WO2003027142A1 - Nouveau recepteur couple a la proteine g - Google Patents

Nouveau recepteur couple a la proteine g Download PDF

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Publication number
WO2003027142A1
WO2003027142A1 PCT/JP2002/009626 JP0209626W WO03027142A1 WO 2003027142 A1 WO2003027142 A1 WO 2003027142A1 JP 0209626 W JP0209626 W JP 0209626W WO 03027142 A1 WO03027142 A1 WO 03027142A1
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Prior art keywords
polypeptide
protein
amino acid
seq
present
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PCT/JP2002/009626
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English (en)
Japanese (ja)
Inventor
Shunichiro Matsumoto
Jun Takasaki
Takeshi Kurama
Tetsu Saito
Masazumi Kamohara
Takatoshi Soga
Hideki Hiyama
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Yamanouchi Pharmaceutical Co., Ltd.
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Priority to JP2003530729A priority Critical patent/JPWO2003027142A1/ja
Publication of WO2003027142A1 publication Critical patent/WO2003027142A1/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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a novel G protein-coupled receptor.
  • G protein-coupled receptors All G protein-coupled receptors known to date form a superfamily that shares a structure that penetrates the cell membrane seven times, with the amino terminal outside the cell and the carboxyl terminal inside the cell. For this reason, they may be collectively referred to as “seven-transmembrane receptors”.
  • G protein-coupled receptors transmit information on various physiologically active substances from cell membranes through the activation of trimeric GTP-binding proteins and the resulting changes in intracellular second messengers. Transfer into the cell. Intracellular second messengers controlled by the trimeric GTP-binding protein are well known, such as cAMP via adenylate cyclase or Ca 2+ via phospholipase C. Many cellular proteins have recently been identified as targets, including channel regulation through GTP-binding proteins and activation of kinases (Gudermann, T. et al., Annu. Rev. Neurosci., 20, 399-427, 1997).
  • bioactive substances that transmit information through G protein-coupled receptors existing bioactive substances such as neurotransmitters, hormones, chemokines, lipid-derived signal transmitters, divalent ions, and proteases Lots of included. Each of these physiologically active substances has a specific G protein-coupled receptor, which transmits that information into cells.
  • G protein-coupled receptors often precede gene cloning, Receptors that do not correspond to the endogenous ligand are called orphan G protein-coupled receptors.
  • Receptors that do not correspond to the endogenous ligand are called orphan G protein-coupled receptors.
  • cAMP or Ca 2+ which is the second messenger of many G protein-coupled receptors
  • GTPase activity which is an indicator of trimeric GTP binding protein activity
  • G protein of GTP yS High-throughput measurement of protein binding enables screening of agonists against orphan G protein-coupled receptors from compound libraries, and discovery of specific agonists and antagonists using the compounds In other words, it is possible to develop therapeutic drugs for specific diseases. Under these circumstances, the discovery of novel G protein-coupled receptors that can be therapeutic targets for new diseases can be regarded as the most important step in creating drugs that act on G protein-coupled receptors.
  • G protein-coupled receptors have been cloned from eukaryotes. For humans, G protein-coupled receptors have been cloned that correspond to more than one hundred endogenous living ligands. To date, hundreds of therapeutic agents targeting these receptors have been utilized (Wilson, J. et al., British J. of Pharmacol., 125, 1387-1392, 1998). Diseases targeted by G protein-coupled receptors are wide-ranging, with G protein-coupled receptors in the central nervous system, circulatory system, inflammatory immune system, digestive system, motor system, or genitourinary system. There are effective drugs that act on the disease (see Stadel, J. et al., Supra). This suggests that agonists or antagonists of G protein-coupled receptors are likely to be therapeutic agents for diseases, and may play an important role in preventing, ameliorating, or treating various diseases There is a need to identify new receptors and elucidate their implications for disease.
  • NPY neuropeptide Y
  • PYY peptide YY
  • PP pancreatic polypeptide
  • NPY-specific receptors ⁇ ⁇ Blomqvist, A. G. and Herzog, H., Trends Neurosci., 20, 294-8, 1997. It is known that there are five different subtypes of NPY family receptors so far, and G protein-coupled receptors (GPCRs) Y1, Y2, ⁇ 4, ⁇ 5, and ⁇ 6 have been identified at the gene level. Have been. Y1 receptor is expressed in blood vessels in peripheral tissues, and causes vasoconstriction by binding to ⁇ . On the other hand, analysis of tissue expression distribution suggests that the ⁇ 5 receptor is involved in feeding (2 Blomqvist, AG, et al., Supra).
  • NPY GPCRs are considered to be therapeutic targets for eating disorders (Blomqvist, AG, et al., Supra). Research is ongoing
  • An object of the present invention is to isolate and identify a gene encoding a novel G protein-coupled receptor necessary for the development of a prophylactic and / or therapeutic agent for eating disorders or obesity, and to express and produce the receptor.
  • An object of the present invention is to provide a screening method for constructing a system and searching for a substance that regulates the receptor activity, which is useful for prevention and / or treatment of eating disorders or obesity.
  • Another object of the present invention is to provide a method for screening a substance that suppresses an ingestion action but does not affect an action other than ingestion.
  • the present inventors have obtained a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 that encodes GPRgl, which is a GPCR, and specifically expresses the hypothalamus that controls the feeding function. . Then, the second messenger of GPRgl was found to be Ca 2+ increase and / or cAMP suppression. Provided a screening system. In addition, GPRglb, which is a GPCR to which a ligand binding to GPRgl binds and is expressed in the hypothalamus and various tissues unrelated to the eating function, and a polynucleotide encoding the same were obtained.
  • the present invention has been completed by providing a method for screening a substance useful for the prevention and / or treatment of Z or obesity.
