WO2005049825A1 - Method of detecting signal transduction by g protein-coupled receptor using germination baculovirus - Google Patents

Method of detecting signal transduction by g protein-coupled receptor using germination baculovirus Download PDF

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WO2005049825A1
WO2005049825A1 PCT/JP2004/017646 JP2004017646W WO2005049825A1 WO 2005049825 A1 WO2005049825 A1 WO 2005049825A1 JP 2004017646 W JP2004017646 W JP 2004017646W WO 2005049825 A1 WO2005049825 A1 WO 2005049825A1
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protein
baculovirus
coupled receptor
gene encoding
ligand
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Japanese (ja)
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Takao Hamakubo
Tatsuhiko Kodama
Toshiko Sakihama
Kazuyuki Masuda
Takefumi Doi
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Toudai Tlo, Ltd.
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • 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
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4719—G-proteins

Definitions

  • the present invention relates to a method for detecting signal transmission of a G protein-coupled receptor using a budding baculovirus. More specifically, the present invention relates to a method for co-expressing a G protein-coupled receptor protein, a G protein and adenylate cyclase in a germinated baculovirus released from a host, and using the germinated baculovirus to produce a G protein. The present invention relates to a method for detecting signal transduction of a conjugated receptor. Background art
  • the paculovirus expression system uses a high expression protein of paculovirus, particularly, a promoter of a polyhedrin gene, to regenerate the target gene in insect cells (such as Sf9 cells). It is a system that wakes up and expresses it in large quantities. A system that introduces a recombinant protein into a polyhedron gene and purifies the expressed protein.
  • the expression system of Baculo is more capable of expressing proteins even if it has a large number of hydrophobic regions such as membrane proteins, compared to expression systems using Escherichia coli or yeast.
  • Baculovirus is a polyhedral virus that has a polyhedral protein and exists in the nucleus, and has another life cycle. The virus germinates as it multiplies and infects other cells or individuals. It becomes a virus (Budded virus) and covers the cell membrane of Sf9 cells and goes out of the cells. At this time, seven transmembrane receptors recombined with the above polyhedrin protein Have been reported by Loisel et al. (Loisel TP, Ansanay H, St-Onge S, Gay B, Boulanger P, Strosberg) to be expressed on the cell membrane of Sf9 and recovered on the budding paculovirus envelope.
  • Loisel TP Loisel TP, Ansanay H, St-Onge S, Gay B, Boulanger P, Strosberg
  • ER endoplasmic reticulum
  • SREBP sterol regulatory element binding protein 2, HMG-CoA (hydroxymethylcurtarylcoenzyme A) reductase, SCAP (SREBP cleavage activating protein), SIP (site 1 protease)
  • HMG-CoA hydroxymethylcurtarylcoenzyme A reductase
  • SCAP SREBP cleavage activating protein
  • SIP site 1 protease
  • G-protein coupled receptor is important as a drug discovery target, and about 700 species have been reported on a genome basis (Venter JG, Adams MD, Myers EW, et al. , Science 291, ppl304-1351, 2001, The sequence of the human genome), and studies on hormonal signal transduction mechanisms are also ongoing (Tate CG, Grissnammer R., Trends in Biotecnnology 1996, 14, pp426-430, Heterologous expression of G—protein—coupled receptors).
  • GPCR has seven transmembrane domains and is conjugated to a trimeric G protein. The type of G protein that is coupled (coupled) during ligand binding is determined for each receptor.
  • leukotriene B4 receptor it is Gi or Gq (Igarashi T, Yokomizo T, Tsutsumi 0, Taketani Y, Shimizu T and Izumi T., Eur. J. Biochem., 259, pp419-425, 1999, Characterization of the leukotiene B4 receptor in porcine leukocytes Separation and reconstitution with heterotrimeric GTP-binding proteins) o
  • Gs is known to be coupled, and according to the report of Loisel et al., Gs derived from Sf 9 cells is It is also presumed to form a complex on the budding virus (Loisel TP, Ansanay H, St-Onge S, Gay B, Boulanger P, Strosberg AD, Marullo S, Bouvier M., Nat Biotechnol.
  • viral adrenergic receptors were coupled to insect cell-derived Gs to express functional membrane receptors.
  • Sf9 is a receptor that is relatively small in amount and is conjugated to another isoform such as Gi (for example, leukotriene B4 receptor), it is highly functional even if expressed as it is. It is difficult to obtain a good receptor.
  • Japanese Patent Application Laid-Open No. 2003-52370 discloses a gene encoding an interacting protein such as a G protein and a gene encoding a membrane-type receptor protein such as a G protein-coupled receptor protein. Culturing a host infected with at least one recombinant baculovirus, and co-expressing the interacting protein and the membrane-type receptor protein in a germinated baculovirus released from the host. A method for expressing a functional membrane-type receptor protein is described. However, there is no report to date on the detection of G protein-coupled receptor (GPCR) signal transduction by measuring ligand-stimulated dependent cAMP production using germinated type Paculovirus. Disclosure of the invention
  • GPCR G protein-coupled receptor
  • An object of the present invention is to solve the above-mentioned problems. That is, the present invention aims to develop a method for detecting GPCR (G protein-coupled receptor) signal transduction by measuring ligand stimulation-dependent cAMP production using budding paculovirus. Issues to be solved.
  • GPCR G protein-coupled receptor
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have developed a technique for expressing a functional GPCR and a G protein into a paculovirus described in JP-A-2003-230370.
  • cAMP production it was demonstrated that signal transduction via the receptor due to ligand binding can be measured.
  • the present invention has been completed based on these findings.
  • At least one recombinant containing a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase is provided.
  • a G protein comprising culturing a host infected with a baculovirus, collecting germinated baculovirus released from the host, contacting the germinated baculovirus with a ligand, and acetating generated cAMP.
  • a recombinant paculovirus containing a gene encoding a G protein-coupled receptor protein a recombinant paculovirus containing a gene encoding a G protein, and adenylate cyclase Culturing a host infected with a recombinant paculovirus containing a gene encoding baculovirus, collecting germinated baculovirus released from the host, contacting the germinated baculovirus with a ligand, and generating cAMP Detect G protein-coupled receptor signaling, including assaying
  • a method is provided for doing so.
  • the host is an insect cell or insect larva.
  • the interaction between a G protein-coupled receptor protein and a ligand is analyzed by contacting a budding baculovirus with a ligand in the presence of a test substance, and then apoptosis of generated cAMP. Substances that promote or inhibit the action can be screened.
  • a substance obtained by the above screening which promotes or inhibits the interaction between a G protein-coupled receptor protein and a ligand.
  • At least one of a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase A germinated paculovirus released from a host infected with a recombinant baculovirus, the germinated paculovirus functionally displaying a G protein-coupled receptor protein, a G protein and adenylate cyclase.
  • a virus is provided.
  • the host is an insect cell or insect larva.
  • Figure 1 shows the results of measuring the activation of effluters in response to ligand stimulation with a virus that co-expresses a receptor, a G protein, and an effector protein (adeni / leic acid cyclase).
  • Co-express dopamine receptor (DRD1), gas j37, adenylate cyclase (ACVI), stimulate with dopamine, activate Ga, bind Go to ACVI, activate ACVI And cAMP is produced.
  • the present invention provides a GPCR by measuring ligand-stimulated dependent cAMP production in a virus using an expression system for a membrane protein complex by a budding paculovirus.
  • the present invention relates to a system for detecting (G protein-coupled receptor) signal transduction.
  • G protein-coupled receptor G protein-coupled receptor
  • dopamine receptor is utilized by utilizing a technique for expressing a functional GPCR and a G protein into a paculovirus described in JP-A-2003-230370.
  • the method for detecting G protein-coupled receptor signal transduction includes a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase. Culturing a host infected with at least one type of recombinant baculovirus, collecting germinated paculovirus released from the host, contacting the germinated baculovirus with a ligand, and assaying cAMP produced. It is a feature.
  • three types of proteins are co-expressed as described above, but the genes encoding the three types of proteins may be contained in the same recombinant baculovirus, or different recombinant baculoviruses may be used. May be included in Bacou Winores.
  • the G protein-coupled receptor referred to in this specification is a receptor capable of interacting with (binding to) a ligand. By coupling information resulting from the interaction with the ligand to the G protein, intracellular Is a protein that can be transmitted to
  • G protein-coupled receptors also abbreviated as GPCRs
  • GPCRs also called seven-transmembrane receptors
  • GPCRs are transmembrane receptors that transmit signals into cells in response to stimuli such as hormones, light, smell, and taste. Body. It is known that about 700 genes, including odor receptors, exist in the human genome. Many of these are regarded as important targets for drug discovery because they are receptors for the hormone ⁇ chemokine.
  • GPCR Activates the trimer G protein by binding to the ligand, and the Go; subunit of the trimer G protein dissociates and interacts with the effector protein to transmit a signal. GPCRs maintain high affinity by coupling to G proteins, and detecting this signal is important for identifying ligands for receptors and screening for inhibitors.
  • G-protein coupled receptors in addition to coupling to trimeric G proteins with a receptor protein, beta 2 Adorenarin receptor beta one arrestin (] 3 - arrestin) Oh Rui G-protein coupled receptors kinase (G- protein coupled receptor kinase, GRK ) interaction with the interaction of the metabotropic glutamate receptor (mGlu) Ho-mer protein and (Homer), 2 adrenoceptor and N a +, H + exchange ⁇ Ko ( Na +, H + exchange factor) (Heuss, C. and Gerber, U.G—protein—independent signaling by G-protein-coupled receptors. Trends Neurosci. (2000) 23, 469-475) Can be
  • RGS Regulators of G-protein signaling
  • Go a subunit called RGS (Regulators of G-protein signaling) protein, such as the binding between RGS 4 and the interleukin 8 B receptor and RGS 12 and the metatropic glutamate receptor (mglu).
  • RGS domains Hepler, JR Emerging roles for RGS proteins in cell signaling. TiPS (1999) 20, 376-382) and G proteins or their conjugated receptors.
  • G proteins include trimer G proteins.
  • Subunits that form a trimer with these a subunits and ⁇ subunits include ⁇ to 335 and ⁇ ⁇ to 0/11, respectively.
  • G protein-coupled receptor protein examples include the following.
  • BLT 1 leuco Toryen B 4
  • ET A angio tensin
  • EDG angio tensin
  • CCR CXCR
  • beta beta 3
  • beta 3 Nepinefurin
  • M u M 2 M 3
  • NK-1 5
  • Y Y
  • neuropeptide Y ⁇ 2
  • VIA vasopressin
  • CB1, CB2 anandamide
  • Dl D2, D3 (dopamine
  • odorant receptor MT1 , MT2, MT3 (melatonin)
  • photoreceptors and the like examples include the following.
  • Metabotropic neurotransmitter / calcium receptor-like G protein-coupled receptor proteins include mgk ⁇ mglu 2 (glutamic acid), GABA B (y-aminobutyric acid), and taste receptors.
  • At least one recombinant paculovirus containing a gene encoding a protein to be expressed as described above is used.
  • Baculovirus a virus that causes disease by infecting insects, is an enveloped virus that has a circular double-stranded DNA as a gene and is susceptible to insects such as Lepidoptera, Hymenoptera, and Diptera.
  • paculoviruses a group of viruses that produce large amounts of inclusion bodies called polyhydric bodies (polyhydra) in the nucleus of infected cells is the nuclear polyhedrosis virus (NPV).
