mM magnesium and 1 mM calcium at a concentration of 10⁸ platelets/ml. The thrombin or test compound is added in a small volume (about 20 μL) in 600 mM NaCl, 10 mM MES pH 6.0, 0.5% PEG 6000 buffer and incubated for 15 minutes at 37°C with a platelet suspension.

Another specific example is a platelet activation/ATP secretion assay wherein platelets are prepared aε above in 480 μl of suspension are added to 20 μl of phosphate buffered saline containing sufficient thrombin to give a final concentration of about 10 nM, or an alternate agonist iε added at its EC₅₀. About 20 μl Chromolume^® reagent (Chronolog Corporation, Havertown, PA) is added. In addition to measuring aggregation, ATP secretion is assessed. These resultε quantitated independently meaεurir<g changeε in luminescence and light transmittance in a chronolog dual channel lumiaggregometer (Chronolog Corp.) . Platelet ATP secretion is measured in a lumiaggregometer as luminescence signal. Potential antagonistε which putatively interact with thrombin are preincubated with the thrombin in 20 μl PBS at room temperature for 5 minutes before addition to the platelets. Preincubation is not necessary for testing agoniεtε or antagonists which interact directly with the receptor.

Antibodies which are immunoreactive with various critical positions on the G-protien thrombin-like receptor may antagonize a G-protein thrombin-like receptor of the present invention. Antibodies include anti-idiotypic antibodies which recognize unique determinantε generally aεsociated with the antigen-binding site of an antibody.

Oligopeptides which bind to the G-protein thrombin-like receptor in competition with thrombin itself but which do not elicit a second mesεenger response, may also be used as antagonist compoundε. Exampleε of oligopeptides include small molecules, for example, small peptides or peptide-like molecules.

Potential antagonist compounds also include thrombin mutants lacking proteolytic activity which compete with native thrombin for the G-protein thrombin-like receptor of the present invention.

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 polynucleotide sequence, which encodeε for the mature polypeptideε of the preεent invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids 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 thrombin-like receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of mRNA moleculeε into G-protein thrombin-like receptor (antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expresεion, CRC Press, Boca Raton, FL (1988)) . The oligonucleotides described above can also be delivered to cells εuch that the antisense RNA or DNA may be expressed in vivo to inhibit production of G-protein thrombin-like receptor.

A soluble form of the G-protein thrombin-like receptor, e.g. noncleavable and/or enhanced binding forms of the extracellular portions of the G-protein thrombin-like receptor may bind circulating thrombin and, therefore, inhibit activation of the receptor. The G-protein thrombin-like receptor antagonists may be employed to inhibit abrupt closure or restenoεis after angioplasty. The antagoniεtε may also be employed to treat unstable angina, myocardial infarction, thrombotic or thromboembolytic stroke, renal diεease and cerebrovascular disease.

The G-protein thrombin-like receptor antagoniεts may also be employed to prevent and/or treat inflammation.

The agonists identified by the screening methodε aε deεcribed above, may be employed to εtimulate platelet aggregation and fibroblaεt proliferation for the purpose of promoting wound sealing and bone remodeling.

The agonist and antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g. , 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 compound, 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 preεcribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological productε, which notice reflectε approval by the agency of manufacture, use or sale for human administration. In addition, the pharamaceutical compositions of the present invention may be employed in conjunction with other therapeutic compounds. The pharmaceutical compoεitions may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuεcular, etc, routeε. The pharmaceutical compoεitionε are adminiεtered in an amount which iε 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 μg/kg body weight and in moεt caεeε they will be adminiεtered in an amount not in excess of about 8 mg/Kg body weight per day. In moεt caεeε, the doεage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.