  • (1) an amino acid sequence represented by SEQ ID NO: 2, and (a) an activity of increasing the amount of intracellular Ca 2+ by being activated in a cell, and / or (b) A polypeptide having an activity of reducing the amount of intracellular cAMP by being activated in cells, or
  • the amino acid sequence represented by SEQ ID NO: 2 contains a substituted, deleted, and / or inserted amino acid sequence of 1 to 5 amino acids, and (a) is activated in a cell. An activity of increasing the amount of intracellular Ca 2+ , and Z or (b) a polypeptide exhibiting an activity of decreasing the amount of intracellular cAMP by being activated in the cell,
  • a method of screening for a substance capable of controlling the activity of the polypeptide comprising: [9] (1) A step of contacting a cell expressing the polypeptide according to [1] or [2] or a cell membrane thereof with a test substance, and whether the polypeptide is activated or not. Analyzing whether or not, and
  • FIG. 1 is a graph showing the reporter activity of GPRgl protein. The luciferase activity value obtained is shown on the Y-axis.
  • ⁇ Gqi5 '' means the result when using the plasmid pEF-B0S-Gqi5
  • the symbol ⁇ GqoJ '' means the result when using the plasmid pEF-BOS-Gqo
  • the symbol ⁇ Gqs '' means the result when using the plasmid pEF-BOS-Gqo.
  • Rasmid pEF-BOS- Gqs means results
  • the symbol ⁇ Gq '' means results using plasmid pEF-BOS-Gq
  • the symbol ⁇ G15 '' means plasmid pEF-BOS- It means the result when G15 is used.
  • GPRgl protein a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 (hereinafter sometimes referred to as GPRgl protein);
  • intracellular Ca 2+ an activity of increasing the amount of intracellular Ca 2+ by containing the amino acid sequence represented by SEQ ID NO: 2 and (a) being activated in cells (hereinafter referred to as intracellular Ca 2+ And (b) a polysaccharide exhibiting an activity of reducing the intracellular cAMP level (hereinafter sometimes referred to as an intracellular cAMP reducing activity) by being activated in Z or (b) cells.
  • Peptide (3) at one or more positions (preferably 1 to 3 positions, particularly preferably 1 position) of the amino acid sequence represented by SEQ ID NO: 2, as a whole 1 to 5, more preferably 1 to 3, Particularly preferably, one amino acid contains a substituted, deleted, and / or inserted amino acid sequence, and exhibits (a) an intracellular Ca 2+ increasing activity and / or (b) an intracellular cAMP decreasing activity.
  • Polypeptide hereinafter referred to as GPRgl protein functionally equivalent variant
  • GPRgl protein homologous polypeptide contains an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by SEQ ID NO: 2; and (a) intracellular Ca 2+ increasing activity, and Z or (b) intracellular cAMP.
  • a polypeptide that exhibits reduced activity hereinafter referred to as GPRgl protein homologous polypeptide
  • GPRgl proteins [Hereinafter, these polypeptides (1) to (4) are collectively referred to as "GPRgl proteins”.
  • GPRglb protein a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 4 (hereinafter sometimes referred to as GPRglb protein);
  • polypeptide comprising an amino acid sequence represented by SEQ ID NO: 4 and binding to a biologically active substance that binds to GPRgl and GPRglb;
  • GPRglb a polypeptide which contains an amino acid sequence in which one amino acid has been substituted, deleted and / or inserted, and which binds to a biologically active substance that binds to GPRgl and GPRglb (hereinafter referred to as GPRglb).
  • GPRglb a polypeptide which contains an amino acid sequence in which one amino acid has been substituted, deleted and / or inserted, and which binds to a biologically active substance that binds to GPRgl and GPRglb
  • GPRglb protein group [Hereinafter, these polypeptides (5) to (8) are collectively referred to as “GPRglb protein group”].
  • GPRgl protein one of the polypeptides of the present invention, that is, a ⁇ polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 '' is a human-derived G protein-coupled receptor consisting of 353 amino acid residues ( GPCR).
  • GPCR 353 amino acid residues
  • W001 / 94582 published December 13, 2001 published after the priority date of the present application describes the amino acid sequence represented by SEQ ID NO: 2. However, there is no description that the polypeptide having the amino acid sequence represented by SEQ ID NO: 2 was obtained, and there is no disclosure or suggestion that the polypeptide controls the eating function. Also published after the priority date of this application
  • W001 / 70978 (published September 27, 2001) describes a sequence that differs from the amino acid sequence represented by SEQ ID NO: 2 by 1 amino acid. However, the fact that the polypeptide comprising the sequence was obtained and the practical use of the polypeptide are not described, and the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 has a feeding function. There is no disclosure or suggestion to control.
  • the GPRgl protein is a GPCR whose second messenger is Ca 2+ increase and / or cAMP suppression, and exhibits intracellular Ca 2+ increasing activity and / or intracellular cAMP decreasing activity.
  • the ability of a polypeptide to exhibit “intracellular Ca 2+ increasing activity and / or intracellular cAMP decreasing activity” is not particularly limited.
  • the following method preferably Can be confirmed by the method described later in Example 5). That is, (1) an expression vector containing a polynucleotide encoding a test polypeptide, and (2) a chimeric protein (for example, Gqi5) of Gq and Gi, which are a kind of G protein, or Gq.
  • An expression vector containing the polynucleotide and (3) a plasmid in which a reporter gene (for example, noresiferase) is linked downstream of a serum response element (SRE) are used in animal cells (eg, HEK293 After introducing the three genes into EBNA cells at the same time, measure the intracellular luciferase activity.
  • a reporter gene for example, noresiferase
  • SRE serum response element
  • control vector not containing a polynucleotide encoding a test polypeptide and (2) a chimeric protein of Gq and Gi, or a polynucleotide encoding Gq
  • intracellular luciferase activity is also measured in animal cells into which the expression vector and (3) a reporter gene linked downstream of the SRE have been transfected simultaneously.
