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WO2000008133A1 - NOUVELLE SEQUENCE D'ADNc D'UN NOUVEAU RECEPTEUR COUPLE A LA PROTEINE G - Google Patents

NOUVELLE SEQUENCE D'ADNc D'UN NOUVEAU RECEPTEUR COUPLE A LA PROTEINE G Download PDF

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WO2000008133A1
WO2000008133A1 PCT/US1999/017388 US9917388W WO0008133A1 WO 2000008133 A1 WO2000008133 A1 WO 2000008133A1 US 9917388 W US9917388 W US 9917388W WO 0008133 A1 WO0008133 A1 WO 0008133A1
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cells
substance
binding
protein
amount
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WO2000008133A9 (fr
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Qingyun Liu
Terrence P. Mcdonald
Ruiping Wang
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Merck and Co Inc
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Merck and Co Inc
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Priority to EP99941986A priority Critical patent/EP1105465A1/fr
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Priority to CA002339347A priority patent/CA2339347A1/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

Definitions

  • This invention relates to a novel human cDNA encoding a G protein- coupled receptor (GPCR) having homology to members of a family of receptors for nucleotides and platelet-activating factor, the protein encoded by the cDNA, and methods of identifying selective agonists and antagonists.
  • GPCR G protein- coupled receptor
  • G-protein coupled receptors are a very large class of membrane receptors that relay information from the exterior to the interior of cells. GPCRs function by interacting with a class of heterotrimeric proteins known as G- proteins. Most GPCRs function by a similar mechanism. Upon the binding of agonist, a GPCR catalyzes the dissociation of guanosine diphosphate (GDP) from the ⁇ subunit of G proteins. This allows for the binding of guanosine triphosphate (GTP) to the ⁇ subunit, resulting in the disassociation of the ⁇ subunit from the ⁇ and ⁇ subunits. The freed ⁇ subunit then interacts with other cellular components, and in the process passes on the extracellular signal represented by the presence of the agonist. Occasionally, it is the freed ⁇ and ⁇ subunits which transduce the agonist signal.
  • GDP guanosine diphosphate
  • GTP guanosine triphosphate
  • GPCRs possess common structural characteristics. They have seven hydrophobic domains, about 20-30 amino acids long, linked by sequences of hydrophilic amino acids of varied length. These seven hydrophobic domains intercalate into the plasma membrane, giving rise to a protein with seven transmembrane domains, an extracellular amino terminus, and an intracellular carboxy terminus (Strader et al., 1994, Ann. Rev. Biochem. 63:101-132; Schertler et al., 1993, Nature 362:770-7721; Dohlman et al., 1991, Ann. Rev. Biochem. 60:653- 688).
  • GPCRs are expressed in a wide variety of tissue types and respond to a wide range of ligands, e.g., protein hormones, biogenic amines, peptides, lipid derived messengers, etc. Given their wide range of expression and ligands, it is not surprising that GPCRs are involved in many pathological states. This has led to great interest in developing modulators of GPCR activity that can be used pharmacologically. For example, Table 1 of Stadel et al., 1997, Trends Pharmacol. Sci. 18:430-437, lists 37 different marketed drugs that act upon GPCRs. Accordingly, there is a great need to understand GPCR function and to develop agents that can be used to modulate GPCR activity.
  • ligands e.g., protein hormones, biogenic amines, peptides, lipid derived messengers, etc. Given their wide range of expression and ligands, it is not surprising that GPCRs are involved in many pathological states. This has led to great interest in developing modulators of
  • the present invention is directed to a novel human cDNA that encodes a G-protein coupled receptor, HG03.
  • the DNA encoding HG03 is substantially free from other nucleic acids and has the nucleotide sequence shown in SEQ.ID.NO.:l.
  • an HG03 protein encoded by the novel cDNA sequence is substantially free from other proteins and has the amino acid sequence shown in SEQ.LD.NO.:2.
  • Figure 1 shows the complete cDNA sequence of HG03 (SEQ.ID.NO.:l).
  • Figure 2 show the complete amino acid sequence of HG03 (SEQ.ID.:2).
  • Figure 3A-C shows the translation of HG03 open reading frame.
  • the nucleotide sequence shown is (SEQ.ID.:1).
  • the amino acid sequence shown is (SEQ.ID.:2).
  • Figure 4 shows the results of a Northern blot of the expression of HG03 mRNA in various human tissues.
