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WO1996039438A1 - Recepteur hibeb69 de proteines g - Google Patents

Recepteur hibeb69 de proteines g Download PDF

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Publication number
WO1996039438A1
WO1996039438A1 PCT/US1995/007180 US9507180W WO9639438A1 WO 1996039438 A1 WO1996039438 A1 WO 1996039438A1 US 9507180 W US9507180 W US 9507180W WO 9639438 A1 WO9639438 A1 WO 9639438A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
receptor
compound
polynucleotide
protein
Prior art date
Application number
PCT/US1995/007180
Other languages
English (en)
Inventor
Yi Li
Jeanine D. Gocayne
Steven M. Ruben
Original Assignee
Human Genome Sciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to AU26989/95A priority Critical patent/AU2698995A/en
Priority to PCT/US1995/007180 priority patent/WO1996039438A1/fr
Publication of WO1996039438A1 publication Critical patent/WO1996039438A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • 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.
  • proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lef owitz, Nature, 351:353-354 (1991)) .
  • 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.
  • G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and pho ⁇ phodiesterase, and actuator proteins, e.g., protein
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the polynucleotide sequences of the present invention.
  • 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.
  • 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.
  • nucleic acid 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.
  • 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.
  • 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.
  • nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • 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.
  • 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- hi ⁇ tidine 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.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polypeptides 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.
  • 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.
  • 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.
  • 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.
  • fragment 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.
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogene- ' ty.
  • 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.
  • 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.
  • 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) .
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • promoter for example: 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.
  • 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.
  • bacterial cells such as E. coli. Streptomvces. Salmonella typhimurium.
  • fungal cells such as yeast
  • insect cells such as Drosophila and Spodoptera Sf9r animal cells
  • 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.
  • 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.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • 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) .
  • 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.
  • 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)) .
  • constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • 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.
  • 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.
  • 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.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , ⁇ -factor, acid phosphatase, or heat shock proteins, among others.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplas ic space or extracellular medium.
  • 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 encoding 1 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 Staphylococcu ⁇ , although others may also be employed a ⁇ a matter of choice.
  • 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) .
  • 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) .
  • pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is induced by appropriate mean ⁇ (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • appropriate mean ⁇ e.g., temperature shift or chemical induction
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical mean ⁇ , and the resulting crude extract retained for further purification.
  • Microbial cells employed in expre ⁇ ion of proteins can be disz ⁇ r ⁇ 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.
  • mammalian cell culture systems can also ba employed to express recombinant protein.
  • mammalian expres ⁇ ion ⁇ y ⁇ tem ⁇ include the COS-7 line ⁇ of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expres ⁇ ing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell line ⁇ .
  • Mammalian expre ⁇ ion vector ⁇ 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 polypeptide ⁇ 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.
  • HPLC high performance liquid chromatography
  • 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) .
  • a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • the polypeptides of the present invention may be glycosylated or may be non-glyco ⁇ ylated.
  • Polypeptide ⁇ of the invention may al ⁇ o include an initial methionine amino acid residue.