  • test polypeptide can be expressed as "intracellular".
  • Ca 2+ increase activity The activity of decreasing the amount of intracellular Ca 2+ and the activity of decreasing Z or the amount of intracellular cAMP by being activated in a cell. "Can be determined.
  • Gq is a G protein that promotes the activity of phospholipase C, and is a member of the G protein subfamily that functions as an amplification factor. is there.
  • the activity of phospholipase C is promoted, for example, the intracellular Ca 2+ concentration increases.
  • GiJ is one of the subfamilies of G proteins that function as an amplifying factor by coupling to a receptor and intracellularly, and suppresses the active production of adenylate cyclase. It is a G protein When the activity of adenylate cyclase is suppressed, for example, the intracellular cAMP concentration decreases.
  • the state in which the polypeptide of the present invention, which is a novel G protein-coupled receptor, is "activated" in a cell refers to a G protein-coupled receptor regardless of the presence or absence of ligand binding. It means a state where a signal is transmitted downstream of the receptor.
  • the absolute amount of the activated G protein-coupled receptor exceeds a certain amount, the polypeptide of the present invention is activated.
  • the G protein-coupled receptor is in equilibrium between the inactive and active forms, and the ligand shifts to the active form when the ligand binds to the G protein-coupled receptor. Even when the receptor is overexpressed, it is known that the absolute amount of the activated G protein-coupled receptor increases, so that it is activated and a signal is transmitted downstream even in the absence of ligand. (Milano, CA et al., Science, 264, 582-586, 1994). That is, even if the ligand is not specified, the signal from the G protein-coupled receptor can be detected by overexpressing the G protein-coupled receptor in the cell even if the ligand is not specified. There are cases. In the experiment described in Example 5 described below, overexpression of the polypeptide of the present invention, even in the absence of a ligand to the polypeptide of the present invention, has the same effect as activation by agonist binding. It is activated in a state.
  • a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 and also exhibiting intracellular Ca 2+ increasing activity and Z or intracellular cAMP decreasing activity includes, for example, a sequence A polypeptide obtained by adding an appropriate marker sequence, etc., to the N-terminus and amino or C-terminus of the amino acid sequence represented by No. 2 (however, intracellular Ca 2+ i addition activity and / or intracellular cAMP decrease) Activity is required).
  • a marker sequence that can be used in the present invention a sequence for confirming expression of a peptide, confirming intracellular localization, or easily performing purification or the like can be used.
  • a FLAG epitope hexar Examples include a histidine 'tag, a hemadaltin' tag, and myc epitope.
  • the origin of the GPRgl proteins of the present invention is not limited to humans.
  • functionally equivalent variants of the GPRgl protein of the present invention include, for example, natural allelic variants in humans of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 (and increased intracellular Ca 2+). Activity and Z or intracellular cAMP decreasing activity) or a polypeptide generated by amino acid substitution such as Single Nucleotide Polymorphism (SNP) (however, intracellular Ca 2+ increasing activity and / or cell Naturally-occurring functional modifications from non-human organisms (eg, non-human mammals (eg, mouse, rat, hamster, or dog)) Body included.
  • SNP Single Nucleotide Polymorphism
  • these natural polypeptides in particular, polypeptides artificially modified by genetic engineering based on the amino acid sequence represented by SEQ ID NO: 2 and the like are included.
  • the GPRgl protein homologous polypeptide of the present invention contains an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by SEQ ID NO: 2 and has an intracellular Ca 2+ increasing activity and
  • the GPRgl protein homologous polypeptide of the present invention is not particularly limited as long as it is a polypeptide exhibiting an intracellular cAMP-reducing activity.
  • Homologous to the amino acid sequence represented by SEQ ID NO: 2 Has an amino acid sequence of more preferably 95% or more, still more preferably 98% or more, and particularly preferably 99% or more, and has an activity of increasing intracellular Ca 2+ and decreasing Z or intracellular cAMP.
  • the term “homology” used herein refers to the bl2seq program (Tatiana A. Tatusova) of the BLAST package [sgi32bit version, version 2.0.12; obtained from the National Center for Biotechnology Information (NCBI)]. , Thomas L. Madden, FEMS Microbiol Lett., 174, 247-250, 1999).
  • the pairwise alignment parameter is "Program name” is "blastpj,
  • the present invention also includes a partial fragment of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and further having a partial activity of increasing intracellular Ca 2+ and / or decreasing intracellular cAMP. It is.
  • GPRglb protein which is one of the polypeptides of the present invention, that is, a “polypeptide comprising an amino acid sequence represented by SEQ ID NO: 4” is a human-derived G protein conjugate consisting of 374 amino acid residues. Type receptor (GPCR). As shown in Example 4 below, GPRglb protein is expressed in various tissues other than the hypothalamus, which controls the feeding function.
  • the GPRglb protein has a high homology (46%) with the GPRgl protein as shown in Example 3 described below, and it is considered that the same physiologically active substance in the living body binds to the GPRgl protein and the GPRglb protein.
  • a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 4 and binding to a biologically active substance that binds to GPRgl and GPRglb includes, for example, an amino acid represented by SEQ ID NO: 4 Polypeptides having an appropriate marker sequence or the like added to the N-terminal and / or C-terminal of the sequence (provided that a biologically active substance that binds to GPRgl and GPRglb must bind).
  • W002 / 06466, published after the priority date of the present application (published on January 24, 2002), describes the amino acid sequence represented by SEQ ID NO: 4.
  • SEQ ID NO: 4 the polypeptide having the amino acid sequence represented by SEQ ID NO: 4 was obtained, and its industrial applicability includes determination of ligands, acquisition of antibodies and antisera.
  • a sequence that facilitates peptide expression confirmation, intracellular localization, or purification can be used.