  • Figure 5 shows the alignment of the amino acid sequence of HG03 with the amino acid sequence of the human platelet activating factor receptor (SEQ.ID.:3). DETAILED DESCRIPTION OF THE INVENTION
  • SEQ.ID.:3 the amino acid sequence of the human platelet activating factor receptor
  • HG03 protein preparation that is substantially free from other proteins will contain, as a percent of its total protein, no more than 10%o, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-HG03 proteins.
  • Whether a given HG03 protein preparation is substantially free from other proteins can be determined by such conventional techniques of assessing protein purity as, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g., silver staining or immunoblotting.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • substantially free from other nucleic acids means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other nucleic acids.
  • an HG03 DNA preparation that is substantially free from other nucleic acids will contain, as a percent of its total nucleic acid, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-HG03 nucleic acids.
  • Whether a given HG03 DNA preparation is substantially free from other nucleic acids can be determined by such conventional techniques of assessing nucleic acid purity as, e.g., agarose gel electrophoresis combined with appropriate staining methods, e.g., ethidium bromide staining, or by sequencing.
  • “Functional equivalent” means a receptor which does not have the exact same amino acid sequence of a naturally occurring G protein-coupled receptor, due to alternative splicing, deletions, mutations, or additions, but retains at least 1%, preferably 10%, and more preferably 25% of the biological activity of the naturally occurring receptor.
  • Such derivatives will have a significant homology with a natural GPCR and genes and cDNA encoding such derivatives can be detected by reduced stringency hybridization with a DNA sequence encoding a natural GPCR.
  • the nucleic acid encoding a functional equivalent has at least about 50% homology at the nucleotide level to a naturally occurring receptor nucleic acid.
  • a polypeptide has "substantially the same biological activity” as HG03 if that polypeptide has a Kd for a ligand that is no more than 5-fold greater than the Kd of HG03 for the same ligand.
  • a “conservative amino acid substitution” refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for as artic acid).
  • GPCR G protein-coupled receptor
  • nucleic acids which encode the HG03 G protein-coupled receptor. These nucleic acids are substantially free from associated nucleic acids. For most cloning purposes, DNA is a preferred nucleic acid.
  • the present invention provides a cDNA molecule substantially free from other nucleic acids having the nucleotide sequence shown in FIGURE 1 as SEQ.ID.NO.:l. Analysis of FIGURE 3A-C revealed that SEQ.ID.NO:l contains an open reading frame at positions 346-1419. Thus, the present invention also provides a cDNA molecule substantially free from other nucleic acids comprising the nucleotide sequence of positions 346-1419 of SEQ.ID.NO.:l . The present invention also provides recombinant DNA molecules comprising the nucleotide sequence of positions 346-1419 of SEQ.ID.NO.:l .
  • HG03 Sequence analysis of the open reading frame of the HG03 cDNA revealed that it encodes a protein of 358 amino acids. Based on its predicted amino acid sequence, HG03 most likely represents a novel GPCR. Northern blot analysis showed that HG03 RNA is highly expressed in the prostate, placenta, and trachea in human with a major transcript of ⁇ 1.8 kb and a minor transcript of ⁇ 8.0 kb. HG03 was also expressed at lower levels in thymus and testis as a transcript of ⁇ 1.8 kb. HG03 appears to be related to the members of receptors for nucleotides and platelet-activating factor.
  • novel DNA sequences of the present invention encoding HG03 in whole or in part, can be linked with other DNA sequences, i.e., DNA sequences to which HG03 is not naturally linked, to form "recombinant DNA molecules" containing HG03.
  • the novel DNA sequences of the present invention can be inserted into vectors which comprise nucleic acids encoding a GPCR or a functional equivalent. These vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes and other forms of episomal or integrated DNA that can encode a GPCR. It is well within the skilled artisan to determine an appropriate vector for a particular gene transfer or other use.
  • cDNA sequences that hybridize to SEQ.ID.NO.:l under stringent conditions.