  • polypeptide ⁇ of the pre ⁇ ent invention may be employed a ⁇ 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 (antagonist ⁇ ) of the receptor polypeptide of the present invention .
  • such screening procedures involve providing appropriate cell ⁇ which expre ⁇ the receptor polypeptide of the pre ⁇ ent invention on the ⁇ urface thereof.
  • Such cells include cells from mammals, yeast, drosophila or E. Coli .
  • a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby express the G-protein thrombin-like receptor.
  • the expres ⁇ ed receptor i ⁇ then contacted with a te ⁇ t compound to ob ⁇ erve binding, ⁇ timulation or inhibition of a functional re ⁇ pon ⁇ e.
  • 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 ⁇ ignal generated by the ligand indicate ⁇ that a compound i ⁇ a potential antagoni ⁇ t for the receptor, i.e., inhibit ⁇ activation of the receptor.
  • the ⁇ creen may be employed for determining a compound which activates the receptor by contacting such cells with compound ⁇ to be ⁇ creened and determining whether ⁇ uch compound generate ⁇ a signal, i.e., activates the receptor.
  • G-protein thrombin-like receptor for example, transfected CHO cells
  • compounds may be contacted with a cell which expres ⁇ e ⁇ 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 .Xe ⁇ qpus 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.
  • 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 antagonist ⁇ 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 ⁇ uch that the cell expre ⁇ ses 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 bind ⁇ to the receptor a ⁇ determined by a reduction of labeled ligand which bind ⁇ to the receptor ⁇ , the binding of labeled ligand to the receptor i ⁇ inhibited.
  • a platelet aggregation a ⁇ ay wherein, wa ⁇ hed human platelets are prepared by the method of Baenzinger, M.G. , Meth. Enzymol., 31:149-155 (1974), or a ⁇ described-by Charo, I.F., et al., J. clin. Invest>, 63:866- 873 (1977) .
  • To induce aggregation approximately 1-20 nM thrombin or EC J Q of an alternate agonist compound is u ⁇ ed to ⁇ timulate aggregation in control reaction ⁇ ; the re ⁇ ults are followed by lumiaggregometry.
  • Agonist compound ⁇ at variou ⁇ concentration ⁇ may be u ⁇ ed in place of thrombin to ⁇ timulate aggregation.
  • Antagoni ⁇ t compound ⁇ are added to the reaction mixture in addition to the thrombin in order tcv-a ⁇ ess 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 10 8 platelets/ml.
  • the thrombin or test compound is added in a small volume (about 20 ⁇ L) 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 a ⁇ above in 480 ⁇ l of suspension are added to 20 ⁇ l of phosphate buffered saline containing sufficient thrombin to give a final concentration of about 10 nM, or an alternate agonist i ⁇ added at its EC 50 . About 20 ⁇ l Chromolume ® reagent (Chronolog Corporation, Havertown, PA) is added. In addition to measuring aggregation, ATP secretion is assessed. These result ⁇ quantitated independently mea ⁇ urir ⁇ g change ⁇ 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 antagonist ⁇ which putatively interact with thrombin are preincubated with the thrombin in 20 ⁇ l PBS at room temperature for 5 minutes before addition to the platelets. Preincubation is not necessary for testing agoni ⁇ t ⁇ 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 determinant ⁇ generally a ⁇ sociated 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 mes ⁇ enger response, may also be used as antagonist compound ⁇ .
  • Example ⁇ 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.
  • the 5' coding portion of the polynucleotide sequence which encode ⁇ for the mature polypeptide ⁇ of the pre ⁇ ent 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.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of mRNA molecule ⁇ into G-protein thrombin-like receptor (antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expres ⁇ ion, CRC Press, Boca Raton, FL (1988)) .
  • the oligonucleotides described above can also be delivered to cells ⁇ uch 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 resteno ⁇ is after angioplasty.
  • the antagoni ⁇ t ⁇ may also be employed to treat unstable angina, myocardial infarction, thrombotic or thromboembolytic stroke, renal di ⁇ ease and cerebrovascular disease.
  • the G-protein thrombin-like receptor antagoni ⁇ ts may also be employed to prevent and/or treat inflammation.
  • the agonists identified by the screening method ⁇ a ⁇ de ⁇ cribed above may be employed to ⁇ timulate platelet aggregation and fibrobla ⁇ t 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.
  • a suitable pharmaceutical carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • 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.
  • 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 pre ⁇ cribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological product ⁇ , which notice reflect ⁇ approval by the agency of manufacture, use or sale for human administration.
  • the pharamaceutical compositions of the present invention may be employed in conjunction with other therapeutic compounds.
  • the pharmaceutical compo ⁇ itions may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramu ⁇ cular, etc, route ⁇ .