  • examples thereof include a FLAG epitope, a hexar histidine tag, a hemadaltune tag, and a myc epitope.
  • the origin of the GPRglb proteins of the present invention is not limited to humans.
  • the functionally equivalent GPRglb protein variant of the present invention includes, for example, a human natural allelic variant of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 (provided that it binds to GPRgl and GPRglb).
  • SN single nucleotide polymorphism
  • the GPRglb protein homologous polypeptide of the present invention contains an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by SEQ ID NO: 4, and has an in vivo physiological binding property to GPRgl and GPRglb.
  • the GPRglb protein homologous polypeptide of the present invention is not particularly limited as long as it is a polypeptide to which the active substance binds, but more preferably has a homology with the amino acid sequence represented by SEQ ID NO: 4.
  • Polypeptides having an amino acid sequence of 95% or more, more preferably 98% or more, particularly preferably 99% or more, and which binds to a bioactive substance in vivo that binds to GPRgl and GPRglb are preferred.
  • the present invention also includes a partial fragment of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4, which binds to a biologically active substance that binds to GPRgl and GPRglb.
  • the polynucleotide of the present invention is not particularly limited as long as it is a polynucleotide encoding the polypeptide of the present invention.
  • the polynucleotide of the present invention includes:
  • a partial fragment of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 and a polypeptide exhibiting intracellular Ca 2+ increasing activity and / or intracellular cAMP decreasing activity '' A polynucleotide that
  • GPRglb protein functional equivalent variant A polynucleotide encoding a GPRglb protein homologous polypeptide; or a partial fragment of a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4; And a polypeptide that binds to a physiologically active substance in vivo that binds to GPRglb "
  • polynucleotide of the present invention a polynucleotide encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4 is preferable, and a polynucleotide consisting of the base sequence represented by SEQ ID NO: 1 or 3 is more preferable. I like it.
  • polynucleotide in the present specification includes both DNA and RNA.
  • the method for producing the polynucleotide of the present invention is not particularly limited. For example, (1) a method using PCR, (2) a conventional genetic engineering technique (that is, transformation using a single cDNA library) Method for selecting a transformant containing a desired cDNA from the transformed transformants), or (3) a chemical synthesis method.
  • a method using PCR for example, (1) a method using PCR, (2) a conventional genetic engineering technique (that is, transformation using a single cDNA library) Method for selecting a transformant containing a desired cDNA from the transformed transformants), or (3) a chemical synthesis method.
  • a conventional genetic engineering technique that is, transformation using a single cDNA library
  • Method for selecting a transformant containing a desired cDNA from the transformed transformants or (3) a chemical synthesis method.
  • the polynucleotide of the present invention can be produced, for example, by the following procedure.
  • mRNA is extracted from human cells or tissues (for example, brain, hypothalamus, or fetal brain) having the ability to produce the polypeptide of the present invention.
  • the polypeptide of the present invention Based on the nucleotide sequence of the polynucleotide encoding, a pair of two primer sets capable of sandwiching the entire length of the mRNA corresponding to the polypeptide of the present invention, or the mRNA region of the-part thereof make a set of two primer sets.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • the polynucleotide of the present invention can be produced, for example, by the following procedure.
  • a single-stranded cDNA is synthesized using reverse transcriptase with the mRNA prepared by the method using PCR as above, and then a double-stranded cDNA is synthesized from the single-stranded cDNA.
  • Methods for selecting a transformant having the desired cDNA from the transformants thus obtained include, for example, the following (1) screening method using a synthetic oligonucleotide probe, and (2) PCR A screening method using the probe thus obtained can be employed.
  • a transformant having a target cDNA can be selected by the following procedure.
  • an oligonucleotide corresponding to all or a part of the polypeptide of the present invention was synthesized, and this was used as a probe (labeled with 32 P or 33 P). After hybridization, search for the obtained positive strain and select it.
  • a nucleotide sequence derived using codon usage can be used, or a plurality of nucleotide sequences obtained by combining possible nucleotide sequences can be used. Can also. In the latter case, the type can be reduced by including inosine.
  • a transformant having a target cDNA can be selected by the following procedure.
  • a sense primer and an antisense primer corresponding to a part of the polypeptide of the present invention.
  • Oligonucleotides of the sense primer are synthesized, and PCR is performed by combining these oligonucleotides to amplify a DNA fragment encoding all or a part of the target polypeptide.
  • cDNA synthesized by reverse transcription reaction from mRNA of a cell producing the polypeptide of the present invention or genomic DNA can be used.
  • the DNA fragment prepared in this manner is labeled with, for example, 32 P or 33 P, and colony hybridization or Braak hybridization is performed using this as a probe to select a transformant having the cDNA of interest. I do.
  • a method for collecting the polynucleotide of the present invention from the obtained target transformant is a known method (for example, 3 Maniatis, T. et al., "Molecular Cloning-A Laboratory”).
  • the polynucleotide of the present invention can be produced by binding DNA fragments produced by the chemical synthesis method.
  • Each DNA can be synthesized using a DNA synthesizer [eg, Oligo 1000M DNA Synthesizer (manufactured by Beckman) or 394 DNA / RA Synthesizer (manufactured by Applied Biosystems)].
  • the polynucleotide of the present invention can be prepared on the basis of information on the polypeptide of the present invention by a conventional method such as the phosphite 'triester method (Humkapiller, M et al., Nature, 10, 105-111, 1984). It can also be produced by chemical synthesis of nucleic acids.
  • the codon for the desired amino acid is known per se and may be selected arbitrarily. For example, it can be determined according to a conventional method in consideration of the frequency of codon usage of the host to be used.
  • nucleic Acids Res. 8, r43-r74, 1981.
  • partial modification of the codons of these nucleotide sequences can be performed by a site-directed mutagenesis method (Mark, DF et al.) Using a primer consisting of a synthetic oligonucleotide encoding the desired modification in a conventional manner. Natl. Acad. Sci. USA, 81, 5662-5666, 1984).