  • a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA is carried out for 2 hr. to overnight at 65°C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65°C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10" cpm of 32p-l a beled probe. Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1 %
  • Another aspect of the present invention includes host cells that have been engineered to contain and/or express DNA sequences encoding HG03.
  • Such recombinant host cells can be cultured under suitable conditions to produce HG03.
  • An expression vector containing DNA encoding HG03 can be used for expression of HG03 in a recombinant host cell.
  • Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines.
  • L cells L-M(TK') (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
  • CHO cells are particularly suitable for expression of the HG03 protein because these cells express a large number of G-proteins. Thus, it is likely that at least one of these G-proteins will be able to functionally couple the signal generated by interaction of HG03 and its ligands, thus transmitting this signal to downstream effectors, eventually resulting in a measurable change in some assayable component, e.g., cAMP level, expression of a reporter gene, hydrolysis of inositol lipids, or intracellular Ca2+ levels.
  • some assayable component e.g., cAMP level
  • expression of a reporter gene e.g., hydrolysis of inositol lipids, or intracellular Ca2+ levels.
  • mammalian expression vectors can be used to express recombinant HG03 in mammalian cells.
  • Commercially available mammalian expression vectors which are suitable include, but are not limited to, pCR2.1
  • HG03 can be purified by conventional techniques to a level that is substantially free from other proteins.
  • this invention includes modified HG03 polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as HG03. It is generally accepted that single amino acid substitutions do not usually alter the biological activity of a protein (see, e.g. , Molecular Biology of the Gene, Watson et al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co., Inc., page 226; and Cunningham & Wells, 1989, Science 244:1081-1085).
  • the present invention includes polypeptides where one amino acid substitution has been made in SEQ.ID.NO.:2 wherein the polypeptides still retain substantially the same biological activity as HG03.
  • the present invention also includes polypeptides where two or more amino acid substitutions have been made in SEQ.ID.NO.:2 wherein the polypeptides still retain substantially the same biological activity as HG03.
  • the present invention includes embodiments where the above-described substitutions are conservative substitutions.
  • the present invention includes embodiments where the above-described substitutions do not occur in the ligand-binding domain of HG03.
  • the present invention also includes C-terminal truncated forms of HG03, particularly those which encompass the extracellular portion of the receptor, but lack the intracellular signaling portion of the receptor.
  • Such truncated receptors are useful in various binding assays described herein, for crystallization studies, and for structure-activity-relationship studies.
  • membrane spanning regions of receptor proteins can be used to inhibit receptor function (Ng et al., 1996, Biochem. Biophys. Res. Comm. 227:200-204; Hebert et al, 1996, J. Biol. Chem. 271, 16384- 16392; Lofts et al, Oncogene 8:2813-2820).
  • the present invention provides peptides derived from the seven membrane spanning regions of HG03 and their use to inhibit HG03 function.
  • Such peptides can include the whole or parts of the receptor membrane spanning domains.
  • the present invention also includes chimeric HG03 proteins.
  • Chimeric HG03 proteins consist of a contiguous polypeptide sequence of HG03 fused in frame to a polypeptide sequence of a non-HG03 protein.
  • the N-terminal domain and seven transmembrane spanning domains of HG03 fused at the C-terminus in frame to a G protein would be a chimeric HG03 protein.
  • the present invention also includes HG03 proteins that are in the form of multimeric structures, e.g., dimers.
  • HG03 proteins that are in the form of multimeric structures, e.g., dimers.
  • Such multimers of other G-protein coupled receptors are known (Hebert et al, 1996, J. Biol. Chem. 271, 16384-16392; Ng et al, 1996, Biochem. Biophys. Res. Comm. 227, 200-204; Romano et al, 1996, J. Biol. Chem. 271, 28612-28616).
  • the present invention also includes isolated forms of HG03 proteins.
  • isolated HG03 protein is meant HG03 protein that has been isolated from a natural source. Use of the term “isolated” indicates that HG03 protein has been removed from its normal cellular environment. Thus, an isolated HG03 protein may be in a cell-free solution or placed in a different cellular environment from that in which it occurs naturally.