  • Polyhedra are composed of a polyhedrin protein with a molecular weight of 31 kDa, and are produced in large quantities at the late stage of infection, in which many virus particles are embedded. I'm crazy. Polyhedra are essential for the virus to survive in nature, but are not necessary for virus growth itself, so even if a foreign gene that is to be expressed in place of the polyhedron gene is inserted, the virus can be transmitted without any problem. And proliferate.
  • baculovirus used in the present invention examples include viruses such as Autographs californica NPV (AcNPV) of NPV subfamily and Bombyx mori NPV (BmNPV) of silkworm. It can be used as a vector.
  • viruses such as Autographs californica NPV (AcNPV) of NPV subfamily and Bombyx mori NPV (BmNPV) of silkworm. It can be used as a vector.
  • AcNPV hosts include Spodoptera frugiperda cells (Sf cells), and BmNPV hosts (infected, passaged cells) include BmN4 cells.
  • Sf cells Spodoptera frugiperda cells
  • BmNPV hosts infected, passaged cells
  • Can be AcNPV-based vectors are preferred because Sf cells have a higher growth rate than BmN4 cells and the like, and AcNPV has the ability to infect human moon-dried cells and human fetal kidney cells. .
  • Spodoptera Frugiperda cell lines Sf9 and Sf21 have been established from ovarian tissues of S. frugiperda larvae, and are available from Invitrogen, Pharmingen (San Diego, CA), ATCC, or the like. In addition, live insect larvae can be used as host cell lines.
  • the method for constructing the recombinant virus used in the present invention may be performed according to a conventional method, and can be performed, for example, by the following procedure.
  • a gene for a protein to be expressed ie, a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase
  • a transfer vector ie, a gene for a protein to be expressed (ie, a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase) are transferred to a transfer vector.
  • a transfer vector ie, a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase
  • the overall size of a transfer vector is generally about several kb to 10 kb, of which about 3 kb is a plasmid-derived skeleton, which is used to initiate resistance to an antibiotic resistance gene such as ampicillin and bacterial DNA replication. Signal.
  • an antibiotic resistance gene such as ampicillin and bacterial DNA replication. Signal.
  • regions 5 and 3 of the polyhedron gene are also included.
  • the transfer vector preferably contains a promoter for expressing the protein gene. Examples of the promoter include a polyhedron gene promoter, a ⁇ 10 gene promoter, and a capsid gene promoter.
  • the type of transfer vector is not particularly limited.
  • Specific examples of the transfer vector include: AcNPV-based transfer vectors include pEVmXIV2, pAcSGl, pVL1392 / 1393, pAcMP2 / 3, pAccP1, AcUW21, pAcDZl, p B lue B ac III, p Ac UW 51, pAcAB 3, p A c 360 S p B lue B a cH i pVT- B ac 33, p A c UW 1 s such as p AcUW42Z43 can be mentioned, BmNPV system transformer Far vectors include ⁇ 283, ⁇ 5, ⁇ 30, ⁇ 1, pBE2, pBK3, pBK52, pBKblue, pBKblue2, and pBF series (obtained from Funakoshi Corporation, Fujisawa Pharmaceutical Co., Ltd., etc.) Possible).
  • the above-mentioned recombinant transfer vector is mixed with the virus and then transferred to a cultured cell to be used as a host, or the above-mentioned recombinant vector is added to a cultured cell to be used as a host previously infected with the virus. Then, homologous recombination is caused between the transfer vector and the viral genomic DNA to construct a recombinant virus.
  • the cultured cells used as hosts include the above-mentioned hosts, and are usually insect cultured cells (Sf9 cells, BmN cells, etc.). Culture conditions are appropriately determined by those skilled in the art. Specifically, when Sf9 cells are used, culture is preferably performed at about 28 ° C. in a medium containing 10% fetal bovine serum.
  • the recombinant virus thus constructed can be purified by a conventional method, for example, plaque assay.
  • the recombinant virus produced in this manner is a nuclear polyhedrosis virus. Since foreign DNA has been substituted or inserted into the gene region of the polyhedron protein of E. coli and cannot form a polyhedron, it can be easily distinguished from a non-recombinant virus.
  • the above-mentioned recombinant baculovirus is infected into an appropriate host (cultured cells such as Spodoptera Frugiperda cell lines Sf9 and Sf21, or insect larvae), and after a certain period of time (for example, 72 After a few hours, extracellular budding virus (BV) is recovered from the culture supernatant by a separation procedure such as centrifugation.
  • an appropriate host cultured cells such as Spodoptera Frugiperda cell lines Sf9 and Sf21, or insect larvae
  • extracellular budding virus BV
  • only one recombinant baculovirus may be infected, or two or more recombinant baculoviruses may be combined and co-infected.
  • the extracellular budding baculovirus can be collected, for example, as follows.
  • the culture of the infected cells is centrifuged at 500 to 1,000 g, and the supernatant containing the extracellular budding baculovirus is recovered.
  • the supernatant can be centrifuged at about 30,000 to 500,000 g to obtain a precipitate containing extracellularly germinated PacuMouth virus.
  • the germinated baculovirus recovered as described above contains a G protein-coupled receptor protein, a G protein, and adenylate cyclase in a state having a physiological activity. Is within the range. That is, the germinated baculovirus is characterized in that it functionally presents a G protein-coupled receptor protein, a G protein, and aduryl cyclase.
  • the germinated baculovirus prepared as described above is used, the germinated baculovirus is brought into contact with a ligand in the presence of a test substance, and the resulting cAMP is conjugated to the G protein-coupled protein.
  • a substance that promotes or inhibits the interaction can be screened.
  • the method of generating cAMP is not particularly limited, and can be appropriately selected by those skilled in the art.
  • the binding of peroxidase-labeled cAMP to an anti-cAMP antibody bound to a solid phase using the principle of Enzymimnoassay is By taking advantage of the competitive inhibition of cAMP produced by activated adenylate cyclase, cAMP can be assayed by measuring the decrease in color development due to the enzymatic reaction.
  • cAMP can also be obtained using commercially available kits such as Amersham cAMP Biotrak Enzyme immunoassay System kit.
  • the test substance to be subjected to the above-mentioned screening includes, for example, peptides, polypeptides, synthetic compounds, fermented microorganisms, extracts from organisms (including plant or animal tissues, microorganisms, or cells), or extracts thereof.
  • Libraries. Libraries include synthetic compound libraries (such as combinatorial libraries) and peptide libraries (such as combinatorial libraries).
  • the chemicals to be screened can be natural or synthetic, and even if a single candidate chemical is tested independently, a mixture of several candidate chemicals (including ) May be tested.
  • it is also possible to screen a fractionated mixture such as a cell extract, to isolate a substance having a desired activity by repeating the fractionation.
  • test substances are preferably substances that are expected to promote or inhibit the interaction between the G protein-coupled receptor protein and the ligand.
  • the screening method of the present invention it is possible to screen for an inhibitor or an activating drug for a G protein-coupled receptor protein.
  • Substances that promote or inhibit the interaction between a G protein-coupled receptor protein and a ligand, which are obtained by the screening method of the present invention, are also within the scope of the present invention.
  • the cells were subcultured on a 10 cm diameter dish. Mass culture was performed in a 1 L spinner-brasco (Wheaton) medium supplemented with 0.001% pluronic F-68 (GIBCO BRL) in complete medium.
  • spinner-brasco Wheaton
  • pluronic F-68 GEBCO BRL
  • the precipitate was suspended in PBS and centrifuged at 800 xg for 10 minutes to remove aggregates, and then centrifuged again at 40,000 xg for 25 minutes.
  • the precipitate obtained was suspended in PBS and the germinated virus fraction ( BV fraction).
  • the expression of DR-D1 in the BV fraction was confirmed by Western Blot using an anti-His antibody (Sigma) recognizing His-tag.
  • the ligand binding ability of the BV fraction was confirmed by a 7,8-3 ⁇ 4-Dopamine (Amersham) binding experiment.
  • DR was added to a binding buffer containing 7, 8-3 ⁇ 4-Dopamine (50 mM Tris-HCl pH 7, 4, 10 mM MgCl 2 , 10 mM NaCl, 0.5% fatty acid-free BSA).
  • the -D1 BV fraction was added to make the reaction solution volume 500 ⁇ 1, and reacted at room temperature for 90 minutes.
  • the reaction solution was overlaid on 500 ⁇ of di-n-butyl phthalate Z-dioctyl phthalate 1: 1 mixture and centrifuged at 15,000 xg for 10 minutes at room temperature to recover the virus as a precipitate. It was.
  • the precipitate washed three times with binding buffer, dried in air, in liquid scintillation counter and foremost, by measuring the amount of tritium contained in the precipitate was calculated the amount of 3 H- dopamine bound to the receptor.
  • the amount of 3 ⁇ 4-dopamine bound increased depending on the concentration of the labeled dopamine added to the reaction solution. Furthermore, the binding of 3 ⁇ 4-dopamine was inhibited by unlabeled dopamine or antagonist.
  • Example 2 Detection of adenylate cyclase activation by G protein associated with binding of G protein-coupled receptor to ligand in budding baculovirus
  • the G protein is activated by specific ligand binding to a coupled receptor, and after activation, interacts with various enzymes, collectively called effector proteins, to regulate the activity of those enzyme molecules.
  • Adenylate cyclase is an effector protein that increases cyclic AMP (cAMP) production by interacting with activated Gs-like G proteins.
  • the dopamine receptor is coupled to the Gs-like G protein, and the Gs-like G protein activated by specific binding of dopamine interacts with adenylate cyclase to increase enzyme activity. It has been reported.
  • AC VI Human type VI adenylate cyclase
  • AC VI Human type VI adenylate cyclase
  • a human fetal brain cDNA library Genbank accession number ⁇ 015270.
  • the cloned AC VI cDNA was inserted into the pECFP-N1 plasmid (Clontech), and the ECFP gene was fused to the 3 ′ end of the AC VI gene.
  • the ECFP-fused ACVI cDNA was further inserted into a pBlueBac4.5 vector (Invitrogen).
  • An ECFP-fused AC VI (AC VI-ECFP) recombinant baculovirus was prepared by the method for preparing a recombinant baculovirus described in Example 1 (1).
  • CAMP production by the germinated baculovirus suspensions prepared for each combination was measured as described below.
  • DR-Dl, Gas, Gj31, Gy2, ACVI-ECFP co-expressed virus fraction 10 zg was added to assay buffer (50 mM HEPES pH 8.0, 0.6 mM EDTA, 5 mM MgGl 2 , 1 mM IBM, 0.01% fatty acid-free BSA, 3 mM phosphoenol pyruvate 3Na, 5 u / ml pyruvate kinase, 5 u / ml myokinase, 1 mM ATP, 50 GTP, 0.02% saponin) to make a total volume of 90 ⁇ 1 and 10 ⁇ of 100 ⁇ M Dopamine (final concentration 10 ⁇ ) or 500 for skolin (final concentration 50 50) was added and reacted at 37 ° C for 30 minutes.
  • assay buffer 50 mM HEPES pH 8.0
  • the reaction was stopped by adding 1/9 volume of Lysis reagent 1A (attached to cAMP Biotrak Enzyme immunoassay System kit manufactured by Amersham).
  • the cAMP produced in the reaction solution was quantified by the ELISA method using the above-described cAMP measurement kit according to the instructions.