  • t Sequencing of DNA obtained by the various methods described above may be performed, for example, by the chemical modification method of Maxam-Gilbert (Maxam, AM and Gilbert, W., "Methods in Enzymology", 65, 499-559, 19). 80) and dideoxynucleotide chain termination (Messing, J. and Vieira, J., Gene, 19, 269-276, 1982).
  • a host cell preferably eukaryote, particularly preferably 293-EBNA cell
  • the polynucleotide can be expressed in each host cell.
  • the present inventors have made it possible to overexpress the polypeptide of the present invention in the cell membrane by using an expression vector capable of adding a signal sequence to the N-terminus of the polypeptide of the present invention.
  • the expression vector of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention.
  • the polynucleotide of the present invention is inserted into a known expression vector appropriately selected according to the host cell to be used. And an expression vector obtained by the above method.
  • the present inventors have made it possible to overexpress the polypeptide of the present invention in the cell membrane by using 293-EBNA cells.
  • the cell of the present invention is also not particularly limited as long as it is transformed with the expression vector of the present invention and contains the polynucleotide of the present invention. It can be a cell that has been integrated into a cell, or it can be a cell that contains a polynucleotide according to the present invention in the form of an expression vector.
  • the cells may be cells expressing the polypeptide of the present invention, or cells not expressing the polypeptide of the present invention.
  • the cell of the present invention can be obtained, for example, by transforming a desired host cell with the expression vector of the present invention.
  • eukaryotic host cells include cells such as vertebrates, insects, and yeast.
  • vertebrate cells include monkey COS cells (Gluzman, Y., Cell, 23). 175-182, 1981), Chinese 'hamster ovary cell (CH0) dihydrofolate reductase-deficient strain (Urlaub, G. and Chasin, LA, Proc. Natl. Acad. Sci. USA, 77, 4216- 4220, 1980), human embryonic kidney-derived HEK293 cells, and 293-EBNA cells (Invitrogen) in which the EBNA-1 gene of Epstein's virus has been introduced into the HEK293 cells.
  • C0 Chinese 'hamster ovary cell
  • CH0 Chinese 'hamster ovary cell
  • LA Proc. Natl. Acad. Sci. USA, 77, 4216- 4220, 1980
  • human embryonic kidney-derived HEK293 cells and 293-EBNA cells (Invitrogen) in which the EBNA
  • a polynucleotide to be expressed normally is used.
  • a promoter having an upstream site, an RNA splice site, a polyadenylation site, and a transcription termination sequence can be used, and if necessary, it can have a replication origin.
  • Examples of the expression vector include, for example, ⁇ 2 (11 1 "(5311113, 3, et al., @ 101. Cell. Biol., 1, 854-864, 1981) having the SV40 early promoter, and extension of human.
  • Examples include pEF-BOS having a factor promoter ( ⁇ Mizushima, S.
  • signal sequence for example, a mugluchen signal sequence
  • a plasmid obtained by introducing a sequence encoding a signal sequence and a FLAG epitope into pEF_B0S can be used.
  • pCEP4 Invitrogen
  • pCEP4 which has an Epstein-Barr virus replication origin and is capable of self-replication in 293-EBNA cells, can be used as an expression vector.
  • COS cell When a COS cell is used as a host cell, it has an SV40 origin of replication as an expression vector, is capable of autonomous growth in COS cells, and has a transcription promoter, a transcription termination signal, and an RNA splice site.
  • pME18S Maruyama, K. and Takebe, Y., Med. Immunol., 20, 27-32, 1990
  • pEF-B0S described above, Mizushima, S. et al.
  • pCDM8 Seed, B., Nature, 329, 840-842, 1987.
  • the expression vector can be prepared, for example, by the DEAE-dextran method (Luthman, H. and Magnusson, G., Nucleic Acids Res., 11, 1295-1308, 1983), the caneoleum phosphate-DNA coprecipitation method.
  • the polypeptide of the present invention is coded.
  • a vector capable of expressing a neo gene functioning as a G418 resistance marker together with an expression vector containing a polynucleotide to be expressed for example, pRSVneo (Sambrook, J. et al., Molecular Cloning-A Laboratory Manual, Cola Spring Harbor Laboratory, NY, 1989) or pSV2-neo (Southern, PJ and Berg, P., J. Mol. Appl. Genet., 1,
  • transformants that stably produce the polypeptide of the present invention can be obtained by selecting G418B "colonies by co-transfection.
  • the cells can be cultured according to a conventional method, and the above-described culture produces the polypeptide of the present invention on the cell surface.
  • a medium such as RPMS-1640 medium or Dulbecco's Modified Eagle's Minimum Essential Medium (DMEM) may be used, and if necessary, fetal bovine serum A medium containing serum components such as (FBS) can be used, and in the case of 293-EBNA cells, Dulbecco's modified Eagle containing serum components such as fetal calf serum (FBS) can be used.
  • G41 for media such as essential media (DMEM) A medium to which 8 has been added can be used.
  • the polypeptide of the present invention which is produced on the cell surface of the cell by culturing the cell of the present invention, comprises various known separation procedures utilizing the physical properties, biochemical properties, and the like of the polypeptide. Can be separated and purified by the method. Specifically, for example, cells expressing the polypeptide of the present invention are cultured by culturing cells expressing the polypeptide of the present invention on the surface thereof, suspending them in a buffer, homogenizing, and centrifuging the cells. A membrane fraction can be obtained.
  • the polypeptide of the present invention can be purified by chromatography, affinity chromatography, high performance liquid chromatography (HPLC) or the like, or dialysis, or a combination thereof.