The G-protein thrombin-like receptor polypeptideε and antagonist or agonist compounds which are polypeptides, may alεo be employed in accordance with the present invention by expresεion of εuch polypeptideε in vivo, which iε often referred to aε "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 RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expresεion of a polypeptide in vivo by, for example, procedureε 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 expresεion of the polypeptide in vivo. Theεe and other methodε for adminiεtering a polypeptide of the preεent invention by εuch method εhould be apparent to those skilled in the art from the teachings of the preεent invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenoviruε which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

Retroviruseε from which the retroviral plaεmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruseε εuch aε Rouε 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 cytomegaloviruε (CMV) promoter deεcribed in Miller, et al./ Biotechniσueε. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such aε eukaryotic cellular promoterε including, but not limited to, the histone, pol III, and /3-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 promoterε, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial viruε (RSV) promoter; inducible promoterε, εuch aε the MMT promoter, the metallothionein promoter; heat shock promoterε; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinaεe promoter,- retroviral LTRε (including the modified retroviral LTRs hereinabove described) ; the β-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, φ-2 , φ-ΑM, PA12, T19-14X, VT-19-17-H2,

GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy. Vol. l, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may tranεduce the packaging cellε through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0₄ precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then adminiεtered to a hoεt.

The producer cell line generateε infectiouε retroviral vector particleε which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vi tro or in vivo. The tranεduced eukaryotic cellε will expreεε -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 cellε, hepatocyteε, fibroblasts, myoblasts, keratinocyteε, endothelial cellε, and bronchial epithelial cellε.

This invention also provides a method of detecting expresεion of the G-protien thrombin-like receptor polypeptide of the preεent invention on the εurface 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 conditionε, 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 receptorε related to the receptor polypeptideε of the present invention. These related receptors may be identified by homology to the G-protein thrombin-like receptor polypeptide of the present invention, by low stringency cross hybridization, or by identifying receptors that interact with related natural or synthetic ligands and or elicit similar behaviors after genetic or pharmacological blockade of the G-protein thrombin-like receptor polypeptides of the present invention.

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. Theεe geneε can be detected by comparing the sequences of the defective 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 expresεion in a functional assay system (e.g., colorimetric asεay, expression on MacConkey plates, complementation experiments, in a receptor deficient εtrain of HEK293 cells) as yet another means to verify or identify mutations. Once "mutant" geneε 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 techniqueε. Nucleic acidε uεed for diagnosis may be obtained from a patient's cells, including but not limited to such aε from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used direct "- 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 insertionε 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 diagnoεtic 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 segmentε may be used as probes to detect specific DNA segmentε. The εenεitivity of thiε method is greatly enhanced when combined with PCR. For example, a sequence primer is used with double stranded PCR product or a single stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedureε with radio labeled nucleotide or by an automatic εequencing 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 SI protection or the chemical cleavage method (e.g. Cotton, et al., PNAS. USA. 85:4397-4401 1985) . In addition, some diseaεes 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 uεed as a reference to identify individuals expressing a decrease of functions associated with receptors of this type.

The present invention alεo relateε to a diagnostic assay for detecting levels of soluble forms of the G-protein thrombin-like receptor polypeptides of the present invention in various tiεεueε. Thromboεis may be detected if an increased level iε determined. Aεsayε 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 radioimmunoassayε, competitive-binding assays, Western blot analysis and preferably as ELISA assay.

An ELISA assay initially comprises preparing an antibody specific to antigens of the G-protein thrombin-like receptor polypeptideε, preferably a monoclonal antibody. In addition a reporter antibody iε prepared againεt the monoclonal antibody. To the reporter antibody iε attached a detectable reagent εuch as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme. A sample is now removed from a host and incubated on a εolid εupport, e.g. a polyεtyrene dish, that binds the proteinε in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the diεh during which time the monoclonal antibodieε attach to any G-protien thrombin-like receptor proteinε attached to the polyεtyrene diεh. All unbound monoclonal antibody iε waεhed out with buffer. The reporter antibody linked to horseradish peroxidaεe iε now placed in the diεh reεulting in binding of the reporter antibody to any monoclonal antibody bound to G- protein thrombin-like 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 thrombin receptor proteins preεent in a given volume of patient εample when compared against a standard curve.