  • GPCR G protein-coupled receptor

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Abstract

It is intended to provide a method of detecting signal transduction by a GPCR (G protein-coupled receptor) by quantifying ligand stimulus-dependent cAMP production with the use of a germination baculovirus. Namely, a method of detecting signal transduction by a G protein-coupled receptor which comprises culturing a host infected with at least one recombinant baculovirus containing a gene encoding a G protein-coupled receptor protein, a gene encoding G protein and a gene encoding adenylate cyclase, collecting a germination baculovirus released from the host, bringing the germination baculovirus into contact with a ligand, and then assaying the cAMP thus formed.

Description

発芽パキュロウィルスを用いた G蛋白質共役型受容体のシグナル伝達の検出方法 技術分野 Method for detecting G protein-coupled receptor signaling using budding paculovirus
本発明は、 発芽バキュロウィルスを用いた G蛋白質共役型受容体のシグナル伝 達を検出する方法に関する。 より詳細には、 本発明は、 宿主から放出される発芽 バキュロウィルス中に G蛋白質共役型受容体蛋白質、 G蛋白質及びアデ二ル酸シ クラーゼを同時発現させ、 この発芽バキュロウィルスを用いて G蛋白質共役型受 容体のシグナル伝達を検出する方法に関する。 背景技術  The present invention relates to a method for detecting signal transmission of a G protein-coupled receptor using a budding baculovirus. More specifically, the present invention relates to a method for co-expressing a G protein-coupled receptor protein, a G protein and adenylate cyclase in a germinated baculovirus released from a host, and using the germinated baculovirus to produce a G protein. The present invention relates to a method for detecting signal transduction of a conjugated receptor. Background art
パキュロウィルス発現系はパキュロウィルスの高発現蛋白質、 特には多角体蛋 白質 （polyhedrin) 遺伝子のプロモーターなどを利用して、 目的遺伝子を昆虫細 胞 （S f 9細胞など） で糸且換えを起こさせて、 大量に発現させる系である。 多角 体遺伝子に組換えタンパク質を導入し、 発現したタンパク質を精製する系バキュ 口の発現系は、 大腸菌やイースト菌を用いる発現系に比べ、 膜蛋白質などの疎水 性領域を多く持つ蛋白質でも発現タンパク質が凝集を作りにくく、 また糖鎖の付 加や金属ィオンの配位などタンパクの機能に必要な翻訳後修飾がはいるなど利点 が多いため膜受容体蛋白質の発現系として多用されている（Tate CG, Grisshammer R. , Trends in Biotechnology 1996, 14, pp426~430, Heterologous expression of G- protein— coupled receptors；及ぴ Grissha蘭 er R, Tate CG, Quarterly Reviews of Biophysics 1995, 28, pp315 - 422， Overexpression of integral membrane proteins for structural studies)  The paculovirus expression system uses a high expression protein of paculovirus, particularly, a promoter of a polyhedrin gene, to regenerate the target gene in insect cells (such as Sf9 cells). It is a system that wakes up and expresses it in large quantities. A system that introduces a recombinant protein into a polyhedron gene and purifies the expressed protein The expression system of Baculo is more capable of expressing proteins even if it has a large number of hydrophobic regions such as membrane proteins, compared to expression systems using Escherichia coli or yeast. Since it has many advantages, such as being difficult to form aggregation and having post-translational modifications required for protein functions such as sugar chain addition and metal ion coordination, it is widely used as a membrane receptor protein expression system (Tate CG) , Grisshammer R., Trends in Biotechnology 1996, 14, pp426 ~ 430, Heterologous expression of G-protein— coupled receptors; and Grissha orchid er R, Tate CG, Quarterly Reviews of Biophysics 1995, 28, pp315-422, Overexpression of integral membrane proteins for structural studies)
バキュロウィルスには多角体蛋白質を被った多角体ウィルスとなって核内に存 在する他にもう一つの生活環があり、 ウィルスが増殖して他の細胞や個体に感染 するために， 発芽型のウィルス （Budded virus) となって S f 9細胞の細胞膜を 被って細胞外にでる。 この際に上記の多角体蛋白質に組換えた 7回膜貫通型受容 体が S f 9の細胞膜に発現され、 発芽したパキュロウィルスのエンベロープ上に 回収されることが Loiselらによって報告された （Loisel TP, Ansanay H, St- Onge S， Gay B, Boulanger P， Strosberg AD, Marullo Bouvier M.， Nat Biotechnol. 1997, Nov. 15 (12) , ppl300- 1304， Recovery of homogeneous and functional beta 2 - adrenergic receptors from extracellular baculovirus particles) 0 王糸田 胞に発現された 7回膜貫通型受容体は糖鎖構造など機能的でないものが多いのに くらべ、 ウィルスエンベロープ上に回収される受容体は機能的な蛋白質のみであ ることが報告されている。 Baculovirus is a polyhedral virus that has a polyhedral protein and exists in the nucleus, and has another life cycle. The virus germinates as it multiplies and infects other cells or individuals. It becomes a virus (Budded virus) and covers the cell membrane of Sf9 cells and goes out of the cells. At this time, seven transmembrane receptors recombined with the above polyhedrin protein Have been reported by Loisel et al. (Loisel TP, Ansanay H, St-Onge S, Gay B, Boulanger P, Strosberg) to be expressed on the cell membrane of Sf9 and recovered on the budding paculovirus envelope. AD, Marullo Bouvier M., Nat Biotechnol. 1997, Nov. 15 (12), ppl300-1304, Recovery of homogeneous and functional beta 2-adrenergic receptors from extracellular baculovirus particles) 0 7 transmembrane expressed in Otoda cells Compared to many non-functional receptors such as sugar chain structures, it is reported that only functional proteins are recovered on the viral envelope.
まに SREBP sterol regulatory element binding protein) 2、 HMG-CoA (ヒ ド ロキシメチルクルタリルコェンザィム A) 還元酵素、 SCAP (SREBP cleavage activating protein)、 SIP ( site 1 protease) などの小胞体 (ER) 莫あるいは ゴルジ体膜に分布する細胞内コレステロールフィードパック調節に関与する膜蛋 白質群も機能を保ったままウィルスエンベロープ上に発現されることが報告され た (Ishihara, G. , Shirai, H.， Yamaguchi, M. , Fukuda, R. , Hamakubo, T. , and Kodama, T.， Atherosclerosis, 151, p290, 2000， Expression of cholesterol regulatory proteins on extracellular baculoviruses)Q The endoplasmic reticulum (ER) such as SREBP sterol regulatory element binding protein) 2, HMG-CoA (hydroxymethylcurtarylcoenzyme A) reductase, SCAP (SREBP cleavage activating protein), SIP (site 1 protease) ) It has been reported that a group of membrane proteins involved in the regulation of intracellular cholesterol feed-pack distributed in giant or Golgi membranes are expressed on the virus envelope while maintaining their functions (Ishihara, G., Shirai, H. , Yamaguchi, M., Fukuda, R., Hamakubo, T., and Kodama, T., Atherosclerosis, 151, p290, 2000, Expression of cholesterol regulatory proteins on extracellular baculoviruses) Q
一方、 G蛋白質共役型受容体 （GPCR; G- protein coupled receptor) は創薬の ターゲットとして重要であり、ゲノムベースで約 7 0 0種と報告され（Venter JG， Adams MD, Myers EW, et al. , Science 291, ppl304 - 1351, 2001, The sequence of the human genome)、 ホルモンのシグナル伝達機構の研究も進んでいる （Tate CG, Grissnammer R. , Trends in Biotecnnology 1996, 14, pp426-430, Heterologous expression of G— protein— coupled receptors)。 GPCR fま膜貫通ドメィンを 7つも ち 3量体 G蛋白質と共役している。 リガンド結合の際に共役 （カップリング） す る G蛋白質 ο;サブュニットの種類は受容体ごとに決まっており， 例えばロイコト リエン B 4受容体の場合は G iあるいは G qである （Igarashi T, Yokomizo T, Tsutsumi 0, Taketani Y, Shimizu T and Izumi T.， Eur. J. Biochem.， 259, pp419-425, 1999, Characterization of the leukotiene B4 receptor in porcine leukocytes Separation and reconstitution with heterotrimeric GTP - binding proteins) o ァドレナリン受容体の場合は G sが共役することが知られており、 Loiselらの報告によると Sf 9細胞由来の G sが共役して発現し、発芽ウィルス上 にもコンプレックスを形成しているとされている（Loisel TP, Ansanay H, St - Onge S, Gay B, Boulanger P， Strosberg AD, Marullo S, Bouvier M. , Nat Biotechnol. 1997, Nov. 15 (12) , ppl300- 1304， Recovery of homogeneous and functional beta 2 - adrenergic receptors from extracellular baculovirus particles) 0 Sf9 細 胞には哺乳類細胞と同様に G蛋白質の各種のアイソフォームが発現しているとさ れているが、 その量は G蛋白質の種類によって差がある （Leopoldt D， Harteneck C, Nurnberg R, Naunyn-Schmiedeberg' s Arch Pharmacol , 356, pp216-224, 1997, G Proteins endogenous ly expressed in Sf9 cells : interactions with mammalian histamine receptors) 0 Gsは Sf 9細胞には比較的多く存在しているため、 Loisel らの系ではウィルスに発現したアドレナリン受容体が昆虫細胞由来の Gs と共役 し機能的な膜受容体が発現されたが、 Sf 9には比較的量が少ない Gi等の他の ァイソフォームと共役する受容体 （例えば、 ロイコトリェン B 4受容体など） の 場合には、 そのまま発現させても親和性の高い機能的な受容体を得ることは困難 である。 On the other hand, G-protein coupled receptor (GPCR) is important as a drug discovery target, and about 700 species have been reported on a genome basis (Venter JG, Adams MD, Myers EW, et al. , Science 291, ppl304-1351, 2001, The sequence of the human genome), and studies on hormonal signal transduction mechanisms are also ongoing (Tate CG, Grissnammer R., Trends in Biotecnnology 1996, 14, pp426-430, Heterologous expression of G—protein—coupled receptors). GPCR has seven transmembrane domains and is conjugated to a trimeric G protein. The type of G protein that is coupled (coupled) during ligand binding is determined for each receptor. For example, in the case of leukotriene B4 receptor, it is Gi or Gq (Igarashi T, Yokomizo T, Tsutsumi 0, Taketani Y, Shimizu T and Izumi T., Eur. J. Biochem., 259, pp419-425, 1999, Characterization of the leukotiene B4 receptor in porcine leukocytes Separation and reconstitution with heterotrimeric GTP-binding proteins) o In the case of adrenergic receptors, Gs is known to be coupled, and according to the report of Loisel et al., Gs derived from Sf 9 cells is It is also presumed to form a complex on the budding virus (Loisel TP, Ansanay H, St-Onge S, Gay B, Boulanger P, Strosberg AD, Marullo S, Bouvier M., Nat Biotechnol. 1997, Nov. 15 (12), ppl300-1304, Recovery of homogeneous and functional beta 2-adrenergic receptors from extracellular baculovirus particles) 0 Sf9 cells are said to express various G protein isoforms, similar to mammalian cells. However, the amount varies depending on the type of G protein (Leopoldt D, Harteneck C, Nurnberg R, Naunyn-Schmiedeberg's Arch Pharmacol, 356, pp216-224, 1997, G Proteins endogenous ly expressed in Sf9 cells: interactions with mammalian histamine recepto (rs) 0 Gs is relatively abundant in Sf 9 cells, so in the Loisel et al. system, viral adrenergic receptors were coupled to insect cell-derived Gs to express functional membrane receptors. However, if Sf9 is a receptor that is relatively small in amount and is conjugated to another isoform such as Gi (for example, leukotriene B4 receptor), it is highly functional even if expressed as it is. It is difficult to obtain a good receptor.