  • isolated does not imply that an isolated HG03 protein is the only protein present, but instead means that an isolated HG03 protein is at least 95% free of non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the HG03 protein.
  • non-amino acid material e.g., nucleic acids, lipids, carbohydrates
  • the specificity of binding of compounds showing affinity for HG03 is shown by measuring the affinity of the compounds for recombinant cells expressing the cloned receptor or for membranes from these cells. Expression of the cloned receptor and screening for compounds that bind to HG03 or that inhibit the binding of a known, radiolabeled ligand of HG03 to these cells, or membranes prepared from these cells, provides an effective method for the rapid selection of compounds with high affinity for HG03.
  • ligands need not necessarily be radiolabeled but can also be nonisotopic compounds that can be used to displace bound radiolabeled compounds or that can be used as activators in functional assays.
  • Compounds identified by the above method are likely to be agonists or antagonists of HG03 and may be peptides, proteins, or non-proteinaceous organic molecules.
  • the present invention include assays by which HG03 agonists and antagonists may be identified.
  • Methods for identifying agonists and antagonists of other receptors are well known in the art and can be adapted to identify agonists and antagonists of HG03.
  • Cascieri et al., 1992, Molec. Pharmacol. 41:1096-1099 describe a method for identifying substances that inhibit agonist binding to rat neurokinin receptors and thus are potential agonists or antagonists of neurokinin receptors.
  • the method involves transfecting COS cells with expression vectors containing rat neurokinin receptors, allowing the transfected cells to grow for a time sufficient to allow the neurokinin receptors to be expressed, harvesting the transfected cells and resuspending the cells in assay buffer containing a known radioactively labeled agonist of the neurokinin receptors either in the presence or the absence of the substance, and then measuring the binding of the radioactively labeled known agonist of the neurokinin receptor to the neurokinin receptor. If the amount of binding of the known agonist is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of the neurokinin receptor.
  • the present invention includes a method for determining whether a substance is a potential agonist or antagonist of HG03 that comprises:
  • step (d) measuring the binding of the labeled agonist to HG03; where if the amount of binding of the known agonist is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of HG03.
  • step (b) is modified in that the cells are stably transfected with the expression vector containing HG03.
  • step (c) is modified in that the cells are not harvested and resuspended but rather the radioactively labeled known agonist and the substance are contacted with the cells while the cells are attached to a substratum, e.g., tissue culture plates.
  • step (c) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
  • the cells are eukaryotic cells.
  • the cells are mammalian cells.
  • the cells are L cells L-M(TK ' ) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or MRC-5 (ATCC CCL 171).
  • the present invention also includes a method for determining whether a substance is capable of binding to HG03, i.e., whether the substance is a potential agonist or an antagonist of HG03, where the method comprises: (a) providing test cells by transfecting cells with an expression vector that directs the expression of HG03 in the cells;
  • HG03 in the test cells with the amount of binding of the substance to control cells that have not been transfected with HG03; wherein if the amount of binding of the substance is greater in the test cells as compared to the control cells, the substance is capable of binding to HG03. Determining whether the substance is actually an agonist or antagonist can then be accomplished by the use of functional assays such as, e.g., the assay involving the use of promiscuous G-proteins described below.
  • step (b) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
  • transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retroviral construct containing HG03, and electroporation.
  • binding of the substance or agonist to HG03 can be measured by employing a labeled substance or agonist.
  • the substance or agonist can be labeled in any convenient manner known to the art, e.g., radioactively, fluorescently, enzymatically.
  • HG03 has an amino acid sequence of SEQ.ID.NO.:2.
