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WO1996039436A1 - Recepteur couple de proteines g humaines (hetgq23) - Google Patents

Recepteur couple de proteines g humaines (hetgq23) Download PDF

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Publication number
WO1996039436A1
WO1996039436A1 PCT/US1995/007137 US9507137W WO9639436A1 WO 1996039436 A1 WO1996039436 A1 WO 1996039436A1 US 9507137 W US9507137 W US 9507137W WO 9639436 A1 WO9639436 A1 WO 9639436A1
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WIPO (PCT)
Prior art keywords
polypeptide
compound
receptor
dna
protein coupled
Prior art date
Application number
PCT/US1995/007137
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English (en)
Inventor
Daniel R. Soppet
Yi Li
Craig A. Rosen
Steven M. Ruben
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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 CA002220978A priority Critical patent/CA2220978A1/fr
Priority to PCT/US1995/007137 priority patent/WO1996039436A1/fr
Priority to CNB951979329A priority patent/CN1157410C/zh
Priority to JP9500363A priority patent/JPH11507812A/ja
Priority to AU29431/95A priority patent/AU716023B2/en
Priority to KR1019970708858A priority patent/KR19990022378A/ko
Priority to EP95925238A priority patent/EP0833846A4/fr
Publication of WO1996039436A1 publication Critical patent/WO1996039436A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • non-naturally occurring synthetic, isolated and/or recombinant G-protein coupled receptor polypeptides which are fragments, consensus fragments and/or sequences having conservative amino acid substitutions, of at least one transmembrane domain of the G- protein coupled receptor of the present invention, such that the receptor may bind G-protein coupled receptor ligands, or which may also modulate, quantitatively or qualitatively, G- protein coupled receptor ligand binding.
  • diagnostic probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the nucleic acid sequences of the present invention.
  • a diagnostic assay for detecting a disease or susceptibility to a disease related to a mutation in a nucleic acid sequence of the present invention.
  • Figure 1 shows the cDNA sequence and the corresponding deduced amino acid sequence of the G-protein coupled receptor of the present invention.
  • the standard one-letter abbreviation for amino acids are used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc. ) .
  • Figure 3 is an illustration of the secondary structural features of the G-protein coupled receptor.
  • the first 7 illustrations set forth the regions of the amino acid sequence which are alpha helices, 'beta sheets, turn regions or coiled regions.
  • the boxed areas are the areas which correspond to the region indicated.
  • the second set of figures illustrate areas of the amino acid sequence which are exposed to intracellular, cytoplasmic or are membrane- spanning.
  • the hydrophilicity part illustrates areas of the protein sequence which are in the lipid bilayer of the membrane and are, therefore, hydrophobic, and areas outside the lipid bilayer membrane which are hydrophilic.
  • the antigenic index corresponds to the hydrophilicity plot, since antigenic areas are areas outside the lipid bilayer membrane and are capable of binding antigens.
  • nucleic acids which encode 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. 97,130 on 4-28-95.
  • a polynucleotide encoding the polypeptide of the present invention was isolated from a cDNA library derived from human endometrial tumor tissue. It is structurally related to the G protein-coupled receptor family. It contains an open reading frame encoding a protein of 364 amino acid residues. The protein exhibits the highest degree of homology to a human EDG-1 protein with 36 % identity and 61 % similarity over a 364 amino acid stretch.
  • Potential ligands to the receptor polypeptide of the present invention include but are not limited to anandamide, serotonin, adrenalin and noradrenalin, platelet activating factor, thrombin, C5a and bradykinin, chemokine, and platelet activating factor.
  • the polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (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.
  • polynucleotides which encode for the mature polypeptides 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 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 variants of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non- naturally occurring variant of the polynucleotides.
  • the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants 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 variar ⁇ s, substitution variants and addition or insertion variants.
  • the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure .1 (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 polypeptides .
  • polynucleotides may also encode for a soluble form of the receptor polypeptide of the present invention 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 polypeptides of the present invention.
  • the marker sequence may be a hexa- histidine 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)) .
  • 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) .
  • Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or similar biological activity.
  • Probes of this type preferably have at lea ⁇ t 20 or 30 bases and may contain, for example, 50 or more bases.
  • the probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene of the present invention including regulatory and promotor regions, exons, and introns.
  • An example of a screen 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 gene 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 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) .
  • the polynucleotide may have at least 20 bases, preferably at least 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has 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:l, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
  • 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 coupled 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 derivative ⁇ of such polypeptide.
  • 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 a 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 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 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.
  • 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 _______________ ,
  • the pre ⁇ ent invention al ⁇ o include ⁇ recombinant constructs comprising one or more of the sequence ⁇ a ⁇ broadly de ⁇ cribed above.
  • the constructs comprise a vector, such a ⁇ 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.
  • suitable vectors and promoters are known to tho ⁇ e of ⁇ kill in the art, and are commercially available. The following vectors are provided by way of example.
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) .
  • 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.
  • the pre ⁇ ent invention relates- to host cells containing the above-described con ⁇ truct ⁇ .
  • the ho ⁇ t cell can be a higher eukaryotic cell, ⁇ uch a ⁇ a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the ho ⁇ t 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 tran ⁇ fection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986) ) .
  • 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 down ⁇ tream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3-pho ⁇ phoglycerate kinase (PGK) , ⁇ -factor, acid phosphatase, or heat shock proteins, among others.
  • PGK 3-pho ⁇ phoglycerate kinase
  • the heterologous structural ⁇ equence is as ⁇ embled in appropriate pha ⁇ e with tran ⁇ lation initiation and termination sequences.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the re ⁇ ulting crude extract retained for further purification.
  • Microbial cell ⁇ employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agent ⁇ , such methods are well know to those skilled in the ar .
  • a ⁇ oluble form of the G-protein coupled receptor e.g. a fragment of the receptor ⁇ , may be u ⁇ ed to inhibit activation of the receptor by binding to the ligand to a polypeptide of the pre ⁇ ent invention and preventing the ligand from interacting with membrane bound G-protein coupled receptors.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al . , Nature, 324:163-166 (1986)) prior to analysis.
  • RNA or cDNA may also be u ⁇ ed for the same purpose.
  • PCR primer ⁇ complementary to the nucleic acid encoding the G-protein coupled receptor protein ⁇ can be used to identify and analyze G-protein coupled receptor mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • the sequences of the present invention are al ⁇ o valuable for chromo ⁇ ome identification.
  • the ⁇ equence i ⁇ 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 chromosomes according to the present invention is an important first step in correlating those sequences with gene associated with disea ⁇ e.
  • polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
  • antibodie ⁇ can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expre ⁇ ion library. Variou ⁇ procedures known in the art may be used for the production of such antibodies and fragments .
  • Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980) .
  • the DNA ⁇ equence encoding GPRC, ATCC # 97,130, is initially amplified using PCR oligonucleotide primers corre ⁇ ponding to the 5' and 3' end sequences of the processed GPRC nucleotide sequence. Additional nucleotides corresponding to the GPRC nucleotide sequence are added to the 5' and 3' sequences respectively.
  • the 5' oligonucleotide primer has the sequence 5' CACAGGATCCCGTGGCTGCCATCTCTACTTC 3' (SEQ ID NO:3) contains a BatnHT restriction enzyme ⁇ ite followed by 17 nucleotides of GPRC coding sequence ⁇ tarting from the presumed second amino acid of the processed protein.
  • the 3' sequence; 5' TCTCAGGTACCGTTCTCTAAACCACAGAGTGGTCA contains complementary sequences to an ASP718 site and is followed by 19 nucleotide ⁇ of GPRC coding sequence.
  • the restriction enzyme site ⁇ correspond to the restriction enzyme sites on the bacterial expression vector pQE-31 (Qiagen, Inc. Chatsworth, CA) .
  • pQE-31 encodes antibiotic resi ⁇ tance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/0) , a ribo ⁇ ome binding ⁇ ite (RBS) , a 6-Hi ⁇ tag and restriction enzyme sites.
  • Clones containing the desired constructs are grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) .