また、 特開 2 0 0 3— 5 2 3 7 0号公報には、 G蛋白質などの相互作用蛋白質 をコードする遺伝子及び G蛋白質共役型受容体蛋白質などの膜型受容体蛋白質を コードする遺伝子を含む少なくとも 1種の組換えバキュロウィルスを感染させた 宿主を培養し、 該宿主から放出される発芽バキュロウィルス中に該相互作用蛋白 質と該膜型受容体蛋白質とを同時発現させることを含む、 機能を有する膜型受容 体蛋白質を発現する方法が記載されている。 しかしながら、 発芽型パキュロウィ ルスを用いてリガンド刺激依存性の cAMP産生を測定することにより、 G蛋白質共 役型受容体 （GPCR) のシグナル伝達を検出できることについては現在までの所、 全く報告がない。 発明の開示 Japanese Patent Application Laid-Open No. 2003-52370 discloses a gene encoding an interacting protein such as a G protein and a gene encoding a membrane-type receptor protein such as a G protein-coupled receptor protein. Culturing a host infected with at least one recombinant baculovirus, and co-expressing the interacting protein and the membrane-type receptor protein in a germinated baculovirus released from the host. A method for expressing a functional membrane-type receptor protein is described. However, there is no report to date on the detection of G protein-coupled receptor (GPCR) signal transduction by measuring ligand-stimulated dependent cAMP production using germinated type Paculovirus. Disclosure of the invention
本発明は上記した問題点を解消することを解決すべき課題とした。 即ち、 本発 明は、発芽型パキュロウィルスを用いてリガンド刺激依存性の cAMP産生を測定す ることにより、 GPCR (G蛋白質共役型受容体） のシグナル伝達を検出する方法を 開発することを解決すべき課題とした。  An object of the present invention is to solve the above-mentioned problems. That is, the present invention aims to develop a method for detecting GPCR (G protein-coupled receptor) signal transduction by measuring ligand stimulation-dependent cAMP production using budding paculovirus. Issues to be solved.
本発明者らは上記課題を解決するために鋭意検討した結果、 特開 2 0 0 3— 5 2 3 7 0号公報に記載したパキュロウィルスへ機能的な GPCR と G蛋白質を発現 する技術を用いて、 ドパミン受容体 Dl、 三量体 G蛋白質の G a s、 G ]3、 G yサブ ユニット、 更にエフェクターであるアデ二ル酸シクラ一ゼの各遺伝子を有するリ コンビナントウィルスの共感染により、 発芽型バキュロウィルスのエンベロープ 上に受容体複合体を再構成し、 リガンドであるドパミン刺激により活性化された G蛋白質とアデ二ル酸シクラーゼとの相互作用を、 活性化アデ二ル酸シクラーゼ による cAMP産生を測定することによって、リガンド結合による受容体を介したシ グナル伝達を測定できることを実証した。 本発明はこれらの知見に基づいて完成 したものである。  The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have developed a technique for expressing a functional GPCR and a G protein into a paculovirus described in JP-A-2003-230370. Co-infection with a recombinant virus having the dopamine receptor Dl, the Gas, G] 3 and Gy subunits of the trimeric G protein, and the effector adenylate cyclase gene. Reconstitutes the receptor complex on the budding baculovirus envelope, and activates the interaction between adipate cyclase and the G-protein activated by dopamine stimulation by the activated adenylate cyclase. By measuring cAMP production, it was demonstrated that signal transduction via the receptor due to ligand binding can be measured. The present invention has been completed based on these findings.
即ち、 本発明によれば、 G蛋白質共役型受容体蛋白質をコードする遺伝子、 G 蛋白質をコードする遺伝子、 及びアデ二ル酸シクラ一ゼをコ一ドする遺伝子を含 む少なくとも 1種の組換えバキュロウィルスを感染させた宿主を培養し、 該宿主 から放出される発芽バキュロウィルスを回収し、 該発芽バキュロウィルスとリガ ンドとを接触させ、 生成する c AM Pをアツセィすることを含む、 G蛋白質共役 型受容体のシグナル伝達を検出する方法が提供される。  That is, according to the present invention, at least one recombinant containing a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase is provided. A G protein comprising culturing a host infected with a baculovirus, collecting germinated baculovirus released from the host, contacting the germinated baculovirus with a ligand, and acetating generated cAMP. Methods for detecting coupled receptor signaling are provided.
本発明の別の態様によれば、 G蛋白質共役型受容体蛋白質をコードする遺伝子 を含む組換えパキュロウィルス、 G蛋白質をコードする遺伝子を含む組換えパキ ュロウィルス、 及びアデ二ル酸シクラ一ゼをコ一ドする遺伝子を含む組換えパキ ュロウィルスを感染させた宿主を培養し、 該宿主から放出される発芽バキュロウ ィルスを回収し、 該発芽バキュロウィルスとリガンドとを接触させ、 生成する c AM Pをァッセィすることを含む、 G蛋白質共役型受容体のシグナル伝達を検出 する方法が提供される。 According to another aspect of the present invention, a recombinant paculovirus containing a gene encoding a G protein-coupled receptor protein, a recombinant paculovirus containing a gene encoding a G protein, and adenylate cyclase Culturing a host infected with a recombinant paculovirus containing a gene encoding baculovirus, collecting germinated baculovirus released from the host, contacting the germinated baculovirus with a ligand, and generating cAMP Detect G protein-coupled receptor signaling, including assaying A method is provided for doing so.
好ましくは、 宿主は昆虫細胞又は昆虫幼虫である。  Preferably, the host is an insect cell or insect larva.
本発明においては、 被験物質の存在下において発芽バキュロウィルスとリガン ドとを接触させ、 生成する c AMPをアツセィすることによって G蛋白質共役型 受容体蛋白質とリガンドとの相互作用を分析し、 該相互作用を促進又は阻害する 物質をスクリーニングすることができる。  In the present invention, the interaction between a G protein-coupled receptor protein and a ligand is analyzed by contacting a budding baculovirus with a ligand in the presence of a test substance, and then apoptosis of generated cAMP. Substances that promote or inhibit the action can be screened.
本発明の別の側面によれば、 上記のスクリーニングにより得られる、 G蛋白質 共役型受容体蛋白質とリガンドとの相互作用を促進又は阻害する物質が提供され る。  According to another aspect of the present invention, there is provided a substance obtained by the above screening, which promotes or inhibits the interaction between a G protein-coupled receptor protein and a ligand.
本発明のさらに別の側面によれば、 G蛋白質共役型受容体蛋白質をコードする 遺伝子、 G蛋白質をコードする遺伝子、 及びアデ二ル酸シクラ一ゼをコ一ドする 遺伝子を含む少なくとも 1種の組換えバキュロウィルスを感染させた宿主から放 出される発芽パキュロウィルスであって、 G蛋白質共役型受容体蛋白質、 G蛋白 質及ぴアデ二ル酸シクラーゼを機能的に提示している発芽パキュロウィルスが提 供される。 好ましくは、 宿主は昆虫細胞又は昆虫幼虫である。 図面の簡単な説明  According to still another aspect of the present invention, at least one of a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase A germinated paculovirus released from a host infected with a recombinant baculovirus, the germinated paculovirus functionally displaying a G protein-coupled receptor protein, a G protein and adenylate cyclase. A virus is provided. Preferably, the host is an insect cell or insect larva. Brief Description of Drawings
図 1は、 受容体 · G蛋白質 ·エフェクター蛋白質 （アデ二/レ酸シクラーゼ） 共 発現ウィルスでのリガンド刺激に伴うエフエタターの活性化を測定した結果を示 す。 ドパミン受容体 （DRD1)、 G a s j3 7 , アデ二レートシクラーゼ （ACVI) を共発 現し、 ドパミンで刺激し、 G aを活性ィ匕し、 G o;が ACVIに結合し、 ACVIが活性化 し、 cAMPが産生する。 発明を実施するための最良の形態  Figure 1 shows the results of measuring the activation of effluters in response to ligand stimulation with a virus that co-expresses a receptor, a G protein, and an effector protein (adeni / leic acid cyclase). Co-express dopamine receptor (DRD1), gas j37, adenylate cyclase (ACVI), stimulate with dopamine, activate Ga, bind Go to ACVI, activate ACVI And cAMP is produced. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明の実施態様及ぴ実施方法について詳細に説明する。  Hereinafter, embodiments and an implementation method of the present invention will be described in detail.
本発明は、 発芽型パキュロウィルスによる膜蛋白質複合体の発現系を用いて、 ウィルスにおけるリガンド刺激依存性の cAMP産生を測定することにより、 GPCR (G蛋白質共役型受容体） のシグナル伝達を検出する系に関するものである。 本明細書の下記実施例においては、 特開 2 0 0 3— 5 2 3 7 0号公報に記載の パキュロウィルスへ機能的な GPCRと G蛋白質を発現する技術を利用して、ドパミ ン受容体 Dl、 三量体 G蛋白質の G a、 サブユニット、 更にエフェクター であるアデニル酸シクラーゼの各遺伝子を有するリコンビナントウィルスの共感 染により、発芽型パキュ口ウィルスのエンベロープ上に受容体複合体を再構成し、 リガンドであるドパミン刺激により活性化された G蛋白質とアデ二ル酸シクラ一 ゼとの相互作用を、活性化アデ二ル酸シクラーゼによる cAMP産生を測定すること によって、 リガンド結合による受容体を介したシグナル伝達を測定することに成 功した。 The present invention provides a GPCR by measuring ligand-stimulated dependent cAMP production in a virus using an expression system for a membrane protein complex by a budding paculovirus. The present invention relates to a system for detecting (G protein-coupled receptor) signal transduction. In the following Examples of the present specification, dopamine receptor is utilized by utilizing a technique for expressing a functional GPCR and a G protein into a paculovirus described in JP-A-2003-230370. Reconstitution of the receptor complex on the envelope of the germinating Pacu Mouth virus by co-infection of a recombinant virus containing the genes of the body Dl, the G a of the trimeric G protein, the subunit, and the effector adenylate cyclase. The interaction between the G protein activated by stimulation of dopamine, a ligand, and adenylate cyclase, and the cAMP production by activated adenylate cyclase were measured to determine the receptor by ligand binding. And succeeded in measuring signal transduction.
本発明による G蛋白質共役型受容体のシグナル伝達を検出する方法は、 G蛋白 質共役型受容体蛋白質をコードする遺伝子、 G蛋白質をコードする遺伝子、 及び アデニル酸シクラーゼをコ一ドする遺伝子を含む少なくとも 1種の組換えバキュ ロウィルスを感染させた宿主を培養し、 該宿主から放出される発芽パキュロウィ ルスを回収し、 該発芽バキュロウィルスとリガンドとを接触させ、 生成する c A M Pをアツセィすることを特徴とするものである。 本発明では、 上記のように 3 種類の蛋白質を共発現させるものであるが、 上記 3種類の蛋白質をコードする遺 伝子は同一の組換えバキュロウィルス中に含めてもよいし、 異なる組換えバキュ 口ウイノレス中に含めてもよい。  The method for detecting G protein-coupled receptor signal transduction according to the present invention includes a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase. Culturing a host infected with at least one type of recombinant baculovirus, collecting germinated paculovirus released from the host, contacting the germinated baculovirus with a ligand, and assaying cAMP produced. It is a feature. In the present invention, three types of proteins are co-expressed as described above, but the genes encoding the three types of proteins may be contained in the same recombinant baculovirus, or different recombinant baculoviruses may be used. May be included in Bacou Winores.