  • the above-described methods can be modified in that, rather than exposing the test cells to the substance, membranes can be prepared from the test cells and those membranes can be exposed to the substance.
  • membranes can be prepared from the test cells and those membranes can be exposed to the substance.
  • Such a modification utilizing membranes rather than cells is well known in the art and is described in, e.g., Hess et al., 1992, Biochem. Biophys. Res. Comm. 184:260-268.
  • the present invention provides a method for determining whether a substance is capable of binding to HG03 comprising:
  • the present invention provides a method for determining whether a substance is capable of binding to HG03 comprising:
  • step (b) exposing the test cells to a ligand of HG03 under conditions such that the ligand binds to the HG03 in the test cells; (c) subsequently or concurrently to step (b), exposing the cells to a substance that is suspected of being capable of binding to HG03;
  • the present invention provides a method for determining whether a substance is capable of binding to HG03 comprising:
  • step (c) subsequently or concurrently to step (b), exposing the membranes from the test cells to a substance;
  • RNA encoding HG03 can be prepared as, e.g., by in vitro transcription using a plasmid containing HG03 under the control of a bacteriophage T7 promoter, and the RNA can be microinjected into Xenopus oocytes in order to cause the expression of HG03 in the oocytes. Substances are then tested for binding to the HG03 expressed in the oocytes. Alternatively, rather than detecting binding, the effect of the substances on the electrophysiological properties of the oocytes can be determined.
  • the present invention includes assays by which HG03 agonists and antagonists may be identified by their ability to stimulate or antagonize a functional response mediated by HG03.
  • HG03 belongs to the class of proteins known as G-protein coupled receptors (GPCRs). GPCRs transmit signals across cell membranes upon the binding of ligand. The ligand-bound GPCR interacts with a heterotrimeric G-protein, causing the G ⁇ subunit of the G-protein to disassociate from the G ⁇ and G ⁇ subunits. The G ⁇ subunit can then go on to activate a variety of second messenger systems.
  • GPCRs G-protein coupled receptors
  • Offermanns described a system in which cells are transfected with expression vectors that result in the expression of one of a large number of GPCRs as well as the expression of one of the promiscuous G-proteins G ⁇ l5 or G ⁇ l6.
  • the GPCR was activated and was able, via G ⁇ l5 or G ⁇ l6, to activate the ⁇ isoform of phospholipase C, leading to an increase in inositol phosphate levels in the cells.
  • the present invention provides a method of identifying antagonists of HG03 comprising:
  • step (b) exposing the cells to an agonist of HG03; (c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG03;
  • Intracellular calcium mobilization is typically assayed in whole cells under a microscope using fluorescent dyes or in cell suspensions via luminescence using the aequorin assay.
  • the cells are eukaryotic cells.
  • the cells are mammalian cells.
  • the cells are L cells L-M(TK") (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or MRC-5 (ATCC CCL 171).
  • the cells are transfected with expression vectors that direct the expression of HG03 and the promiscuous G-protein in the cells.
  • step (b) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
  • the promiscuous G-protein is selected from the group consisting of G ⁇ l5 or G ⁇ l6.
  • Expression vectors containing G ⁇ l5 or G ⁇ l6 are known in the art. See, e.g., Offermanns; Buhl et al, 1993, FEBS Lett. 323:132-134; Amatruda et al, 1993, J. Biol. Chem. 268:10139-10144.
  • the above -de scribed assay can be easily modified to form a method to identify antagonists of HG03.
  • Such a method is also part of the present invention and comprises:
  • step (c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG03;
  • the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells.
  • the cells are L cells L-M(TK") (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
  • conditions under which steps (b) and (c) of the method are practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
  • the cells are transfected with expression vectors that direct the expression of HG03 and the promiscuous G-protein in the cells.
  • the promiscuous G-protein is selected from the group consisting of G ⁇ l5 or G ⁇ l6.
  • HG03 has an amino acid sequence of SEQ.ID.NO.:2.