  • a solubilizer as mild as possible (eg, CHAPS, Triton II-100, or dichitonin)
  • CHAPS Triton II-100, or dichitonin
  • the polypeptide of the present invention By expressing the polypeptide of the present invention by fusing it in-frame with a marker sequence, confirmation of expression of the polypeptide of the present invention, confirmation of intracellular localization, or purification can be achieved. It will be easier.
  • the marker sequence include a FLAG epitope, a hexhistidine tag, a hemagglutinin tag, and a myc epitope.
  • a protease for example, Enterokinase, Factor Xa, or thrombin
  • a muscarinic acetylcholine receptor is linked to a hexa-histidine tag by a thrombin recognition sequence (Hayashi, MK and Haga, T., J. Biochem., 120, 1232-1238, 1996).
  • a substance capable of controlling the activity of the GPRgl protein group of the present invention can be screened.
  • GPRgl protein which is one of the polypeptides of the present invention, is expressed in the hypothalamus, which controls the feeding function, and is considered to be a GPCR that controls the feeding function. Therefore, a substance capable of controlling the activity of the GPRgl protein is useful as an active ingredient of a therapeutic and / or prophylactic agent for eating disorders and / or obesity.
  • cells expressing the GPRgl protein group or the cell membrane itself can be used as a screening tool for substances for treating eating disorders and Z or obesity. it can.
  • the “cells expressing the GPRgl protein group” are referred to as the screening cells of the present invention.
  • test substance which can be subjected to screening using the substance for treating an eating disorder and / or obesity of the present invention, which is screened using the screening tool is not particularly limited, but may be, for example, a substance registered in a chemical file.
  • Known compounds including peptides
  • compounds obtained by combinatorial 'chemistry technology (Terrett, NK et al., Tetrahedron, 51, 8135-8137, 1995)
  • phage' display method Felici , F. et al., J. Mol. Biol., 222, 301-310, 1991) and the like.
  • culture supernatants of microorganisms, natural components derived from plants or marine organisms, or animal tissue extracts can also be used as test substances for screening.
  • a compound (including a peptide) selected by the substance screening tool for the treatment of eating disorders and / or obesity of the present invention (including a peptide) is chemically or biologically modified (such as a peptide). Inclusive) can be used.
  • the method for screening a substance for treating an eating disorder and z or obesity comprises: a cell expressing the GPRgl protein group (preferably GPRgl protein) of the present invention so as to function as a receptor; As long as including a step of contacting with a test substance and a step of analyzing whether the GPRgl protein group is activated or not, for example, a chimeric protein of Gq and Gi ( For example, a screening method using Gqi5) or Gq can be mentioned.
  • GPRgl protein group preferably GPRgl protein
  • Examples of the screening cells of the present invention include, for example, (1) an expression vector containing a polynucleotide encoding a group of GPRgl proteins, and (2) a chimeric protein of Gq and Gi, which are a kind of G a protein (for example, Gqi5) or an expression vector containing a polynucleotide encoding Gq, and (3) a plasmid in which a reporter gene (eg, luciferase) is ligated downstream of the serum response element (SRE).
  • Animal cells for example, HEK293-EBNA cells into which the cells have been introduced can be used.
  • the screening cell of the present invention By contacting the screening cell of the present invention with a test substance and analyzing (ie, measuring or detecting) the luciferase activity in the cell, it is determined whether or not the GPRgl protein group is activated. To analyze. If the luciferase activity in the cells increases when the screening cell of the present invention is brought into contact with a test substance, it can be determined that the test substance is an agonist against the GPRgl protein group of the present invention. it can. On the other hand, when the test cell is brought into contact with the screening cell of the present invention, if the luciferase activity in the cell is suppressed, it can be determined that the test substance is an antagonist to the GPRgl protein group of the present invention. it can.
  • An expression vector containing the polynucleotide and (3) a plasmid in which a reporter gene is linked downstream of the SRE are subjected to the same operation using control cells into which the three have been transfected simultaneously. It is preferable to confirm that luciferase activity in control cells is not increased or suppressed.
  • the screening method using a chimeric protein of Gq and Gi or Gq is more preferably performed according to the conditions described in Example 5 described later. That is, a 24-well plate coated with collagen type I (for example,
  • Collagen- Typel- the Coated 24 well plate lASAHI TECHNO GLASS Co., Ltd. were seeded with HEK293-EBNA cells so that 7Xl0 4 cells per Ueru After culturing for 24 hours, (1) plasmid pEF- B0S-SSF -GPRgl or plasmid DEF-B0S (empty vector as control) (100ng per gel) (2) Each plasmid pEF-BOS-Gqi5, pEF-BOS-Gqo, pEF-B0S_Gqs, pEF_B0S-Gq, or pEF_B0S_G15 One of the two types of plasmids (100 ng / l well) and (3) Plasmid pSRE-luc (STRATAGENE) (20 ng / l well) were transfected with a gene transfer reagent (for example, FuGENE TM 6 Transfection Reagent; Roche Diagnostics) 3) to introduce genes at
  • the test substance is added, and after 24 hours from the gene transfer, the medium is discarded, and the luciferase-attay system is used. According to the method of (Wako Pure Chemical Industries), the intracellular luciferase activity is measured. For details of each plasmid used, see Example 5 described later.
  • the activity of the GPRgl protein group is reduced.
  • Substances which can be controlled and which do not affect the activity of the GPRglb protein group ie, do not inhibit or inhibit the activity of the GPRglb protein group
  • Such a substance is a more preferable substance as a substance for treating an eating disorder and obesity or obesity because it can specifically control only the eating function.
  • a substance that does not affect the activity of the GPRglb protein group can be selected by, for example, excluding a substance that binds to the GPRglb protein group by a binding experiment using the GPRglb protein group.
  • the present invention will be specifically described by way of examples, but these do not limit the scope of the present effort.
  • the method can be carried out according to a known method (the above-mentioned 3 Maniatis, T et al., "Molecular Cloning-A Laboratory Manual”).