The sequences of the present invention are alεo valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular siteε on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphismε) are presently available for marking chromosomal location. The mapping of DNAs to chromoεomeε 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 cDNAt 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 procesε. These primers are then uεed 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 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 εpecific chromoεomeε 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, preεcreening with labeled flow-εorted chromoεomeε and preselection by hybridization to construct chromosome specific-cDNA librarieε. Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphaεe chromosomal spread can be uεed to provide a preciεe 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 phyεical poεition 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 diseaεe.

With current reεolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the diseaεe could be one of between 50 and 500 potential cauεative geneε. (This assumes 1 megabase mapping resolution and one gene per 20 kb) .

The polypeptides, their fragmentε 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 fragmentε, or the product of an Fab expresεion library. Variouε procedureε known in the art may be used for the production of such antibodies and fragments.

Antibodies generated againεt 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 polypeptideε to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itεelf. In thiε 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 tiεεue expreεsing that polypeptide.

For preparation of monoclonal antibodies, any technique which provideε antibodieε produced by continuous cell line cultures can be used. Exampleε include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the triotna technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodieε and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) .

Techniqueε deεcribed for the production of εingle chain antibodieε (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of thiε invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of thiε invention.

The preεent invention will be further deεcribed with reference to the following exampleε; however, it is to be understood that the present invention is not limited to εuch exampleε. All partε or amounts, unless otherwise specified, are by weight.

In order to facilitate understanding of tiie following exampleε certain frequently occurring methodε and/or terms will be deεcribed. "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 be constructed from available plasmidε in accord with published procedures. In addition, equivalent plasmids to those deεcribed are known in the art and will be apparent to the ordinarily skilled artisan.

"Digeεtion" 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 artiεan. For analytical purpoεeε, typically l μg of plaεmid or DNA fragment iε used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg 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 timeε of about 1 hour at 37'C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to iεolate the deεired fragment.

Size separation of the cleaved fragments is performed uεing 8 percent polyacrylamide gel deεcribed by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980) .

"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide εtrandε 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 preεence of a kinaεe. A εynthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146) . Unless otherwise provided, ligation may be accomplished uεing known buffers and conditions with 10 units to T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed as αeεcribed in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973) .

Example 1 Bacterial Expression and Purification of G-protien thrombin- like Receptor

The DNA sequence encoding for the G-protein thrombin- like receptor, ATCC # _ iε initially amplified using PCR oligonucleotide primers correεponding to the 5' and sequences of the processed G-protein thrombin-like receptor protein (minus the signal peptide sequence) and the vector sequences 3' to the gene. Additional nucleotides corresponding to the G-protein thrombin-like receptor were added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence CXlXIiAATTCCTCCATGAATGGCCrTGAAGTG contains a EcoRI restriction enzyme site followed by 18 nucleotides of the G- protein thrombin-like receptor coding sequence εtarting from the preεumed terminal amino acid of the proceεεed protein codon. The 3' sequence CX-ΩAAGC TCXlfTCACΑGCrCTGAC TGGC contains complementary sequences to a Hindlll site and is followed by 18 nucleotides encoding the G-protein thrombin- like receptor. The restriction enzyme siteε correεpond to the reεtriction enzyme sites on the bacterial expresεion vector pQE-9. (Qiagen, Inc. 9259 Eton Avenue, Chatεworth, CA, 91311) . pQE-9 encodeε antibiotic reεiεtance (Amp^r) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a ribosome binding site (RBS) , a 6- Hiε tag and reεtriction enzyme siteε. pQE-9 was then digested with EcoRI and Hindlll. The amplified εequences 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 available from Qiagen under the trademark M15/rep 4 by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,

(1989) . M15/rep4 contains multiple copieε of the plaεmid pREP4, which expreεεeε the lad repressor and also confers kanamycin resistance (Kan^r) . Transformantε are identified by their ability to grow on LB plateε and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired constructs were grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml and Kan (25 ug/ml) . The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") waε then added to a final concentration of 1 mM. IPTG induces by inactivating the lad represεor 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 HCl. After clarification, solubilized G-protein thrombin-like receptor was purified from thiε 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) . The G-protein thrombin-like receptor was eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpoεe of renaturation adjuεted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this solution for 12 hours the protein was dialyzed to 10 mmolar sodium phoεphate.