本明細書で言う G蛋白質共役型受容体とは、 リガンドと相互作用 （結合） する 能力を有する受容体であって、 リガンドとの相互作用に起因する情報を G蛋白質 と共役することにより細胞内に伝達することができる蛋白質である。  The G protein-coupled receptor referred to in this specification is a receptor capable of interacting with (binding to) a ligand. By coupling information resulting from the interaction with the ligand to the G protein, intracellular Is a protein that can be transmitted to
G蛋白質共役型受容体 （GPCRとも略記される） は、 7回膜貫通型受容体とも呼 ばれ、 ホルモンや光、 におい、 味などの刺激に反応してシグナルを細胞内に伝達 する膜型受容体である。 ヒトゲノム上ににおい受容体を含めて 700種類程度の遺 伝子が存在していることが知られている。 これらの多くは、 ホルモンゃケモカイ ンの受容体であることから、創薬のターゲットとして重要視されている。 GPCRは リガンドと結合して三量体 G蛋白質を活性化し、 三量体 G蛋白質の G o;サブュニ ットは解離して、エフェクター蛋白質と相互作用を起こしてシグナルを伝播する。 GPCRは G蛋白質と共役することによって高親和性を保っており、 このシグナルを 検出することは、 受容体に対するリガンドの特定や阻害剤のスクリ一ユングに重 要である。 G protein-coupled receptors (also abbreviated as GPCRs), also called seven-transmembrane receptors, are transmembrane receptors that transmit signals into cells in response to stimuli such as hormones, light, smell, and taste. Body. It is known that about 700 genes, including odor receptors, exist in the human genome. Many of these are regarded as important targets for drug discovery because they are receptors for the hormone ゃ chemokine. GPCR Activates the trimer G protein by binding to the ligand, and the Go; subunit of the trimer G protein dissociates and interacts with the effector protein to transmit a signal. GPCRs maintain high affinity by coupling to G proteins, and detecting this signal is important for identifying ligands for receptors and screening for inhibitors.
Gタンパク質共役型受容体においては、 三量体 Gタンパク質と受容体タンパク 質との共役に加えて、 β 2ァドレナリン受容体と β一アレスチン（]3 - arrestin)あ るいは Gタンパク質共役型受容体キナーゼ（G- protein coupled receptor kinase, GRK)との相互作用、 メタボトロピックグルタミン酸受容体（mglu) とホー マータンパク質 (Homer)との相互作用、 2アドレナリン受容体と N a +、 H+ 交 換因子 (Na+, H+ exchange factor) との相互作用 など (Heuss, C. and Gerber, U. G— protein— independent signaling by G - protein - coupled receptors. Trends Neurosci. (2000) 23, 469 - 475)が挙げられる。 In G-protein coupled receptors, in addition to coupling to trimeric G proteins with a receptor protein, beta 2 Adorenarin receptor beta one arrestin (] 3 - arrestin) Oh Rui G-protein coupled receptors kinase (G- protein coupled receptor kinase, GRK ) interaction with the interaction of the metabotropic glutamate receptor (mGlu) Ho-mer protein and (Homer), 2 adrenoceptor and N a +, H + exchange換因Ko ( Na +, H + exchange factor) (Heuss, C. and Gerber, U.G—protein—independent signaling by G-protein-coupled receptors. Trends Neurosci. (2000) 23, 469-475) Can be
また、 メタポトロピックグルタミン酸受容体 （mglu) と RGS 4、 インターロイ キン 8 B受容体と RGS 1 2 との結合など、 RGS (Regulators of G- protein signaling)タンパク質とよばれる G o;サブュニットに結合する RGS ドメインを持 つ一群の鱼白質 (Hepler, J. R. Emerging roles for RGS proteins in cell signalling. TiPS (1999) 20, 376-382) と G タンパク質あるいはその共役型受 容体との相互作用がある。  In addition, it binds to Go; a subunit called RGS (Regulators of G-protein signaling) protein, such as the binding between RGS 4 and the interleukin 8 B receptor and RGS 12 and the metatropic glutamate receptor (mglu). There is an interaction between a group of proteins with RGS domains (Hepler, JR Emerging roles for RGS proteins in cell signaling. TiPS (1999) 20, 376-382) and G proteins or their conjugated receptors.
G蛋白質の具体例としては、 3量体 G蛋白質が挙げられる。 G蛋白質の aサブ ュニットとして次のものが挙げられる。 Gsクラスの Gs o;、 Golf er Gi クラスの Gil a N Gi2 a、 Gi3 o;、 Gol CK、 Go2 a、 Gtl o;、 Gt2 α s Ggust o;。 Gq クラスの Gq a、 Gll a、 G14 a、 G15 a、 G16 o;。 G12クラスの G12 a;、 G13 a。 またこれら aサ プユニットと三量体を形成する および γサブユニットとして、 それぞれ ΐ か ら ]3 5までおよぴ γ ΐから 0/ 11まで挙げられる。 Specific examples of G proteins include trimer G proteins. The following are examples of the a subunit of the G protein. Gil a N Gi2 a of Gs o ;, Golf er Gi class of Gs class, Gi3 o ;, Gol CK, Go2 a, Gtl o ;, Gt2 α s Ggust o ;. Gq a, Gll a, G14 a, G15 a, G16 o; of the Gq class. G12 a; G13 a of the G12 class. Subunits that form a trimer with these a subunits and γ subunits include ら to 335 and γ ΐ to 0/11, respectively.
G蛋白質共役型受容体蛋白質 （そのリガンドを括弧内に示す） の具体例として は、 以下のものが挙げられる。 ( 1 )口ドプシン Z/3アドレナリン受容体様 Gタンパク質共役型受容体タンパク 質として、 BLT 1 (ロイコ トリェン B4)， ETA, ETB (エンドセリン）， ATI (アンギオ テンシン）, EDG (スフインゴシンリン酸）, CCR, CXCR (ケモカイン）， い α 2、 βい β 2、 β 3 (ノルェピネフリン）、 Mu M2, M3 (アセチルコリン）、 5— HT1A (セロ トニン）、 NK-1 (サブスタンス！5)、 Y, (ニューロペプチド Y)、 Β Β2 (ブラジキニ ン）、 VIA (バソプレツシン）、 CB1, CB2 (アナンダマイ ド） ， Dl， D2, D3 (ドーパ ミン）， におい受容体， MT1, MT2, MT3 (メラトニン），光受容体などが挙げられる。Specific examples of the G protein-coupled receptor protein (the ligand is shown in parentheses) include the following. (1) as mouth Dopushin Z / 3-adrenergic receptor-like G protein-coupled receptor protein, BLT 1 (leuco Toryen B 4), ET A, ET B ( endothelin), ATI (angio tensin), EDG (Sufuingo forest acid), CCR, CXCR (chemokine), have alpha 2, beta have beta 2, beta 3 (Noruepinefurin), M u M 2, M 3 ( acetylcholine), 5-HT 1A (serotonin), NK-1 ( 5 ), Y, (neuropeptide Y), 、 2 (bradykinin), VIA (vasopressin), CB1, CB2 (anandamide), Dl, D2, D3 (dopamine), odorant receptor, MT1 , MT2, MT3 (melatonin), photoreceptors and the like.
( 2 ) グルカゴン /VIP (Vasoactive intestinal peptide) /カルシトニン受容体様 G タンパク質共役型受容体タンパク質として、 カルシトニン受容体 （カルシトニ ン ) 、 VIP VIP2 ( Vasoactive intestinal peptide ) 、 CRF1(2) Glucagon / VIP (Vasoactive intestinal peptide) / calcitonin receptor-like G protein-coupled receptor protein, calcitonin receptor (calcitonin), VIP VIP 2 (Vasoactive intestinal peptide), CRF1
(corticotropin- releasing factor) , PTH受容体 （パラトルモン） などが挙げ られる。 (corticotropin-releasing factor), PTH receptor (paratolmon) and the like.
( 3 )代謝型神経伝達物質/カルシウム受容体様 Gタンパク質共役型受容体タン パク質として、 mgk^ mglu2 (グルタミン酸）、 GABAB ( y -ァミノ酪酸）， 味受容体 などが挙げられる。 (3) Metabotropic neurotransmitter / calcium receptor-like G protein-coupled receptor proteins include mgk ^ mglu 2 (glutamic acid), GABA B (y-aminobutyric acid), and taste receptors.
(Gether, U. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocrine Reviews (2000) 21, 90 - 113 ) ( 1998 Receptor and Ion Channel Nomenclature Supplement, Trends in Pharmacological Science, 1998)  (Gether, U. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors.Endocrine Reviews (2000) 21, 90-113) (1998 Receptor and Ion Channel Nomenclature Supplement, Trends in Pharmacological Science, 1998)
本発明では、 上記したような発現させるための蛋白質をコードする遺伝子を含 む少なくとも 1種の組換えパキュロウィルスを使用する。  In the present invention, at least one recombinant paculovirus containing a gene encoding a protein to be expressed as described above is used.
昆虫に感染して病気を起こすウィルスであるバキュロウィルスは、 環状の二本 鎖 D NAを遺伝子としてもつエンベロープウィルスで、 鱗翅目、 膜翅目おょぴ双 翅目などの昆虫に感受性を示す。 パキュロウィルスの中で、 感染細胞の核内に多 角体 （ポリヒ ドラ） と呼ばれる封入体を大量につくる一群のウィルスが核多角体 病ウィルス （N P V) である。 多角体は、 分子量 3 1 k D aのポリヘドリンタン パクより構成され、 感染後期に大量につくられその中に多数のウィルス粒子を埋 め込んでいる。 多角体はウィルスが自然界で生存するためには必須であるが、 ゥ ィルスの増殖そのものには必要ないので、 多角体遺伝子の代わりに発現させたい 外来遺伝子を挿入してもウィルスは全く支障なく感染し増殖する。 Baculovirus, a virus that causes disease by infecting insects, is an enveloped virus that has a circular double-stranded DNA as a gene and is susceptible to insects such as Lepidoptera, Hymenoptera, and Diptera. Among the paculoviruses, a group of viruses that produce large amounts of inclusion bodies called polyhydric bodies (polyhydra) in the nucleus of infected cells is the nuclear polyhedrosis virus (NPV). Polyhedra are composed of a polyhedrin protein with a molecular weight of 31 kDa, and are produced in large quantities at the late stage of infection, in which many virus particles are embedded. I'm crazy. Polyhedra are essential for the virus to survive in nature, but are not necessary for virus growth itself, so even if a foreign gene that is to be expressed in place of the polyhedron gene is inserted, the virus can be transmitted without any problem. And proliferate.
本発明で用いられるバキュロウィルスとしては、 NPVのキンゥヮバ亜科のォ ートグラファ 'カリフォルニ力 （Autographs californica) NPV (AcNPV) やカイコのボンビックス .モリ （Bombyx mori ) NPV (BmNP V) などのゥ ィルスがベクターとして用いることができる。  Examples of the baculovirus used in the present invention include viruses such as Autographs californica NPV (AcNPV) of NPV subfamily and Bombyx mori NPV (BmNPV) of silkworm. It can be used as a vector.