  • Another embodiment of the present invention are methods of expressing HG03 in recombinant systems and of identifying agonists and antagonists of HG03, and for counter-screening.
  • HG03 proteins and DNA encoding HG03 proteins have utility in that they can be used as "minus targets" in screens design to identify compounds that specifically interact with other G-protein coupled receptors.
  • the present invention also includes antibodies to the HG03 protein.
  • Such antibodies may be polyclonal antibodies or monoclonal antibodies.
  • the antibodies of the present invention are raised against the entire HG03 protein or against suitable antigenic fragments of the protein that are coupled to suitable carriers, e.g., serum albumin or keyhole limpet hemocyanin, by methods well known in the art. Methods of identifying suitable antigenic fragments of a protein are known in the art. See, e.g., Hopp & Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824-3828; and Jameson & Wolf, 1988, CABIOS (Computer Applications in the Biosciences) 4:181-186.
  • HG03 protein or an antigenic fragment is injected on a periodic basis into an appropriate non-human host animal such as, e.g., rabbits, sheep, goats, rats, mice.
  • an appropriate non-human host animal such as, e.g., rabbits, sheep, goats, rats, mice.
  • the animals are bled periodically and sera obtained are tested for the presence of antibodies to the injected antigen.
  • the injections can be intramuscular, intraperitoneal, subcutaneous, and the like, and can be accompanied with adjuvant.
  • HG03 protein or an antigenic fragment is injected into an appropriate non- human host animal as above for the production of polyclonal antibodies.
  • the animal is generally a mouse.
  • the animal's spleen cells are then immortalized, often by fusion with a myeloma cell, as described in Kohler & Milstein, 1975, Nature 256:495-497.
  • Gene therapy may be used to introduce HG03 polypeptides into the cells of target organs.
  • Nucleotides encoding HG03 polypeptides can be ligated into viral vectors which mediate transfer of the nucleotides by infection of recipient cells. Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, and polio virus based vectors. Alternatively, nucleotides encoding HG03 polypeptides can be transferred into cells for gene therapy by non- viral techniques including receptor-mediated targeted transfer using ligand-nucleotide conjugates, hpofection, membrane fusion, or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo gene therapy. Gene therapy with HG03 polypeptides will be particularly useful for the treatment of diseases where it is beneficial to elevate HG03 activity.
  • HG03 The full-length coding sequence of HG03 was isolated by multiple rounds of RCCA from a prostate cDNA library. Originally, the HG03 gene was named AOMF15 but was designated later HG03.
  • the primer pair, F216 +R369 was initially used to scan the 1 -2.5KB and 2.5KB prostate cDNA libraries. Positive pools were identified where nested insert-vector PCRs were then carried out using the following combinations of primers: first round PCR reactions, F216+543R, F216+873F, and R369+543R, R369+873F; nested second round PCR reaction, F325+578R, R280+383F.
  • the list of primers used for the isolation of HG03 The list of primers used for the isolation of HG03:
  • AOMF15.R280 CAGGCTTTTAGATGAATCTGCA (SEQ . ID .NO . : 6)
  • AOMF15.R369 AGCATCAGGGTTGTTGTGGC (SEQ. ID .NO. : 7)
  • HG03.37R GCGTGTCAGGAAACACTTGG (SEQ . ID . NO . : 8 )
  • HG03_FL243F AAAGAAATCAAACCAGGAATAACC (SQ . ID .NO . : 9)
  • HG03 FL1429R CTTTGTACATATCGATTCCAACACAC (SEQ . ID .NO . : 10 )
  • PBS . 873 F CCCAGGCTTTACACTTTATGCTTCC SEQ . ID . NO . : 11 )
  • PCR reactions were carried out with AmpliTaq (Perkin Elmer, CA and Taq extender (Stratagene) under the conditions suggested by the supplier of Taq extender. The PCR fragments were sequenced and assembled. Complete sequence was obtained by using primers 37R and 1118R by primer walking.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention porte sur l'ADNc codant pour un nouveau récepteur couplé à la protéine G humaine, le HG03, ainsi que sur la protéine codée par ledit ADNc, et sur des procédés d'identification des agonistes et antagonistes du HG03.
PCT/US1999/017388 1998-08-06 1999-08-02 NOUVELLE SEQUENCE D'ADNc D'UN NOUVEAU RECEPTEUR COUPLE A LA PROTEINE G Ceased WO2000008133A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP99941986A EP1105465A1 (fr) 1998-08-06 1999-08-02 NOUVELLE SEQUENCE D'ADNc D'UN NOUVEAU RECEPTEUR COUPLE A LA PROTEINE G
JP2000563760A JP2002526036A (ja) 1998-08-06 1999-08-02 新規なＧタンパク質共役受容体ｃＤＮＡ配列
CA002339347A CA2339347A1 (fr) 1998-08-06 1999-08-02 Nouvelle sequence d'adnc d'un nouveau recepteur couple a la proteine g