Example 2 Expression of Recombinant G-protein Thrombin-like receptor in COS cells

The expression of plasmid, thrombin receptor HA iε 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 G-protien thrombin-like 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 aε previously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767) . The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is deεcribed aε follows:

The DNA sequence encoding for the G-protein thrombin- like receptor, ATCC # , was constructed by PCR cloned using t^'w o p r i m e r s : t h e 5 ' p r i m e r GTCOΛGCTTGCC1ACC-ATGAATGGCCTTGAAGTG contains a Hind III εite followed by 18 nucleotideε of coding sequence starting from the initiation codon; the 3' sequence (CTAGCTCGAGTC-AAGOnAGTCTGGGACOTCGTAT^^

ACT) contains complementary sequences to Xhol site, translation stop codon, HA tag and the laεt 18 nucleotideε of the G-protein thrombin-like receptor coding εequence (n„c including the εtop codon) . Therefore, the PCR product containε a Hind III site, G-protein thrombin-like receptor coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xhol site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digested with Hind III and Xhol restriction enzyme and ligated. The ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning Systemε, 11099 North Torrey Pineε Road, La Jolla, CA 92037) the tranεformed culture waε plated on ampicillin media plates and resiεtant colonieε were selected. Plasmid DNA was isolated from tranεformantε and examined by restriction analysis for the presence of the correct fragment. For expresεion of the recombinant G-protein thrombin-like receptor, COS cellε were tranεfected with the expresεion vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatiε, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)) . The expression of the G-protein thrombin-like receptor HA protein was detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Presε, (1988)) . Cellε were labelled for 8 hourε with ³⁵S-cyεteine two dayε poεt tranεfection. Culture media were then collected and cells were lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) . (Wilεon, I. et al., Id. 37:767 (1984)) . Both cell lysate and culture media were precipitated with a HA specific monoclonal antibody. Proteinε precipitated were analyzed on 15% SDS-PAGE gelε.

Example 3 Cloning and expression of G-protein thrombin-like receptor using the baculoviruε expression system

The DNA sequence encoding the full length G-protein thrombin-like receptor protein, ATCC # , waε amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:

The 5 ' primer has the sequence 5 ' - CGGGATCCCTCCATGAATGGCCTTGAAGTG-3' and contains a BamHl restriction enzyme site (in bold) followed by 4 nucleotides reεembling an efficient εignal for the initiation of translation in eukaryotic cells (J. Mol. Biol. 1987, 196. 947-950, Kozak, M.) , and just behind the first 18 nucleotides of the G-protein thrombin-like receptor gene (the initiation codon for translation "ATG" is underlined) .

The 3 ' primer has the seque ce 5' -CXIΩGATCCCGCTC1ACΑGCTCTGACTTGGC and contains the cleavage site for the restriction endonuclease BamHl 18 nucleotides complementary to the 3' non-translated sequence of the G- protein thrombin-like receptor gene. The amplified sequences were isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.) .' The fragment was then digested with the endonucleaseε BamHl and then purified aε deεcribed above. Thiε fragment iε designated F2.

The vector pRGl (modification of pVL941 vector, diεcuεsed below) iε uεed for the expression of the G-protein thrombin-like receptor protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. -:87, A manual of methodε for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) . This expreεsion vector containε the εtrong polyhedrin promoter of the Autographa californica nuclear polyhedroεiε virus (AcMNPV) followed by the recognition sites for the restriction endonucleases BamHl. The polyadenylation εite of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant viruses the beta-^alactosidaεe gene from E.coli is inserted in the same orientation aε 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 pRGl such aε pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D. , Virology, 170:31-39).