AcNPVの宿主 （感染、 継代細胞） としてはスポドプテラ ·フルギぺルダ (Spodoptera frugiperda ) 細胞 （S f細胞） などが挙げられ、 BmNPVの宿 主 （感染、 継代細胞） としては BmN4細胞などが挙げられる。 S f細胞は、 B mN 4細胞などに比べ増殖速度が速いこと、 また、 AcNPVはヒト月干細胞およ びヒト胎児腎細胞などにも感染する能力を有することから、 AcNPV系のベタ ターが好ましい。  AcNPV hosts (infected, passaged cells) include Spodoptera frugiperda cells (Sf cells), and BmNPV hosts (infected, passaged cells) include BmN4 cells. Can be AcNPV-based vectors are preferred because Sf cells have a higher growth rate than BmN4 cells and the like, and AcNPV has the ability to infect human moon-dried cells and human fetal kidney cells. .
宿主としては、 Spodoptera Frugiperda細胞系統 Sf9および Sf21などが S. frugiperda幼虫の卵巣組織から確立しており、Invitrogen社あるいは Pharmingen 社 （San Diego, CA)、 又は ATCCなどから入手可能である。 さらに、 生きている昆 虫幼虫を宿主細胞系として使用することもできる。  As a host, Spodoptera Frugiperda cell lines Sf9 and Sf21 have been established from ovarian tissues of S. frugiperda larvae, and are available from Invitrogen, Pharmingen (San Diego, CA), ATCC, or the like. In addition, live insect larvae can be used as host cell lines.
本発明で用いる組換えウィルスを構築する方法は、 常法に従って行えばよく、 例えば次の手順で行うことができる。  The method for constructing the recombinant virus used in the present invention may be performed according to a conventional method, and can be performed, for example, by the following procedure.
先ず、 発現させたい蛋白質の遺伝子 （即ち、 G蛋白質共役型受容体蛋白質をコ ードする遺伝子、 G蛋白質をコードする遺伝子、 及びアデ二ル酸シクラ一ゼをコ 一ドする遺伝子） をトランスファーベクターに挿入して組換えトランスファーべ クタ一を構築する。  First, a gene for a protein to be expressed (ie, a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase) are transferred to a transfer vector. To construct a recombinant transfer vector.
トランスファーベクターの全体の大きさは一般的には数 k b〜l 0 k b程度で あり、 そのうちの約 3 k bはプラスミ ド由来の骨格であり、 アンピシリン等の抗 生物質耐性遺伝子と細菌の DNA複製開始のシグナルを含んでいる。 通常のトラ ンスファーベクターではこの骨格以外に、 多角体遺伝子の 5， 領域と 3， 領域を それぞれ数 k bずつ含み、 以下に述べるようなトランスフエクシヨンを行った際 に、 この配列間で目的遺伝子と多角体遺伝子との間で相同糸且換えが引き起こる。 また、 トランスファーベクターには蛋白質遺伝子を発現させるためのプラモータ 一を含むことが好ましい。プロモーターとしては、多角体遺伝子のプロモーター、 ρ 10遺伝子のプロモーター、 キヤプシド遺伝子のプロモーターなどが挙げられ る。 The overall size of a transfer vector is generally about several kb to 10 kb, of which about 3 kb is a plasmid-derived skeleton, which is used to initiate resistance to an antibiotic resistance gene such as ampicillin and bacterial DNA replication. Signal. In a normal transfer vector, in addition to this backbone, regions 5 and 3 of the polyhedron gene are also included. When transfection is carried out as described below, containing several kb each, homologous recombination occurs between the target gene and the polyhedron gene between these sequences. The transfer vector preferably contains a promoter for expressing the protein gene. Examples of the promoter include a polyhedron gene promoter, a ρ10 gene promoter, and a capsid gene promoter.
トランスファーベクターの種類は特に限定されない。 トランスファーベクター の具体例としては、 A c NPV系トランスファーベクターとしては、 pEVmX I V2、 pAc SG l、 p VL 1392/ 1393、 pAcMP 2/3、 p A c 】 P 1、 Ac UW 21、 pAcDZ l、 p B l u e B a c I I I , p Ac UW 51、 pAcAB 3、 p A c 360S p B l u e B a cH i pVT— B a c 33、 p A c UW 1 s p AcUW42Z43などが挙げられ、 BmNPV系トラ ンスファーベクターとしては、 ρΒΚ283、 ρ ΒΚ5、 ρΒΒ 30、 ρΒΕ 1、 pBE 2、 p BK3、 p BK52、 pBKb l u e、 pBKb l u e 2、 p B F シリーズ （以上、 フナコシ株式会社、 藤沢薬品工業株式会社等から入手可能） な どが挙げられる。 The type of transfer vector is not particularly limited. Specific examples of the transfer vector include: AcNPV-based transfer vectors include pEVmXIV2, pAcSGl, pVL1392 / 1393, pAcMP2 / 3, pAccP1, AcUW21, pAcDZl, p B lue B ac III, p Ac UW 51, pAcAB 3, p A c 360 S p B lue B a cH i pVT- B ac 33, p A c UW 1 s such as p AcUW42Z43 can be mentioned, BmNPV system transformer Far vectors include ρΒΚ283, ρΒΚ5, ρΒΒ30, ρΒΕ1, pBE2, pBK3, pBK52, pBKblue, pBKblue2, and pBF series (obtained from Funakoshi Corporation, Fujisawa Pharmaceutical Co., Ltd., etc.) Possible).
次に、 組換えウィルスを作製するために、 上記の組換えトランスファーベクタ 一をウィルスと混合した後、 宿主として用いる培養細胞に移入するか、 あるいは 予めウィルスで感染させた宿主として用いる培養細胞に上記のトランスファーべ クタ一を移入し、 糸且換えトランスファーベクターとウィルスゲノム DNAとの間 に相同組み換えを起こさせ、 組み換えウィルスを構築する。  Next, in order to produce a recombinant virus, the above-mentioned recombinant transfer vector is mixed with the virus and then transferred to a cultured cell to be used as a host, or the above-mentioned recombinant vector is added to a cultured cell to be used as a host previously infected with the virus. Then, homologous recombination is caused between the transfer vector and the viral genomic DNA to construct a recombinant virus.
ここで宿主として用いる培養細胞とは、 上記した宿主が挙げられ、 通常、 昆虫 培養細胞 （S f 9細胞や BmN細胞など） である。 培養条件は、 当業者により適 宜決定されるが、 具体的には S f 9細胞を用いた場合は 10%ゥシ胎児血清を含 む培地で、 28 °C前後で培養することが好ましい。 このようにして構築された組 み換えウィルスは、 常法、 例えばプラークアツセィなどによって精製することが できる。 なお、 このようにして作製された組換えウィルスは、 核多角体病ウィル スの多角体蛋白質の遺伝子領域に外来の D N Aが置換または挿入されており多角 体を形成することができないため、 非組換えウィルスと容易に区別することが可 能である。 Here, the cultured cells used as hosts include the above-mentioned hosts, and are usually insect cultured cells (Sf9 cells, BmN cells, etc.). Culture conditions are appropriately determined by those skilled in the art. Specifically, when Sf9 cells are used, culture is preferably performed at about 28 ° C. in a medium containing 10% fetal bovine serum. The recombinant virus thus constructed can be purified by a conventional method, for example, plaque assay. The recombinant virus produced in this manner is a nuclear polyhedrosis virus. Since foreign DNA has been substituted or inserted into the gene region of the polyhedron protein of E. coli and cannot form a polyhedron, it can be easily distinguished from a non-recombinant virus.
本発明の方法では、 前記の組換えバキュロウィルスを、 上記した適当な宿主 (Spodoptera Frugiperda細胞系統 Sf9および Sf21などの培養細胞、 又は昆虫幼 虫など） に感染させ、 一定時間後 （例えば、 7 2時間後等） に培養上清から細胞 外発芽ウィルス （budded virus, BV) を遠心などの分離操作によって回収する。 なお、 組換えバキュロウィルスは 1種類のみ感染させてもよいし、 2種類以上の 組換えパキュロウィルスを組み合わせて共感染させてもよい。  In the method of the present invention, the above-mentioned recombinant baculovirus is infected into an appropriate host (cultured cells such as Spodoptera Frugiperda cell lines Sf9 and Sf21, or insect larvae), and after a certain period of time (for example, 72 After a few hours, extracellular budding virus (BV) is recovered from the culture supernatant by a separation procedure such as centrifugation. In addition, only one recombinant baculovirus may be infected, or two or more recombinant baculoviruses may be combined and co-infected.
細胞外発芽バキュロウィルスの回収は、 例えば、 以下のように行うことができ る。  The extracellular budding baculovirus can be collected, for example, as follows.
先ず感染細胞の培養液を 5 0 0〜1， O O O gで遠心分離して、 細胞外発芽バ キュロウィルスを含む上清を回収する。 この上清を約 3 0， 0 0 0〜 5 0， 0 0 0 gで遠心分離して細胞外発芽パキュ口ウィルスを含む沈殿物を得ることができ る。 上記のようにして回収される発芽パキュロウィルスは、 G蛋白質共役型受容 体蛋白質、 G蛋白質、 及びアデ二ル酸シクラーゼを生理活性を有する状態で含ん でおり、 当該発芽バキュロウィルス自体も本発明の範囲内である。 即ち、 当該発 芽バキュロウィルスは、 G蛋白質共役型受容体蛋白質、 G蛋白質及ぴァデュル酸 シクラーゼを機能的に提示していることを特徴とする。  First, the culture of the infected cells is centrifuged at 500 to 1,000 g, and the supernatant containing the extracellular budding baculovirus is recovered. The supernatant can be centrifuged at about 30,000 to 500,000 g to obtain a precipitate containing extracellularly germinated PacuMouth virus. The germinated baculovirus recovered as described above contains a G protein-coupled receptor protein, a G protein, and adenylate cyclase in a state having a physiological activity. Is within the range. That is, the germinated baculovirus is characterized in that it functionally presents a G protein-coupled receptor protein, a G protein, and aduryl cyclase.
本発明においては、上記のようにして調製した発芽バキュロウィルスを使用し、 被験物質の存在下において発芽バキュ口ウィルスとリガンドとを接触させ、 生成 する c AM Pをアツセィすることによって、 G蛋白質共役型受容体蛋白質とリガ ンドとの相互作用を分析し、 これにより該相互作用を促進又は阻害する物質をス クリーユングすることができる。  In the present invention, the germinated baculovirus prepared as described above is used, the germinated baculovirus is brought into contact with a ligand in the presence of a test substance, and the resulting cAMP is conjugated to the G protein-coupled protein. By analyzing the interaction between the type receptor protein and the ligand, a substance that promotes or inhibits the interaction can be screened.
生成する c AM Pのアツセィ方法は特に限定されず、 当業者であれば適宜選択 することができる。 一例としては、 ェンザィムィムノアツセィの原理を用いて、 固相に結合させた抗 c AM P抗体へのペルォキシダーゼ標識 c AM Pの結合を、 活性化アデニル酸シクラーゼにより生成された c AM Pが競合的に阻害すること を利用して、 酵素反応による発色の減少を測定することにより、 c AM Pをアツ セィすることができる。なお、 c AM Pのアツセィは、例えば、 Amersham社製 cAMP Biotrak Enzyme immunoassay Systemキットなどの巿販のキットを使用して行つこ ともできる。 The method of generating cAMP is not particularly limited, and can be appropriately selected by those skilled in the art. As an example, the binding of peroxidase-labeled cAMP to an anti-cAMP antibody bound to a solid phase using the principle of Enzymimnoassay is By taking advantage of the competitive inhibition of cAMP produced by activated adenylate cyclase, cAMP can be assayed by measuring the decrease in color development due to the enzymatic reaction. Note that cAMP can also be obtained using commercially available kits such as Amersham cAMP Biotrak Enzyme immunoassay System kit.