  • the pharmaceutical compo ⁇ ition ⁇ are admini ⁇ tered in an amount which i ⁇ effective for treating and/or prophylaxis of the specific indication.
  • the pharmaceutical compositions will be administered in an amount of at least about 10 ⁇ g/kg body weight and in mo ⁇ t ca ⁇ e ⁇ they will be admini ⁇ tered in an amount not in excess of about 8 mg/Kg body weight per day.
  • the do ⁇ age is from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
  • G-protein thrombin-like receptor polypeptide ⁇ and antagonist or agonist compounds which are polypeptides may al ⁇ o be employed in accordance with the present invention by expres ⁇ ion of ⁇ uch polypeptide ⁇ in vivo, which i ⁇ often referred to a ⁇ "gene therapy.”
  • 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.
  • a polynucleotide DNA or RNA
  • 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.
  • cells may be engineered in vivo for expres ⁇ ion of a polypeptide in vivo by, for example, procedure ⁇ 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 expres ⁇ ion of the polypeptide in vivo.
  • the ⁇ e and other method ⁇ for admini ⁇ tering a polypeptide of the pre ⁇ ent invention by ⁇ uch method ⁇ hould be apparent to those skilled in the art from the teachings of the pre ⁇ ent invention.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenoviru ⁇ which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • Retroviruse ⁇ from which the retroviral pla ⁇ mid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruse ⁇ ⁇ uch a ⁇ Rou ⁇ Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • 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 cytomegaloviru ⁇ (CMV) promoter de ⁇ cribed in Miller, et al./ Biotechni ⁇ ue ⁇ . Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such a ⁇ eukaryotic cellular promoter ⁇ including, but not limited to, the histone, pol III, and /3-actin promoters) .
  • CMV cytomegaloviru ⁇
  • 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.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoter ⁇ , such as the cytomegalovirus (CMV) promoter; the respiratory syncytial viru ⁇ (RSV) promoter; inducible promoter ⁇ , ⁇ uch a ⁇ the MMT promoter, the metallothionein promoter; heat shock promoter ⁇ ; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kina ⁇ e promoter,- retroviral LTR ⁇ (including the modified retroviral LTRs hereinabove described) ; the ⁇ -actin promoter; and human growth hormone promoters.
  • the promoter also may be the native
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ⁇ -2 , ⁇ - ⁇ M, 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 tran ⁇ duce the packaging cell ⁇ through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then admini ⁇ tered to a ho ⁇ t.
  • the producer cell line generate ⁇ infectiou ⁇ retroviral vector particle ⁇ which include the nucleic acid sequence(s) encoding the polypeptides.
  • retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vi tro or in vivo.
  • the tran ⁇ duced eukaryotic cell ⁇ will expre ⁇ -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 cell ⁇ , hepatocyte ⁇ , fibroblasts, myoblasts, keratinocyte ⁇ , endothelial cell ⁇ , and bronchial epithelial cell ⁇ .
  • This invention also provides a method of detecting expres ⁇ ion of the G-protien thrombin-like receptor polypeptide of the pre ⁇ ent invention on the ⁇ urface 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 condition ⁇ , 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 receptor ⁇ related to the receptor polypeptide ⁇ 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.
  • the ⁇ e gene ⁇ 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.
  • mutant receptor genes into a suitable vector for expres ⁇ ion in a functional assay system (e.g., colorimetric as ⁇ ay, expression on MacConkey plates, complementation experiments, in a receptor deficient ⁇ train of HEK293 cells) as yet another means to verify or identify mutations.
  • a functional assay system e.g., colorimetric as ⁇ ay, expression on MacConkey plates, complementation experiments, in a receptor deficient ⁇ train of HEK293 cells
  • Nucleic acid ⁇ u ⁇ ed for diagnosis may be obtained from a patient's cells, including but not limited to such a ⁇ 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.
  • 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.
  • deletions and insertion ⁇ 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 diagno ⁇ tic 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.
  • cloned DNA segment ⁇ may be used as probes to detect specific DNA segment ⁇ . The ⁇ en ⁇ itivity of thi ⁇ method is greatly enhanced when combined with PCR.
  • 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 procedure ⁇ with radio labeled nucleotide or by an automatic ⁇ equencing procedure with fluorescent-tags.
  • 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 disea ⁇ es 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 u ⁇ ed as a reference to identify individuals expressing a decrease of functions associated with receptors of this type.
  • the present invention al ⁇ o relate ⁇ to a diagnostic assay for detecting levels of soluble forms of the G-protein thrombin-like receptor polypeptides of the present invention in various ti ⁇ ue ⁇ . Thrombo ⁇ is may be detected if an increased level i ⁇ determined.
  • a ⁇ say ⁇ 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 radioimmunoassay ⁇ , 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 polypeptide ⁇ , preferably a monoclonal antibody.