The plasmid was digested with the restriction enzymes BamHl and then dephosphorylated using calf intestinal phoεphataεe by procedureε 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 HBlOl cells were then transformed and bacteria identified that contained the plasmid (pBacthrombin receptor) with the thrombin receptor gene using the enzyme BamHl. The sequence of the cloned fragment was confirmed by DNA sequencing. ••

5 μg of the plasmid pBacthrombin receptor were co¬ transfected with 1.0 μg of a commercially available linearized baculovirus ("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)). lμg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacthrombin receptor were mixed in a sterile well of a microtiter plate containing 50 μl of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD) . Afterwards 10 μl Lipofectin plus 90 μl Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then the tranεfection mixture waε 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 waε rocked back and forth to mix the newly added solution. The plate was then incubated for 5 hours at 27°C. After 5 hours the transfection εolution was removed from the plate and 1 ml of Grace'ε inεect medium supplemented with 10% fetal calf serum waε added. The plate was put back into an incubator and cultivation continued at 27°C for four days.

After four days the supernatant waε collected and a plaque assay performed similar as deεcribed by Summerε and Smith (supra) . As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaitherεburg) was used which allows an easy isolation of blue εtained plaques. (A detailed description of a "plaque assay" can alεo be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologieε Inc., Gaitherεburg, page 9- 10) .

Four dayε after the εerial dilution of the viruεes was added to the cellε, blue εtained plaqueε were picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses was then resuεpended in an Eppendorf tube containing 200 μl of Grace's medium. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculoviruses was used to infect Sf9 cellε seeded in 35 mm disheε. Four dayε later the supernatan s of these culture disheε were harveεted and then εtored at 4°C.

Sf9 cellε were grown in Grace's medium εupplemented with 10% heat-inactivated FBS. The cellε were infected with the recombinant baculoviruε v-thrombin receptor at a multiplicity of infection (MOD of 2. Six hourε later the medium was removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc. , Gaithersburg) . 42 hours later 5 μCi of ³³S-methionine and 5 μCi ³⁵S cysteine (Amersham) were added. The cells were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins viεualized by SDS-PAGE and autoradiograph .

Example 4 Expression via Gene Therapy

Fibroblastε are obtained from a εubject by εkin biopεy. The reεulting tiεεue is placed in tisεue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tiεsue culture flask, approximately ten pieces are placed in each flaεk. The flaεk is turned upside down, cloεed tight and left at room temperature over night. After 24 hours at room temperature, the flaεk is inverted and the chunks of tiεεue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added. This is then incubated at 37°C for approximately one week. At this time, freεh media iε added and εubsequently 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 (1988V flanked by the long terminal repeatε of the Moloney murine sarcoma virus, is digested with EcoRI and Hindlll and subsequently treated with calf intestinal phoεphatase. The linear vector is fractionated on agarose gel and purified, using glasε beadε.

The cDNA encoding a polypeptide of the preεent 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 Hindlll site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the EcoRI and Hindlll fragment are added together, in the presence of T4 DNA ligase. The reεulting mixture iε maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is uεed to tranεform bacteria HBlOl, which are then plated onto agar- containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted. 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 iε then added 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 tranεduced producer cellε, and subsequently, the media is harvested from a 10 cm plat-, of confluent producer cells. The sper- media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cellε nd this media is then uεed to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblaεts will be infected and no εelection is required. If the titer is very low, then it iε necessary to use a retroviral vector that has a selectable marker, such as neo or his.

The engineered fibroblastε are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblaεtε now produce the protein product.

Numerous modifications and variationε of the preεent invention are poεεible in light of the above teachingε and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than aε particularly deεcribed.