上記したスクリーニングに供される被験物質としては、 例えばペプチド、 ポリ ペプチド、 合成化合物、 微生物発酵物、 生物体 （植物又は動物の組織、 微生物、 又は細胞などを含む） からの抽出物、 あるいはそれらのライブラリーが挙げられ る。 ライブラリ一としては、 合成化合物ライプラリー （コンビナトリアルライブ ラリーなど）、ペプチドライブラリー（コンビナトリアルライブラリーなど） など が挙げられる。 スクリーニングに供される化学物質は、 天然物でも合成物でもよ く、 また候補となる単一の化学物質を独立に試験しても、 いくつかの候補となる 化学物質の混合物（ライプラリーなどを含む）について試験をしてもよい。また、 細胞抽出物のような混合物を分画したものについてスクリーニングを行い、 分画 を重ねて、 所望の活性を有する物質を単離することも可能である。  The test substance to be subjected to the above-mentioned screening includes, for example, peptides, polypeptides, synthetic compounds, fermented microorganisms, extracts from organisms (including plant or animal tissues, microorganisms, or cells), or extracts thereof. Libraries. Libraries include synthetic compound libraries (such as combinatorial libraries) and peptide libraries (such as combinatorial libraries). The chemicals to be screened can be natural or synthetic, and even if a single candidate chemical is tested independently, a mixture of several candidate chemicals (including ) May be tested. In addition, it is also possible to screen a fractionated mixture such as a cell extract, to isolate a substance having a desired activity by repeating the fractionation.
これらの被験物質は、 G蛋白質共役型受容体蛋白質とリガンドとの相互作用を 促進又は阻害することが予想される物質であることが好ましい。  These test substances are preferably substances that are expected to promote or inhibit the interaction between the G protein-coupled receptor protein and the ligand.
本発明のスクリーニング方法により、 G蛋白質共役型受容体蛋白質に対する阻 害薬または活性化薬物をスクリーニングすることが可能である。 本発明のスクリ 一ユング方法により得られる、 G蛋白質共役型受容体蛋白質とリガンドとの相互 作用を促進又は阻害する物質も本発明の範囲内である。  According to the screening method of the present invention, it is possible to screen for an inhibitor or an activating drug for a G protein-coupled receptor protein. Substances that promote or inhibit the interaction between a G protein-coupled receptor protein and a ligand, which are obtained by the screening method of the present invention, are also within the scope of the present invention.
以下の実施例により本発明をさらに具体的に説明するが、 本発明は実施例によ つて限定されるものではない。 実施例  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. Example
実施例 1： ドパミン受容体 D1を発現する組換えパキュロウィルスの調製 Example 1 Preparation of Recombinant Paculovirus Expressing Dopamine Receptor D1
( 1 ) 細胞の培養と感染、 および発芽型パキュロウィルスの収集 ヒ ト胎児脳 cDNA から PCR法により増幅して得られたヒ ト ドパミン受容体 D1 (DR - D1)全長 cDNA ( Genbank accession number 丽 000794 ) を pBlueBacHis2C (Invitrogen)へ組み込み、 pBluBac- His- DR- D1を作成した。 Sf 9細胞（Invitrogen) は完全培地 （10%ゥシ胎児血清 （Sigma；)、 100 units/mlペニシリンおよび 100 μ g/mlストレプトマイシンを含む Grace ' s supplemented media (GIBCO BRL) ) で 27°Cで 10 cm径ディッシュに継代培養した。 大量培養は 1 L容量のスピナ一ブラ スコ （Wheaton) にて完全培地に 0. 001% pluronic F- 68 (GIBCO BRL) を添加して 行なった。 組み換えバキュロウィルスの作成は説明書 （Bac- N- BlueTM Transfection Kit, Invitrogen) に従い、 Sf9細月包に Bac— N— Blue DNA (ApMNPV由 来） と pBluBac- His- DR - Dlとを共感染させ組み換えウィルスを作成した。 (1) Cell culture and infection, and collection of budding paculovirus The full-length human dopamine receptor D1 (DR-D1) cDNA (Genbank accession number 丽 000794) obtained by amplifying the human fetal brain cDNA by PCR was incorporated into pBlueBacHis2C (Invitrogen), and pBluBac-His-DR-D1 It was created. Sf9 cells (Invitrogen) were incubated in complete medium (Grace's supplemented media (GIBCO BRL) containing 10% fetal calf serum (Sigma;), 100 units / ml penicillin and 100 μg / ml streptomycin) at 27 ° C. The cells were subcultured on a 10 cm diameter dish. Mass culture was performed in a 1 L spinner-brasco (Wheaton) medium supplemented with 0.001% pluronic F-68 (GIBCO BRL) in complete medium. For the preparation of recombinant baculovirus, follow the instructions (Bac-N-BlueTM Transfection Kit, Invitrogen) and co-infect Sf9 cells with Bac-N-Blue DNA (from ApMNPV) and pBluBac-His-DR-Dl. Created a recombinant virus.
( 2 ) 発芽型バキュロウィルスにおける受容体発現の Western Blotによる解析 Sf9細胞を 1 L容量のスピナ一フラスコ （Wheaton) に 2xl06個 / ml濃度で 500 ml培養し、上記 （ 1 ) で作成した組み換えウィルスを M0I=5で感染させ、 72時間 後の培養液を実験に用いた。 培養液は 800 xg、 10分間遠心し、 沈澱および上清に 分離した。 800 xgの遠心後の上清を 40, 000 xg、 25分遠心し、 沈澱を PBSに懸濁 後、 再度 40，000 xg、 25分遠心した。 沈澱を PBSに懸濁し， 800 xg， 10分遠心 して凝集物を除いた後に、 再度、 40, 000 xg、 25分遠心して得られた沈澱を PBS に懸濁して、発芽型ウィルス画分（BV画分） とした。 BV画分における DR- D1の発 現は His- tagを認識する抗 His抗体 （Sigma) を用いた Western Blotにより確認 した。 (2) Budding analysis Sf9 cells by Western Blot receptor expression in baculovirus and 500 ml culture to 1 L volume of spinner one flask (Wheaton) 2Xl0 6 cells / ml concentration was prepared in the above (1) recombinant The virus was infected at M0I = 5, and the culture solution 72 hours later was used for the experiment. The culture was centrifuged at 800 xg for 10 minutes and separated into a precipitate and a supernatant. The supernatant after 800 xg centrifugation was centrifuged at 40,000 xg for 25 minutes. The precipitate was suspended in PBS, and then centrifuged again at 40,000 xg for 25 minutes. The precipitate was suspended in PBS and centrifuged at 800 xg for 10 minutes to remove aggregates, and then centrifuged again at 40,000 xg for 25 minutes. The precipitate obtained was suspended in PBS and the germinated virus fraction ( BV fraction). The expression of DR-D1 in the BV fraction was confirmed by Western Blot using an anti-His antibody (Sigma) recognizing His-tag.
( 3 ) 発芽型バキュロウィルスにおける受容体に対するリガンド結合能解析 (3) Analysis of ligand-binding ability of budding baculovirus to receptor
BV画分のリガンド結合能は、 7, 8- ¾- Dopamine (Amersham) 結合実験により確 認した。 ¾- Dopamine結合実験は、 7, 8- ¾- Dopamineを含む binding buffer (50 mM Tris-HCl pH 7, 4、 10 mM MgCl2、 10 mM NaCl、 0. 5 % fatty acid-free BSA) に DR-D1 BV画分を加えて反応液量 500 · 1 とし、 室温で 90分、 反応させて行なつ た。 反応液をフタル酸ジ -n-ブチル Zフタル酸ジォクチル 1 : 1混合液 500 μ ΐ上に 重層し、 15, 000 xg, 10 分間、 室温にて遠心して、 ウィルスを沈澱として回収し た。 沈澱を binding bufferで 3回洗浄、 風乾後、 液体シンチレーシヨンカウンタ 一で、 沈澱物中に含まれるトリチウムの量を測定することにより、 受容体と結合 した3 H- dopamineの量を算出した。 ¾- dopamineの結合量は、反応液中に加えた標 識 dopamine の濃度依存的に増加した。 更にこの ¾-dopamine の結合は、 非標識 dopamineまたはアンタゴニストにより、 阻害された。 以上より、 発芽型バキュ口 ウィルス上に発現した DR- D1は、 リガンド結合活性を有することが確認された。 実施例 2 ：発芽型バキュロウィルスにおける Gタンパク質共役型受容体とリガン ドの結合に伴う Gタンパク質によるアデニル酸シクラーゼ活性化の検出 The ligand binding ability of the BV fraction was confirmed by a 7,8-¾-Dopamine (Amersham) binding experiment. In the 実 験 -Dopamine binding experiment, DR was added to a binding buffer containing 7, 8-¾-Dopamine (50 mM Tris-HCl pH 7, 4, 10 mM MgCl 2 , 10 mM NaCl, 0.5% fatty acid-free BSA). The -D1 BV fraction was added to make the reaction solution volume 500 · 1, and reacted at room temperature for 90 minutes. The reaction solution was overlaid on 500 μΐ of di-n-butyl phthalate Z-dioctyl phthalate 1: 1 mixture and centrifuged at 15,000 xg for 10 minutes at room temperature to recover the virus as a precipitate. It was. The precipitate washed three times with binding buffer, dried in air, in liquid scintillation counter and foremost, by measuring the amount of tritium contained in the precipitate was calculated the amount of 3 H- dopamine bound to the receptor. The amount of ¾-dopamine bound increased depending on the concentration of the labeled dopamine added to the reaction solution. Furthermore, the binding of ¾-dopamine was inhibited by unlabeled dopamine or antagonist. From the above, it was confirmed that DR-D1 expressed on the budding baculovirus had ligand binding activity. Example 2: Detection of adenylate cyclase activation by G protein associated with binding of G protein-coupled receptor to ligand in budding baculovirus
G タンパク質は、 共役する受容体への特異的リガンド結合により活 1·生化され、 活性化後にエフェクタータンパク質と総称される各種酵素と相互作用しそれら酵 素分子の活性を調節する。アデ二ル酸シクラーゼは活性型 Gs様 Gタンパク質と相 互作用することによりサイクリック AMP (cAMP) 産生を上昇させるエフェクター タンパク質である。 ドパミン受容体は Gs様 Gタンパク質と共役し、 ドパミンの特 異的結合によつて活性型となった Gs様 Gタンパク質はアデ二ル酸シクラ一ゼと相 互作用し酵素活性を上昇させることが報告されている。  The G protein is activated by specific ligand binding to a coupled receptor, and after activation, interacts with various enzymes, collectively called effector proteins, to regulate the activity of those enzyme molecules. Adenylate cyclase is an effector protein that increases cyclic AMP (cAMP) production by interacting with activated Gs-like G proteins. The dopamine receptor is coupled to the Gs-like G protein, and the Gs-like G protein activated by specific binding of dopamine interacts with adenylate cyclase to increase enzyme activity. It has been reported.