  • Example 1 Isolation of a gene encoding a novel GPCR protein
  • the full-length cDNA encoding the novel GPCR proteins (GPRgl and GPRglb) of the present invention was obtained by PCR according to the following procedure.
  • cDNA derived from the hypothalamus of human brain was used as type III.
  • Type I cDNA derived from the hypothalamus of the human brain is prepared by purchasing mRNA (Clontech) from the above tissue and performing a reverse transcription reaction using a reverse transcription reaction reagent (Super Script; GIBCO). did.
  • an oligonucleotide having a base sequence represented by SEQ ID NO: 5 was used as a forward primer
  • an oligonucleotide having a base sequence represented by SEQ ID NO: 6 was used as a reverse primer.
  • PCR was carried out using Pyrobest DNA polymerase (Takara Shuzo) in the presence of 5% formamide at 94 ° C (2 minutes), 96 ° C (5 seconds) and 72 ° C (1 5 cycles of 5 minutes), 5 cycles of 96 ° C (5 seconds) and 70 ° C (1.5 minutes), and 5 cycles of 96 ° C (5 seconds) and 68 ° C ( (5 minutes) was repeated 20 times.
  • This base sequence has an open reading frame consisting of 1062 bases (1st to 1062th base sequence represented by SEQ ID NO: 1).
  • human testis-derived cDNA (Marathon Ready cDNA; Clontech) was used as type II.
  • an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 7 was used as a forked primer
  • an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 8 was used.
  • the PCR was carried out using Pyrobest DNA polymerase (Takara Shuzo Co., Ltd.), and a cycle consisting of 96 ° C (5 seconds) and 72 ° C (1.5 minutes) was performed after 94 ° C (2 minutes).
  • This nucleotide sequence has an open reading frame consisting of 1125 nucleotides (1st to 1125th of the nucleotide sequence represented by SEQ ID NO: 1).
  • GPRglb protein showed the highest homology (25%) to the B1 bradykinin receptor (P48748; 352 amino acid residues) among known GPCRs, but the amino acid sequence No identical molecules were present. from this result, GPRglb protein was found to be a novel GPCR.
  • the bl2seq program (Tatiana) of the BLAST package [sgi32bit version, Purgeyon 2.0.12 obtained from the National Center for Biotechnology Information (NCBI)]
  • the GPRgl protein and GPRglb protein were 46 ° /. It was found to have a high homology with. It is considered that the same physiologically active substance in the living body binds to GPRgl protein and GPRglb protein.
  • Example 2 To analyze the tissue expression distribution of the GPRgl gene, as in Example 1, commercially available mRNA from human tissues (Clontech) was purchased, and a reverse transcription reaction reagent (Super Script; GIBC0) was used. By performing a reverse transcription reaction, cDNAs derived from human tissues were prepared. Each of the synthesized cDNAs was converted into type III and subjected to PCR using Tag DNA polymerase (SIGMA) in the presence of 5% dimethyl sulfoxide (DMS0).
  • SIGMA Tag DNA polymerase
  • an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 9 was used
  • the reverse primer an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 10 was used.
  • a cycle consisting of 94 ° C (30 seconds), 55 ° C (30 seconds), and 72 ° C (1 minute) was repeated 40 times.
  • a DNA fragment of about 0.5 kbp was amplified with cDNAs derived from the hypothalamus, whole brain, and fetal brain, which are known to control the feeding function.
  • corpus callosum cerebellum, frontal lobe, heart, placenta, lung, bronchi, liver, skeletal muscle, kidney, pancreas, small intestine, stomach, spleen, bone marrow, thymus, thyroid, salivary gland, adrenal gland, breast, prostate, testis, No amplification was observed for cDNAs from the uterus, fetal kidney, fetal liver, and fetal lung.
  • the cDNA derived from each human tissue previously prepared to analyze the tissue expression distribution of the GPRgl gene was designated as type III, and LA-Taq DNA polymerase was used.
  • PCR was performed using Ze (Takara Shuzo).
  • An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 11 was used as the foreprimer, and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 12 was used as the reverse primer.
  • a cycle consisting of 94 ° C (for 30 seconds), 50 ° C (for 30 seconds), and 72 ° C (for 1 minute) was repeated 35 times after 94 ° C (for 2 minutes).
  • a DNA fragment of about 0.5 kbp was found in the amygdala, caudate nucleus, hippocampus, corpus callosum, substantia nigra, thalamus, cerebellum, frontal lobe, hypothalamus, spinal cord, pituitary, whole brain, heart, and placenta , Lung, bronchi, liver, skeletal muscle, kidney, pancreas, small intestine, stomach, spleen, bone marrow, thymus, thyroid, salivary gland, adrenal gland, breast, prostate, testis, uterus, fetal brain, fetal kidney, fetal liver, and fetus Amplified with cDNA from lung.
  • the full-length cDNA of GPRgl cloned in Example 1 was used as a forward primer, an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 13, and a reverse primer consisting of an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 14.
  • Amplification was carried out by PCR using pie mouth DNA polymerase (Takara Shuzo).
  • the PCR product was treated with the restriction enzymes Xbal and Spel, and inserted into the Xbal site of pEF-B0S (described above in Mizushima, S and et al.) (Hereinafter, the obtained plasmid was replaced with plasmid pEF-BOS-SSF-GPRgl). Name).
  • pEF-BOS-SSF-GPRgl a GPRgl protein having a signal sequence added to the N-terminus can be expressed, so that the target polypeptide can be expressed at a high frequency in a cell membrane.
  • plasmid pEF-BOS-Gqi5 5 amino acids (EYNLV; SEQ ID NO: 15) on the C-terminal side of Gq (GenBank Acc No. XM—040974) were converted to Gi C-terminal.
