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WO2000008133A9 - 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

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Publication number
WO2000008133A9
WO2000008133A9 PCT/US1999/017388 US9917388W WO0008133A9 WO 2000008133 A9 WO2000008133 A9 WO 2000008133A9 US 9917388 W US9917388 W US 9917388W WO 0008133 A9 WO0008133 A9 WO 0008133A9
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WO
WIPO (PCT)
Prior art keywords
cells
substance
binding
protein
amount
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US1999/017388
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English (en)
Other versions
WO2000008133A1 (fr
Inventor
Qingyun Liu
Terrence P Mcdonald
Ruiping Wang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
Original Assignee
Merck and Co 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 Merck and Co Inc filed Critical Merck and Co Inc
Priority to EP99941986A priority Critical patent/EP1105465A1/fr
Priority to JP2000563760A priority patent/JP2002526036A/ja
Priority to CA002339347A priority patent/CA2339347A1/fr
Publication of WO2000008133A1 publication Critical patent/WO2000008133A1/fr
Anticipated expiration legal-status Critical
Publication of WO2000008133A9 publication Critical patent/WO2000008133A9/fr
Ceased legal-status Critical Current

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Classifications

    • 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

  • 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).
  • 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.ID.NO.:2.
  • Figure 2 show the complete amino acid sequence of HG03 (SEQ.ID.:2).
  • 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 polyacryl amide gel electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g., silver staining or immunoblotting.
  • SDS-PAGE sodium dodecyl sulfate polyacryl amide 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.
  • GPCR G protein-coupled receptor
  • 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.
  • 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.: 1.
  • the present invention also provides recombinant DNA molecules comprising the nucleotide sequence of positions 346-1419 of SEQ.ID.NO.: 1.
  • 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 rf ⁇ 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.: 1 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-labeled probe. Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1%
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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).
  • 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.
  • transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retroviral construct containing HG03, and electroporation.
  • HG03 has an amino acid sequence of SEQ.ID.NO. :2.
  • the present invention provides a method for determining whether a substance is capable of binding to HG03 comprising:
  • 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.
  • 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), CN-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), ⁇ IH/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 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-described 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-(b) 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), CN-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), ⁇ IH/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.
  • HG03 has an amino acid sequence of SEQ.ID.NO.:2.
  • 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.
  • 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, lipofection, 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:
  • A0MF15 R280 CAGGCTTTTAGATGAATCIGCA — (SEQ - D . NO-_: 6 ) AOMF15 .
  • HG03_FL243 F AAAGAAATCAAACCAGGAATAACC ( SQ . ID . NO . : 9 )
  • HG03 FL1429R CTTTGTACATATCGATTCCAACACAC SEQ . ID . NO . : 10
  • PBS . 873F 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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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using 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 WO2000008133A1 (fr) 2000-02-17
WO2000008133A9 true WO2000008133A9 (fr) 2001-12-20

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EP (1) EP1105465A1 (fr)
JP (1) JP2002526036A (fr)
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Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6181600A (en) * 1999-07-29 2001-02-19 Helix Research Institute Guanosine triphosphate-binding protein coupled receptors, genes thereof and production and use of the same
WO2001038348A2 (fr) * 1999-11-26 2001-05-31 Merck Patent Gmbh Recepteur htogh35 couple a la proteine g
EP1248842A1 (fr) * 1999-12-16 2002-10-16 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
AU2002217455A1 (en) * 2000-12-20 2002-07-01 Takeda Chemical Industries Ltd. Novel g protein-coupled receptor protein and dna thereof
AU2002257927A1 (en) * 2001-05-16 2002-11-25 Paradigm Therapeutics Limited G-protein coupled receptor
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

Family Cites Families (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

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JP2002526036A (ja) 2002-08-20
EP1105465A1 (fr) 2001-06-13
CA2339347A1 (fr) 2000-02-17
WO2000008133A1 (fr) 2000-02-17

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