  • the 0/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250.
  • the cell ⁇ are grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG Isopropyl-B-D- thiogalacto pyranoside
  • IPTG induce ⁇ by inactivating the lad repressor, clearing the P/O leading to increased gene expression.
  • Cells are grown an extra 3 to 4 hours.
  • Cells are then harvested by centrifugation.
  • the cell pellet is ⁇ olubilized in the chaotropic agent 6 Molar Guanidine HCl .
  • solubilized GPRC is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al . , J. Chromatography 411:177-184 (1984)) .
  • GPRC is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM ⁇ odium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this ⁇ olution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate .
  • the expres ⁇ ion of plasmid, GPRC HA was derived from a vector pcDNA3/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resi ⁇ tance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation ⁇ ite.
  • a DNA fragment encoding the entire GPRC 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 was directed under the CMV promoter.
  • the HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I.
  • HA tag The infusion of HA tag to the target protein allows ea ⁇ y detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the PCR product contains a Hindlll site, GPRC coding sequence followed by HA tag fused in frame, a translation termination ⁇ top codon next to the HA tag, and an Xhol ⁇ ite.
  • the PCR amplified DNA fragment and the vector, pcDNA3/Amp were dige ⁇ ted with Hindlll and Xhol restriction enzymes and ligated.
  • the ligation mixture was transformed into E. coli strain DH5 ⁇ , the transformed cultur. wa ⁇ plated on ampicillin media plate ⁇ and resistant colonies were selected. Pla ⁇ mid DNA wa ⁇ i ⁇ olated from transformants and examined by restriction analysis for the presence of the correct fragment .
  • C0S7 cells were transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniati ⁇ , Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)) .
  • Cells were labelled for 8 hours with 35 S-cysteine two days post transfection.
  • the polyadenylation site of the simian virus (SV)40 was used for efficient polyadenylation.
  • the beta-galactosida ⁇ e gene from E.coli was inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene.
  • the polyhedrin ⁇ equence ⁇ were flanked at both ⁇ ides by viral sequences for the cell-mediated homologous recombination of cotran ⁇ fected wild-type viral DNA.
  • Many other baculoviru ⁇ vector ⁇ could be u ⁇ ed in place of pA2 such as pAc373, pVL941, PRGl and pAcIMl (Luckow, V.A. and Summers, M.D. , Virology, 170:31-39) .
  • the DNA was then isolated from a 1% agaro ⁇ e gel u ⁇ ing the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) . This vector DNA was designated V2.
  • the virus were added to the cells and blue ⁇ tained plaque ⁇ were picked with the tip of an Eppendorf pipette.
  • the agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing 200 ⁇ l of Grace's medium.
  • the agar was removed by a brief centrifugation and the supernatant containing the recombinant baculovirus was used to infect Sf9 cells seeded in 35 mm dishe ⁇ .
  • the supernatants of these culture dishes were harvested and then ⁇ tored at 4°C.
  • Fibroblasts are obtained from a subject by skin biopsy.
  • the resulting ti ⁇ sue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tis ⁇ ue culture flask, approximately ten pieces are placed in each flask.
  • the fla ⁇ k is turned upside down, closed tight and left at room temperature over night . After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fre ⁇ h media (e.g., Ham' ⁇ F12 media, with 10% FBS, penicillin and streptomycin, is added. This is then incubated at 37°C for approximately one week.
  • fre ⁇ h media e.g., Ham' ⁇ F12 media, with 10% FBS, penicillin and streptomycin
  • the cDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5' and 3' end sequence ⁇ re ⁇ pectively.
  • the 5' primer contains an EcoRI site and the 3' primer further include ⁇ a Hindlll site.
  • Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and Hindlll fragment are added together, in the presence of T4 DNA ligase.
  • the resulting mixture is maintained under conditions appropriate for ligation of the two fragments .
  • the ligation mixture is used to transform bacteria 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 cell ⁇ are grown in ti ⁇ sue 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 is 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) .
  • the engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
  • the fibroblasts now produce the protein product.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • GGAACCGCCC CACCGTGGTG GCGGCCGCCC AGAACTAGTG GATCCCCCGG GCTGCAGGAA 60 TTCGGCACGA GCAGACACAC TTGCTTTGGT TTACAGATCC AGTGAAGTGA AAAATCAGAA 120 CTAGAAACGT ATGCACCTTC CTAGCAGCAA AGCCGCTTCT GCGTTCTTCG CAGCCTCCAG 180
  • ATCCCCATCC CTTCTGAAAG TAGGAAGTTG GAGCTCTTGC AATGGAATTC AAGAACAGAC 1920
  • Val Asn Arg Arg Phe Hi ⁇ Phe Pro lie Tyr Tyr Leu Met Ala Asn
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:7: TTCACCACCT ACCTGGATCC ACAGAGCTGT CATGGCTGCC 40