  • the plasmid was constructed by substituting the 5 amino acid (DCGLF; SEQ ID NO: 16) into pEF-B0S (hereinafter, the constructed plasmid is referred to as plasmid pEF-BOS-Gqi5).
  • a chimeric protein Gqo of Gq and Go was also cloned into plasmid pEF-BOS (resulting plasmids were pEF-BOS-Gqo, pEF_B0S-Gqs, pEF_B0S-Gq, and pEF-B0S-G15, respectively).
  • the plasmid pEF-BOS-Gqi5 is a type II plasmid containing a DNA encoding Gq (the above-mentioned nature), and an oligonucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 17 as a forward primer.
  • a PCR product obtained by amplifying a nucleotide using an oligonucleotide having the nucleotide sequence of SEQ ID NO: 18 as a reverse primer is treated with the restriction enzyme Spel, and inserted into the Xbal site of the plasmid pEF-BOS.
  • Spel restriction enzyme
  • Plasmid pEF-BOS-Gq is expressed as type III, a plasmid containing cDNA encoding Gq, as a forward primer, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 17 as a reverse primer, and as a reverse primer, as SEQ ID NO: 19.
  • the PCR product amplified using the oligonucleotide consisting of the nucleotide sequence to be prepared was treated with the restriction enzyme Spel and inserted into the Xbal site of the plasmid pEF-B0S.
  • Plasmid pEF-BOS-G15 is represented as SEQ ID NO: 21 as a type I plasmid, a plasmid containing a cDNA encoding G15 as a forward primer, an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 20 as a reverse primer, and a reverse primer as SEQ ID NO: 21.
  • the PCR product amplified using the oligonucleotide having the base sequence to be prepared was treated with the restriction enzyme Xbal, and was inserted into the Xbal site of the plasmid pEF-B0S to prepare.
  • plasmid PEF_B0S-SSF-GPRgl or plasmid pEF-B0S (empty vector as control) 100 ng per liter
  • plasmid pEF-B0S-Gqi5, pEF-B0S-Gqo, pEF- BOS-Gqs, pEF ⁇ B0S-Gq, or pEF-BOS-G15 Any one type of plasmid (100 ng / l) and (3) Plasmid pSRE-luc (STRATAGENE) (20 ng / l) And a gene transfer reagent (FuGENE TM 6 Transfection Reagent; Roche
  • polypeptide of the present invention a polynucleotide encoding the same, an expression vector containing the polynucleotide, and a cell containing the expression vector are useful for screening for a substance effective as a therapeutic agent for eating disorders and Z or obesity. Useful. Sequence listing free text

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Abstract

La présente invention concerne un gène codant pour un nouveau récepteur couplé à la protéine G, qui est utile pour la mise au point d'agents de prévention et/ou de traitement de troubles de l'alimentation ou de l'obésité, ledit gène étant isolé et identifié et un système de production d'expression de ce récepteur étant élaboré. L'invention a également pour objet un procédé de criblage destiné à rechercher une substance régulant l'activité du récepteur, qui est utile pour prévenir et/ou traiter les troubles de l'alimentation ou l'obésité.
PCT/JP2002/009626 2001-09-21 2002-09-19 Nouveau recepteur couple a la proteine g WO2003027142A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004074841A3 (fr) * 2003-02-14 2004-11-18 Regeneron Pharma Proteines de type kor3 et methodes de modulation de l'activite mediee par kor3l

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WO2001070978A2 (fr) * 2000-03-20 2001-09-27 Curagen Corporation Nouveaux polypeptides et acides nucleiques codant ces polypeptides
WO2001094582A1 (fr) * 2000-06-02 2001-12-13 Takeda Chemical Industries, Ltd. Nouvelle proteine de recepteur couple a la proteine g et adn pour cette proteine de recepteur
WO2001096592A2 (fr) * 2000-06-14 2001-12-20 Facultes Universitaires Notre-Dame De La Paix Detection inverse pour l'identification et/ou la quantification des sequences nucleotidiques cibles sur des biopuces
WO2001098330A2 (fr) * 2000-06-20 2001-12-27 Euroscreen S.A. Lignee cellulaire recombinee exprimant gpcrx11 en tant que recepteur fonctionnel valide par l'angiopeptine et utile dans le criblage d'agonistes et antagonistes
WO2002006466A1 (fr) * 2000-07-14 2002-01-24 Takeda Chemical Industries, Ltd. Nouvelle proteine gr et son adn
WO2002010387A2 (fr) * 2000-07-27 2002-02-07 Incyte Genomics, Inc. Récepteurs couplés à la protéine g

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070978A2 (fr) * 2000-03-20 2001-09-27 Curagen Corporation Nouveaux polypeptides et acides nucleiques codant ces polypeptides
WO2001094582A1 (fr) * 2000-06-02 2001-12-13 Takeda Chemical Industries, Ltd. Nouvelle proteine de recepteur couple a la proteine g et adn pour cette proteine de recepteur
WO2001096592A2 (fr) * 2000-06-14 2001-12-20 Facultes Universitaires Notre-Dame De La Paix Detection inverse pour l'identification et/ou la quantification des sequences nucleotidiques cibles sur des biopuces
WO2001098330A2 (fr) * 2000-06-20 2001-12-27 Euroscreen S.A. Lignee cellulaire recombinee exprimant gpcrx11 en tant que recepteur fonctionnel valide par l'angiopeptine et utile dans le criblage d'agonistes et antagonistes
WO2002006466A1 (fr) * 2000-07-14 2002-01-24 Takeda Chemical Industries, Ltd. Nouvelle proteine gr et son adn
WO2002010387A2 (fr) * 2000-07-27 2002-02-07 Incyte Genomics, Inc. Récepteurs couplés à la protéine g

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004074841A3 (fr) * 2003-02-14 2004-11-18 Regeneron Pharma Proteines de type kor3 et methodes de modulation de l'activite mediee par kor3l

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