  • a reporter antibody i ⁇ prepared again ⁇ t the monoclonal antibody.
  • a detectable reagent ⁇ uch as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme.
  • a sample is now removed from a host and incubated on a ⁇ olid ⁇ upport, e.g. a poly ⁇ tyrene dish, that binds the protein ⁇ 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.
  • the monoclonal antibody is incubated in the di ⁇ h during which time the monoclonal antibodie ⁇ attach to any G-protien thrombin-like receptor protein ⁇ attached to the poly ⁇ tyrene di ⁇ h. All unbound monoclonal antibody i ⁇ wa ⁇ hed out with buffer.
  • 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 pre ⁇ ent in a given volume of patient ⁇ ample when compared against a standard curve.
  • the sequences of the present invention are al ⁇ o valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphism ⁇ ) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromo ⁇ ome ⁇ according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • 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 proces ⁇ . These primers are then u ⁇ ed 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.
  • sublocalization can be achieved with panels of fragments from ⁇ pecific chromo ⁇ ome ⁇ 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, pre ⁇ creening with labeled flow- ⁇ orted chromo ⁇ ome ⁇ and preselection by hybridization to construct chromosome specific-cDNA librarie ⁇ .
  • Fluorescence in situ hybridization of a cDNA clone to a metapha ⁇ e chromosomal spread can be u ⁇ ed to provide a preci ⁇ e chromosomal location in one step.
  • FISH Fluorescence in situ hybridization
  • the phy ⁇ ical po ⁇ ition of the sequence on the chromosome can be correlated with genetic map data.
  • genetic map 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) .
  • a cDNA precisely localized to a chromosomal region associated with the disea ⁇ e could be one of between 50 and 500 potential cau ⁇ ative gene ⁇ . (This assumes 1 megabase mapping resolution and one gene per 20 kb) .
  • polypeptides, their fragment ⁇ 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 fragment ⁇ , or the product of an Fab expres ⁇ ion library. Variou ⁇ procedure ⁇ known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated again ⁇ t 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 polypeptide ⁇ to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides it ⁇ elf. In thi ⁇ 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 ti ⁇ ue expre ⁇ sing that polypeptide.
  • Example ⁇ 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 Antibodie ⁇ and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) .
  • 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 plasmid ⁇ in accord with published procedures.
  • equivalent plasmids to those de ⁇ cribed are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Dige ⁇ tion” 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 arti ⁇ an.
  • For analytical purpo ⁇ e ⁇ typically l ⁇ g of pla ⁇ mid or DNA fragment i ⁇ used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer.
  • Incubation time ⁇ 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 i ⁇ olate the de ⁇ ired fragment.
  • Size separation of the cleaved fragments is performed u ⁇ ing 8 percent polyacrylamide gel de ⁇ cribed by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980) .
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide ⁇ trand ⁇ 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 pre ⁇ ence of a kina ⁇ e. A ⁇ ynthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase 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 u ⁇ ing known buffers and conditions with 10 units to T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • 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 ⁇ tarting from the pre ⁇ umed terminal amino acid of the proce ⁇ ed protein codon.
  • the 3' sequence CX- ⁇ AAGC TCXlfTCAC ⁇ GCrCTGAC TGGC contains complementary sequences to a Hindlll site and is followed by 18 nucleotides encoding the G-protein thrombin- like receptor.
  • the restriction enzyme site ⁇ corre ⁇ pond to the re ⁇ triction enzyme sites on the bacterial expres ⁇ ion vector pQE-9.
  • pQE-9 encode ⁇ antibiotic re ⁇ i ⁇ tance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a ribosome binding site (RBS) , a 6- Hi ⁇ tag and re ⁇ triction enzyme site ⁇ .
  • pQE-9 was then digested with EcoRI and Hindlll. The amplified ⁇ equences 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,
  • M15/rep4 contains multiple copie ⁇ of the pla ⁇ mid pREP4, which expre ⁇ e ⁇ the lad repressor and also confers kanamycin resistance (Kan r ) .
  • Transformant ⁇ are identified by their ability to grow on LB plate ⁇ 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.
  • IPTG Isopropyl-B-D-thiogalacto pyranoside
  • IPTG induces by inactivating the lad repres ⁇ or 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 thi ⁇ 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
  • the G-protein thrombin-like receptor was eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpo ⁇ e of renaturation adju ⁇ ted 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 pho ⁇ phate.