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Abstract

La présente invention concerne des polypeptides récepteurs couplés de protéines G humaines et l'ADN (ARN) codant de tels polypeptides, ainsi qu'un procédé de production de ces polypeptides par des techniques de recombinaison. Elle concerne également des procédés d'utilisation desdits polypeptides pour l'identification d'antagonistes et d'agonistes à ces polypeptides ainsi que des procédés d'application thérapeutique des agonistes et des antagonistes dans le traitement, respectivement, de la sous-expression et de la surexpression des polypeptides récepteurs couplés de protéines G. L'invention a en outre pour objet des méthodes de diagnostic permettant de détecter une mutation dans les séquences d'acides nucléiques des récepteurs couplés de protéines G, de même qu'une modification de la concentration de la forme soluble des récepteurs.
PCT/US1995/007137 1995-06-05 1995-06-05 Recepteur couple de proteines g humaines (hetgq23) WO1996039436A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
CA002220978A CA2220978A1 (fr) 1995-06-05 1995-06-05 Recepteur couple de proteines g humaines (hetgq23)
PCT/US1995/007137 WO1996039436A1 (fr) 1995-06-05 1995-06-05 Recepteur couple de proteines g humaines (hetgq23)
CNB951979329A CN1157410C (zh) 1995-06-05 1995-06-05 人g蛋白偶联受体(hetgq23)
JP9500363A JPH11507812A (ja) 1995-06-05 1995-06-05 ヒトｇ−タンパク質結合性受容体（ｈｅｔｇｑ２３）
AU29431/95A AU716023B2 (en) 1995-06-05 1995-06-05 Human G-protein coupled receptor (HETGQ23)
KR1019970708858A KR19990022378A (ko) 1995-06-05 1995-06-05 사람 ｇ-단백질 결합된 수용체(ｈｅｔｇｑ23)
EP95925238A EP0833846A4 (fr) 1995-06-05 1995-06-05 Recepteur couple de proteines g humaines (hetgq23)

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CA002220978A CA2220978A1 (fr) 1995-06-05 1995-06-05 Recepteur couple de proteines g humaines (hetgq23)
PCT/US1995/007137 WO1996039436A1 (fr) 1995-06-05 1995-06-05 Recepteur couple de proteines g humaines (hetgq23)
CNB951979329A CN1157410C (zh) 1995-06-05 1995-06-05 人g蛋白偶联受体(hetgq23)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2759375A1 (fr) * 1997-02-13 1998-08-14 Inst Nat Sante Rech Med Polypeptide a activite de recepteur ob25 specifique des cellules myelinisantes chez le rat, application au criblage de medicaments et medicaments
FR2759374A1 (fr) * 1997-02-13 1998-08-14 Inst Nat Sante Rech Med Polypeptide a activite de recepteur ob25 specifique des cellules myelinisantes chez le rat, application au criblage de medicaments et medicaments
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US6485922B1 (en) * 1997-10-10 2002-11-26 Atairgin Technologies, Inc. Methods for detecting compounds which modulate the activity of an LPA receptor

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6140060A (en) * 1996-12-12 2000-10-31 Chun; Jerold J. M. Cloned lysophosphatidic acid receptors
FR2759375A1 (fr) * 1997-02-13 1998-08-14 Inst Nat Sante Rech Med Polypeptide a activite de recepteur ob25 specifique des cellules myelinisantes chez le rat, application au criblage de medicaments et medicaments
FR2759374A1 (fr) * 1997-02-13 1998-08-14 Inst Nat Sante Rech Med Polypeptide a activite de recepteur ob25 specifique des cellules myelinisantes chez le rat, application au criblage de medicaments et medicaments
WO1998036060A1 (fr) * 1997-02-13 1998-08-20 Institut National De La Sante Et De La Recherche Medicale (Inserm) Utilisation a des fins de diagnostic et therapeutiques d'un polypeptide a activite de recepteur ob25 exprime par les cellules myelinisantes
US6485922B1 (en) * 1997-10-10 2002-11-26 Atairgin Technologies, Inc. Methods for detecting compounds which modulate the activity of an LPA receptor

Also Published As

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EP0833846A4 (fr) 1999-07-14
CN1157410C (zh) 2004-07-14
CN1193981A (zh) 1998-09-23
CA2220978A1 (fr) 1996-12-12
EP0833846A1 (fr) 1998-04-08

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