( 1 ) アデ二ル酸シクラーゼ組み換えバキュ口ウィルスの作成  (1) Preparation of recombinant adjuate cyclase baculovirus
ヒ ト VI型アデニル酸シクラーゼ （AC VI) cDNAは、 ヒ ト胎児脳 cDNAライブラ リーより PCRによってクローユングした（Genbank accession number 丽 015270)。 蛍光蛋白 ECFP とアデ二ル酸シクラーゼを融合発現するため、 クローユングした AC VI cDNAを pECFP- N1プラスミド （Clontech) に組込み、 ECFP遺伝子を AC VI 遺伝子 3 '末端に融合させた。 さらにこの ECFP融合 ACVI cDNAを pBlueBac4. 5 ベクター （Invitrogen) へ組込んだ。 実施例 1 (1) で示した組換えバキュロウィ ルスの作成法により、 ECFP融合 AC VI (AC VI- ECFP) 組換えバキュロウィルスを 調製した。  Human type VI adenylate cyclase (AC VI) cDNA was cloned by PCR from a human fetal brain cDNA library (Genbank accession number 丽 015270). In order to express the fluorescent protein ECFP and adenylate cyclase in a fused manner, the cloned AC VI cDNA was inserted into the pECFP-N1 plasmid (Clontech), and the ECFP gene was fused to the 3 ′ end of the AC VI gene. The ECFP-fused ACVI cDNA was further inserted into a pBlueBac4.5 vector (Invitrogen). An ECFP-fused AC VI (AC VI-ECFP) recombinant baculovirus was prepared by the method for preparing a recombinant baculovirus described in Example 1 (1).
( 2 ) 受容体、 G蛋白質、 アデ二ル酸シクラ一ゼを共発現する発芽型バキュロウ ィルスにおけるリガンド刺激依存性 cAMP産生の検出 2xl06細胞/ ml濃度の Sf9細胞 200 mlに、 DR-D1およぴラット Ga s、 Gj31+Gy 2 (Gj31 と Gy2 遺伝子を同一ウィルスゲノム上に直列に持つウィルス）、 AC VI- ECFPの各組換えパキュ口ウィルスをそれぞれ M0I=2.5:1.5:0.5: :4.0で共感染 させ、 72時間後に BV画分を調製し TBSへ懸濁した。 また同様に、 Ga sを除い た DR- D1および G]31+G"y2、 AC VI- ECFPの各組換えバキュロウィルスをそれぞれ M0I=2.5: :0.5: :4.0で Sf9細胞に共感染させ、 発芽型バキュ口ウィルスを調製し た。 更に、 野生型バキュロウィルスを M0I=2で Sf 9細胞に感染させ、 発芽型バキ ュロウィルスを調製した。 (2) Detection of ligand-stimulated dependent cAMP production in budding baculoviruses co-expressing receptor, G protein and adenylate cyclase 2Xl0 6 Sf9 cells 200 ml of cells / ml concentration, DR-D1 Oyopi rats Ga s, Gj31 + Gy 2 ( Gj31 and Gy2 genes viruses having in series on the same viral genome), each of the AC VI- ECFP Recombinant pacific viruses were co-infected with M0I = 2.5: 1.5: 0.5 :: 4.0, respectively. After 72 hours, a BV fraction was prepared and suspended in TBS. Similarly, Sf9 cells were co-infected with DR-D1 and G] 31 + G "y2 excluding Gas and AC VI-ECFP recombinant baculovirus at M0I = 2.5 :: 0.5 :: 4.0, respectively. A budding baculovirus was prepared, and a wild-type baculovirus was infected to Sf9 cells at M0I = 2 to prepare a budding baculovirus.
各組み合わせで調製した発芽型バキュロウィルス懸濁液による cAMP産生は、下 記のようにして測定した。 DR- Dl, Gas, Gj31, Gy2, ACVI - ECFP共発現ウィルス 画分 10 z gを assay buffer (50 mM HEPES pH8.0, 0.6 mM EDTA, 5 mM MgGl2, 1 mM IBM, 0.01% fatty acid- free BSA, 3 mM phosphoenol pyruvate 3Na, 5 u/ml pyruvate kinase, 5 u/ml myokinase, 1 mM ATP, 50 GTP, 0.02% saponin) に 力 tlえて、 総体積を 90μ1 とし、 更に 10 μΐの 100 μ M dopamine (終濃度 10 μ Μ) , あるいは 500 for skolin (終濃度 50 Μ) を添加して、 37 °Cで 30分 間、 反応させた。 反応は 1/9容の Lysis reagent 1A (Amersham社製 cAMP Biotrak Enzyme immunoassay System キットに付属）を加えることにより、 停止させた。 反 応液中に産生された cAMPは上記の cAMP測定キットを用いて、説明書に従い ELISA 法により定量した。 CAMP production by the germinated baculovirus suspensions prepared for each combination was measured as described below. DR-Dl, Gas, Gj31, Gy2, ACVI-ECFP co-expressed virus fraction 10 zg was added to assay buffer (50 mM HEPES pH 8.0, 0.6 mM EDTA, 5 mM MgGl 2 , 1 mM IBM, 0.01% fatty acid-free BSA, 3 mM phosphoenol pyruvate 3Na, 5 u / ml pyruvate kinase, 5 u / ml myokinase, 1 mM ATP, 50 GTP, 0.02% saponin) to make a total volume of 90 μ1 and 10 μΐ of 100 μM Dopamine (final concentration 10 μΜ) or 500 for skolin (final concentration 50 50) was added and reacted at 37 ° C for 30 minutes. The reaction was stopped by adding 1/9 volume of Lysis reagent 1A (attached to cAMP Biotrak Enzyme immunoassay System kit manufactured by Amersham). The cAMP produced in the reaction solution was quantified by the ELISA method using the above-described cAMP measurement kit according to the instructions.
DR-D1および Ga s、 G]3、 、 AC VI- ECFPの組換えバキュロウィルスを共感 染させ得られた発芽型バキュロウィルスについて 10 /zMドパミン （Sigma)による 刺激を行なった場合、 cAMP産生の上昇が確認された （図 1)。 一方、 野生型の発芽 型バキュロウィルスでは 10 μΜドパミン刺激による cAMP産生の上昇は認められ なかった （図 1)。 更に Ga sを除く DRD1、 Gj3+Gy、 および AC VI- ECFPの組換 えバキュ口ウィルスを共感染させて得られた発芽型バキュロウィルスでもドパミ ン刺激による cAMP産生の上昇は認められなかった。以上のことから、発芽型バキ ュロウィルスにおけるドパミンの DR-D1受容体への特異的結合による Go; sおよ ぴ AC VI- ECFPの相互作用、すなわち特異的リガンド刺激による受容体活性化後の シグナル伝達現象の検出が可能であることが確認された。 産業上の利用可能性 When stimulated with 10 / zM dopamine (Sigma), sprouted baculovirus obtained by co-infection of DR-D1 and Gas, G] 3, ACVI-ECFP recombinant baculovirus, An increase was confirmed (Figure 1). On the other hand, in wild-type budding baculovirus, cAMP production was not increased by stimulation with 10 μΜ dopamine (Fig. 1). Furthermore, no increase in cAMP production due to dopamine stimulation was observed in germinated baculoviruses obtained by co-infection with recombinant baculoviruses of DRD1, Gj3 + Gy, and ACVI-ECFP excluding Gas. Based on the above, the specific binding of dopamine to the DR-D1 receptor in budding baculoviruses た It was confirmed that it was possible to detect the AC VI-ECFP interaction, that is, the signal transduction phenomenon after receptor activation by specific ligand stimulation. Industrial applicability
本発明によれば、発芽型バキュロウィルスを用いてリガンド刺激依存性の cAMP 産生を測定することにより、 GPCR (G蛋白質共役型受容体） のシグナル伝達を簡 便に検出することが可能になった。  According to the present invention, it has become possible to easily detect GPCR (G protein-coupled receptor) signal transduction by measuring ligand stimulation-dependent cAMP production using a budding baculovirus. .

Claims

請求の範囲 . The scope of the claims .
1 . G蛋白質共役型受容体蛋白質をコードする遺伝子、 G蛋白質をコードす る遺伝子、 及ぴアデ二ル酸シクラーゼをコードする遺伝子を含む少なくとも 1種 の組換えバキュロウィルスを感染させた宿主を培養し、 該宿主から放出される発 芽バキュロウィルスを回収し、該発芽バキュロウィルスとリガンドとを接触させ、 生成する c AM Pをアツセィすることを含む、 G蛋白質共役型受容体のシグナル 伝達を検出する方法。 1. Culturing a host infected with at least one recombinant baculovirus containing a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase Recovering the germinated baculovirus released from the host, contacting the germinated baculovirus with a ligand, and detecting the signal transduction of the G protein-coupled receptor, including attenuating the generated cAMP. how to.
2 . G蛋白質共役型受容体蛋白質をコードする遺伝子を含む組換えバキュロ ウィルス、 G蛋白質をコードする遺伝子を含む糸且換えバキュロウィルス、 及ぴァ デニル酸シクラ一ゼをコ一ドする遺伝子を含む組換えパキュロウィルスを感染さ せた宿主を培養し、 該宿主から放出される発芽バキュロウィルスを回収し、 該発 芽パキュロウィルスとリガンドとを接触させ、 生成する c AMPをアツセィする ことを含む、 G蛋白質共役型受容体のシグナル伝達を検出する方法。  2. Includes a recombinant baculovirus containing a gene encoding a G protein-coupled receptor protein, a recombinant baculovirus containing a gene encoding a G protein, and a gene encoding adenylate cyclase Culturing a host infected with the recombinant paculovirus, collecting germinated baculovirus released from the host, contacting the germinated paculovirus with a ligand, and assaying cAMP produced. A method for detecting G protein-coupled receptor signal transduction.
3 . 宿主が昆虫細胞又は昆虫幼虫である、 請求項 1又は 2に記載の方法。 3. The method according to claim 1, wherein the host is an insect cell or an insect larva.
4 . 被験物質の存在下において発芽パキュロウィルスとリガンドとを接触さ せ、 生成する c AM Pをアツセィすることによって G蛋白質共役型受容体蛋白質 とリガンドとの相互作用を分析し、 該相互作用を促進又は阻害する物質をスクリ 一ユングする、 請求項 1カゝら 3の何れかに記載の方法。 4. Contact the budding paculovirus with the ligand in the presence of the test substance, and analyze the interaction between the G protein-coupled receptor protein and the ligand by assaying the generated cAMP. The method according to any one of claims 1 to 3, wherein a substance that promotes or inhibits lipase is screened.
5 . 請求項 4に記載の方法により得られる、 G蛋白質共役型受容体蛋白質と リガンドとの相互作用を促進又は阻害する物質。  5. A substance which promotes or inhibits the interaction between a G protein-coupled receptor protein and a ligand, which is obtained by the method according to claim 4.
6 . G蛋白質共役型受容体蛋白質をコードする遺伝子、 G蛋白質をコードす る遺伝子、 及びアデ二ル酸シクラーゼをコードする遺伝子を含む少なくとも 1種 の組換えパキュロウィルスを感染させた宿主から放出される発芽バキュ口ウィル スであって、 G蛋白質共役型受容体蛋白質、 G蛋白質及びアデ二ル酸シクラーゼ を機能的に提示している発芽パキュロウィルス。  6. Release from a host infected with at least one recombinant paculovirus, including a gene encoding a G protein-coupled receptor protein, a gene encoding a G protein, and a gene encoding adenylate cyclase A germinated baculovirus that is functionally presenting a G protein-coupled receptor protein, a G protein and adenylate cyclase.
7 . 宿主が昆虫細胞又は昆虫幼虫である、 請求項 6に記載の発芽パキュ口ゥ イノレス。 7. The germinated pacu lip according to claim 6, wherein the host is an insect cell or an insect larva. Innores.
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