  • plasmid, thrombin receptor HA i ⁇ 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 a ⁇ 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 de ⁇ cribed a ⁇ 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 GTCO ⁇ GCTTGCC1ACC-ATGAATGGCCTTGAAGTG contains a Hind III ⁇ ite followed by 18 nucleotide ⁇ 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 la ⁇ t 18 nucleotide ⁇ of the G-protein thrombin-like receptor coding ⁇ equence (n instructc including the ⁇ top codon) . Therefore, the PCR product contain ⁇ 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 System ⁇ , 11099 North Torrey Pine ⁇ Road, La Jolla, CA 92037
  • Plasmid DNA was isolated from tran ⁇ formant ⁇ and examined by restriction analysis for the presence of the correct fragment.
  • COS cell ⁇ were tran ⁇ fected with the expres ⁇ ion vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniati ⁇ , 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.
  • Cell ⁇ were labelled for 8 hour ⁇ with 35 S-cy ⁇ teine two day ⁇ po ⁇ t tran ⁇ fection. 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) .
  • RIPA buffer 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5
  • Both cell lysate and culture media were precipitated with a HA specific monoclonal antibody. Protein ⁇ precipitated were analyzed on 15% SDS-PAGE gel ⁇ .
  • the 5 ' primer has the sequence 5 ' - CGGGATCCCTCCATGAATGGCCTTGAAGTG-3' and contains a BamHl restriction enzyme site (in bold) followed by 4 nucleotides re ⁇ embling an efficient ⁇ ignal 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' -CXI ⁇ GATCCCGCTC1AC ⁇ GCTCTGACTTGGC 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 endonuclease ⁇ BamHl and then purified a ⁇ de ⁇ cribed above. Thi ⁇ fragment i ⁇ designated F2.
  • the vector pRGl (modification of pVL941 vector, di ⁇ cu ⁇ sed below) i ⁇ u ⁇ ed 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 method ⁇ for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) .
  • This expre ⁇ sion vector contain ⁇ the ⁇ trong polyhedrin promoter of the Autographa californica nuclear polyhedro ⁇ i ⁇ virus (AcMNPV) followed by the recognition sites for the restriction endonucleases BamHl.
  • the polyadenylation ⁇ ite of the simian virus (SV)40 is used for efficient polyadenylation.
  • the beta- ⁇ alactosida ⁇ e gene from E.coli is inserted in the same orientation a ⁇ 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 a ⁇ 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 pho ⁇ phata ⁇ e by procedure ⁇ known in the art. The DNA was then isolated from a 1% agarose gel as described above. This vector DNA is designated V2.
  • 5 ⁇ g of the plasmid pBacthrombin receptor were co ⁇ transfected with 1.0 ⁇ g of a commercially available linearized baculovirus ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
  • BaculoGoldTM virus DNA and 5 ⁇ g of the plasmid pBacthrombin receptor were mixed in a sterile well of a microtiter plate containing 50 ⁇ l of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD) .
  • plaque assay performed similar as de ⁇ cribed by Summer ⁇ and Smith (supra) .
  • an agarose gel with "Blue Gal” (Life Technologies Inc., Gaither ⁇ burg) was used which allows an easy isolation of blue ⁇ tained plaques.
  • a detailed description of a "plaque assay” can al ⁇ o be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologie ⁇ Inc., Gaither ⁇ burg, page 9- 10) .
  • Sf9 cell ⁇ were grown in Grace's medium ⁇ upplemented with 10% heat-inactivated FBS.
  • the cell ⁇ were infected with the recombinant baculoviru ⁇ v-thrombin receptor at a multiplicity of infection (MOD of 2.
  • MOD multiplicity of infection
  • the medium was removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc. , Gaithersburg) .
  • the cells were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins vi ⁇ ualized by SDS-PAGE and autoradiograph .
  • Fibroblast ⁇ are obtained from a ⁇ ubject by ⁇ kin biop ⁇ y.
  • the re ⁇ ulting ti ⁇ ue is placed in tis ⁇ ue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a ti ⁇ sue culture flask, approximately ten pieces are placed in each fla ⁇ k.
  • the fla ⁇ k is turned upside down, clo ⁇ ed tight and left at room temperature over night. After 24 hours at room temperature, the fla ⁇ k is inverted and the chunks of ti ⁇ ue 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.
  • fresh media e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin
  • the cDNA encoding a polypeptide of the pre ⁇ ent 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 re ⁇ ulting mixture i ⁇ maintained under conditions appropriate for ligation of the two fragments.
  • the ligation mixture is u ⁇ ed to tran ⁇ form 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.
  • DMEM Dulbecco's Modified Eagles Medium
  • CS calf serum
  • penicillin and streptomycin The MSV vector containing the gene i ⁇ 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 tran ⁇ duced producer cell ⁇ , 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 cell ⁇ nd this media is then u ⁇ ed 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 fibrobla ⁇ ts will be infected and no ⁇ election is required. If the titer is very low, then it i ⁇ necessary to use a retroviral vector that has a selectable marker, such as neo or his.
  • the engineered fibroblast ⁇ are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
  • the fibrobla ⁇ t ⁇ now produce the protein product.