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US9557198P 1998-08-06 1998-08-06
US60/095,571 1998-08-06

Publications (2)

Publication Number Publication Date
WO2000008133A1 true WO2000008133A1 (fr) 2000-02-17
WO2000008133A9 WO2000008133A9 (fr) 2001-12-20

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PCT/US1999/017388 Ceased WO2000008133A1 (fr) 1998-08-06 1999-08-02 NOUVELLE SEQUENCE D'ADNc D'UN NOUVEAU RECEPTEUR COUPLE A LA PROTEINE G

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EP (1) EP1105465A1 (fr)
JP (1) JP2002526036A (fr)
CA (1) CA2339347A1 (fr)
WO (1) WO2000008133A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001009322A1 (fr) * 1999-07-29 2001-02-08 Helix Research Institute Recepteurs couples a la proteine de liaison guanosine triphosphate, genes correspondants, et leur production et utilisation
WO2001044474A1 (fr) * 1999-12-16 2001-06-21 Millennium Pharmaceuticals, Inc. Recepteur 2871, recepteur couple a la proteine g et ses procedes d'utilisation
WO2001077325A1 (fr) * 2000-04-12 2001-10-18 Takeda Chemical Industries, Ltd. Nouvelle proteine de recepteur couple aux proteines g et adn de celle-ci
WO2001038348A3 (fr) * 1999-11-26 2001-11-08 Merck Patent Gmbh Recepteur htogh35 couple a la proteine g
WO2002050266A1 (fr) * 2000-12-20 2002-06-27 Takeda Chemical Industries, Ltd. Nouvelle proteine receptrice couplee a la proteine g et son adn
WO2002092624A3 (fr) * 2001-05-16 2003-05-08 Paradigm Therapeutics Ltd Recepteur
EP1363655A4 (fr) * 2000-12-18 2005-04-06 Merck & Co Inc Molecules d'acides nucleiques isolees codant un recepteur humain et murin couple a la proteine g - gpr54 -, proteines codees, cellules transformees par ces molecules et utilisations correspondantes
CN114766546A (zh) * 2021-12-29 2022-07-22 淮阴工学院 地衣芽孢杆菌hg03在防治葡枝根霉所致水蜜桃采后软腐病中的应用及应用方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4985352A (en) * 1988-02-29 1991-01-15 The Trustees Of Columbia University In The City Of New York DNA encoding serotonin 1C (5HT1c) receptor, isolated 5HT1c receptor, mammalian cells expressing same and uses thereof
US5144007A (en) * 1988-11-03 1992-09-01 La Jolla Cancer Research Foundation Thyroid hormone receptor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4985352A (en) * 1988-02-29 1991-01-15 The Trustees Of Columbia University In The City Of New York DNA encoding serotonin 1C (5HT1c) receptor, isolated 5HT1c receptor, mammalian cells expressing same and uses thereof
US5144007A (en) * 1988-11-03 1992-09-01 La Jolla Cancer Research Foundation Thyroid hormone receptor

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001009322A1 (fr) * 1999-07-29 2001-02-08 Helix Research Institute Recepteurs couples a la proteine de liaison guanosine triphosphate, genes correspondants, et leur production et utilisation
WO2001038348A3 (fr) * 1999-11-26 2001-11-08 Merck Patent Gmbh Recepteur htogh35 couple a la proteine g
WO2001044474A1 (fr) * 1999-12-16 2001-06-21 Millennium Pharmaceuticals, Inc. Recepteur 2871, recepteur couple a la proteine g et ses procedes d'utilisation
WO2001077325A1 (fr) * 2000-04-12 2001-10-18 Takeda Chemical Industries, Ltd. Nouvelle proteine de recepteur couple aux proteines g et adn de celle-ci
EP1363655A4 (fr) * 2000-12-18 2005-04-06 Merck & Co Inc Molecules d'acides nucleiques isolees codant un recepteur humain et murin couple a la proteine g - gpr54 -, proteines codees, cellules transformees par ces molecules et utilisations correspondantes
WO2002050266A1 (fr) * 2000-12-20 2002-06-27 Takeda Chemical Industries, Ltd. Nouvelle proteine receptrice couplee a la proteine g et son adn
WO2002092624A3 (fr) * 2001-05-16 2003-05-08 Paradigm Therapeutics Ltd Recepteur
CN114766546A (zh) * 2021-12-29 2022-07-22 淮阴工学院 地衣芽孢杆菌hg03在防治葡枝根霉所致水蜜桃采后软腐病中的应用及应用方法
CN114766546B (zh) * 2021-12-29 2024-03-26 淮阴工学院 地衣芽孢杆菌hg03在防治葡枝根霉所致水蜜桃采后软腐病中的应用及应用方法

Also Published As

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WO2000008133A9 (fr) 2001-12-20
EP1105465A1 (fr) 2001-06-13
CA2339347A1 (fr) 2000-02-17
JP2002526036A (ja) 2002-08-20

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