Claims

WHAT IS CLAIMED IS:

1. An iεolated polynucleotide comprising a member selected from the group consisting of:

(a) a polynucleotide encoding the polypeptide as set forth in Figure 1;

(b) a polynucleotide encoding a mature polypeptide encoded bv the DNA contained in ATCC Depoεit No. ;

(c) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a) or (b) ,- and

(d) a polynucleotide fragment of the polynucleotide of (a) , (b) , or (c) .

2. The polynucleotide of Claim 1 encoding the polypeptide aε set forth in Figure 1;

3. A vector containing the polynucleotide of Claim 1.*

4. A host cell transformed or transfected with the vector of Claim 3.

5. A process for producing a polypeptide comprising: expressing from the host cell of Claim 4 the polypeptide encoded by said polynucleotide.

6. A procesε for producing cellε capable of expreεsing a polypeptide comprising transforming or transfecting the cellε with the vector of Claim 3.

7. A receptor polypeptide selected from the group consisting of:

(i) a polypeptide having the deduced amino acid sequence of Figure 1 and fragments, analogε and derivativeε thereof; and (ii) a polypeptide encoded by the cDNA of ATCC

Depoεit No. and fragments, analogs and derivatives of said polypeptide.

8. The polypeptide of Claim 7 wherein the polypeptide has the deduced amino acid sequence of Figure 1.

9. An antibody against the polypeptide of claim 7.

10. A compound which activates the polypeptide of claim 7.

11. A compound which inhibitε activation the polypeptide of claim 7.

12. A method for the treatment of a patient having need to activate a G-protein thrombin-like receptor compriεing* adminiεtering to the patient a therapeutically effective amount of the compound of claim 10.

13. A method for the treatment of a patient having need to inhibit a G-protein thrombin-like receptor compriεing: adminiεtering to the patient ^'a therapeutically effective amount of the compound of claim 11.

14. -The method of claim 12 wherein εaid compound iε a polypeptide and a therapeutically effective amount of the compound iε adminiεtered by providing to the patient DNA encoding εaid agoniεt and expreεεing εaid agonist in vivo.

15. The method of claim 13 wherein εaid compound iε a polypeptide and a therapeutically effective amount of the compound iε adminiεtered by providing to the._patient DNA encoding εaid antagonist and expressing said antagonist in vivo.

16. A method for identifying compounds which bind to and activate the receptor polypeptide of claim 7 comprising: contacting a cell expresεing on the εurface thereof the receptor polypeptide, said receptor being asεociated with a εecond component capable of providing a detectable εignal in response to the binding of a compound to εaid receptor polypeptide, with a compound under conditionε εufficient to permit binding of the compound to the receptor polypeptide; and identifying if the compound iε capable of receptor binding by detecting the εignal produced by εaid second component.

17. A method for identifying compounds which bind to and inhibit activation of the polypeptide of claim 7 comprising: contacting a cell expresεing on the surface thereof the receptor polypeptide, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor polypeptide, with a compound to be screened under conditions to permit binding to the receptor polypeptide; and determining whether the compound inhibits activation of the polypeptide by detecting the absence of a signal generated from the interaction of the compound with the polypeptide.

18. A method for identifying compounds which bind to and activate the polypeptide of claim 7 comprising: contacting a cell expreεsing on the surface thereof the receptor polypeptide, said receptor being asεociated with a εecond component capable of providing a detectable signal in response to the binding of a compound to said receptor polypeptide, with a compound to be screened under conditionε to permit binding to the receptor polypeptide; and determining whether the compound activates the polypeptide by detecting the presence of a signal generated from the interaction of the compound with the polypeptide.

19. A process for diagnosing in a patient a disease or a susceptibility to a disease related to an under-expression of the polypeptide of claim 7 comprising: determining a mutation in the nucleic acid sequence encoding said polypeptide in a sample derived from a patient.

20. A diagnostic procesε comprising: analyzing for the presence of the polypeptide of claim 7 in a sample derived from a hoεt.