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Abstract

La présente invention concerne des polypeptides récepteurs de type thrombine de protéines G humaines et l'ADN (ARN) codant de tels polypeptides, ainsi qu'une procédure de production de ces polypeptides par des techniques de recombinaison. L'invention concerne également des procédés d'utilisation desdits polypeptides pour l'identification d'antagonistes et d'agonistes de ces polypeptides et des procédés d'utilisation thérapeutique des agonistes et antagonistes. L'invention a en outre pour objet des méthodes de diagnostic permettant de détecter une mutation dans les séquences d'acide nucléique des récepteurs de type thrombine de protéines G, de même que le niveau de la forme soluble des récepteurs dans un échantillon tiré d'un hôte.
PCT/US1995/007180 1995-06-06 1995-06-06 Recepteur hibeb69 de proteines g WO1996039438A1 (fr)

Priority Applications (2)

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AU26989/95A AU2698995A (en) 1995-06-06 1995-06-06 G-protein receptor hibeb69
PCT/US1995/007180 WO1996039438A1 (fr) 1995-06-06 1995-06-06 Recepteur hibeb69 de proteines g

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PCT/US1995/007180 WO1996039438A1 (fr) 1995-06-06 1995-06-06 Recepteur hibeb69 de proteines g

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WO1996039438A1 true WO1996039438A1 (fr) 1996-12-12

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0860502A1 (fr) * 1997-02-24 1998-08-26 Smithkline Beecham Corporation Clone HDPBI30 de cDNA codant pour un nouveau récepteur 7-transmembrane humain
GB2360586A (en) * 2000-02-18 2001-09-26 Glaxo Group Ltd Assays for and modulators of cysteinyl leukotriene-like receptors
US6737252B2 (en) 1997-07-25 2004-05-18 Schering Corporation 7 transmembrane receptor family member BLRX
WO2005040829A3 (fr) * 2003-10-21 2005-06-23 Bayer Healthcare Ag Agents de diagnostic et de traitement des maladies associees au recepteur 17 couple aux proteines g (gpr17)
WO2006045476A3 (fr) * 2004-10-21 2006-07-20 Univ Degli Studi Milano Modulateurs du recepteur gpr17, procede de criblage et utilisations de ceux-ci

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FEBS LETTERS, Vol. 271, issued October 1990, EVA et al., "Molecular Cloning of a Novel G Protein-Coupled Receptor that May Belong to the Neuropeptide Receptor Family", pages 81-84. *
FEBS LETTERS, Vol. 284, No. 2, issued June 1991, MEYERHOF et al., "Molecular Cloning of a Novel Putative G-Protein Coupled Receptor Expressed During Rat Spermiogenesis", pages 155-160. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 87, issued April 1990, ROSS et al., "RTA, a Candidate G Protein-Coupled Receptor: Cloning, Sequencing and Tissue Distribution", pages 3052-3056. *
SCIENCE, Vol. 244, issued 05 May 1989, LIBERT et al., "Selective Amplification and Cloning of Four New Members of the G Protein-Coupled Receptor Family", pages 568-572. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 265, No. 16, issued 05 June 1990, HLA et al., "An Abundant Transcript Induced in Differentiating Human Endothelial Cells Encodes a Polypeptide with Structural Similarities to G-Protein-Coupled Receptors", pages 9308-9316. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0860502A1 (fr) * 1997-02-24 1998-08-26 Smithkline Beecham Corporation Clone HDPBI30 de cDNA codant pour un nouveau récepteur 7-transmembrane humain
US6221627B1 (en) 1997-02-24 2001-04-24 Smithkline Beecham Corporation cDNA clone HDPB130 that encodes a novel human 7-transmembrane receptor
US6737252B2 (en) 1997-07-25 2004-05-18 Schering Corporation 7 transmembrane receptor family member BLRX
US7307146B2 (en) 1997-07-25 2007-12-11 Schering Corporation Dnaxccr10
GB2360586A (en) * 2000-02-18 2001-09-26 Glaxo Group Ltd Assays for and modulators of cysteinyl leukotriene-like receptors
WO2005040829A3 (fr) * 2003-10-21 2005-06-23 Bayer Healthcare Ag Agents de diagnostic et de traitement des maladies associees au recepteur 17 couple aux proteines g (gpr17)
WO2006045476A3 (fr) * 2004-10-21 2006-07-20 Univ Degli Studi Milano Modulateurs du recepteur gpr17, procede de criblage et utilisations de ceux-ci
US8158593B2 (en) 2004-10-21 2012-04-17 Universita' Degli Studi Di Milano GPR17 modulators, method of screening and uses thereof

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