WO2005049825A1 - Methode de detection de transduction de signaux par un recepteur couple a la proteine g a l'aide d'un baculovirus de germination - Google Patents

Methode de detection de transduction de signaux par un recepteur couple a la proteine g a l'aide d'un baculovirus de germination 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

L'invention concerne une méthode de détection de la transduction d'un signal par un GPCR (récepteur couplé à la protéine G) par quantification de la production d'AMPc dépendant du stimulus d'un ligand à l'aide d'un baculovirus de germination. L'invention concerne une méthode de détection de la transduction d'un signal par un récepteur couplé à la protéine G, consistant à mettre en culture un hôte infecté par au moins un baculovirus de recombinaison contenant un gène codant pour une protéine du récepteur couplé à la protéine G, un gène codant pour la protéine G et un gène codant pour l'adénylate cyclase, à récupérer un baculovirus de germination libéré par l'hôte, à mettre le baculovirus de germination en contact avec un ligand, puis à doser l'AMPc ainsi formée.
PCT/JP2004/017646 2003-11-19 2004-11-19 Methode de detection de transduction de signaux par un recepteur couple a la proteine g a l'aide d'un baculovirus de germination WO2005049825A1 (fr)

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JP2005515702A JPWO2005049825A1 (ja) 2003-11-19 2004-11-19 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の検出方法

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JP2003-388842 2003-11-19
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003052370A (ja) * 2001-08-16 2003-02-25 Sentan Kagaku Gijutsu Incubation Center:Kk 発芽バキュロウィルスを用いた機能を有する膜型受容体蛋白質の発現法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003052370A (ja) * 2001-08-16 2003-02-25 Sentan Kagaku Gijutsu Incubation Center:Kk 発芽バキュロウィルスを用いた機能を有する膜型受容体蛋白質の発現法

Non-Patent Citations (3)

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Title
CORDEAUX, Y. ET AL.: "Agonist Regulation of D2 Dopamine Receptor/G Protein Interaction", J.BIOL.CHEM., vol. 276, no. 31, 2001, pages 28667 - 28675, XP002983184 *
MASUDA, K. ET AL.: "A Combinatorial G Protein-coupled Receptor Reconstitution System on Budded Baculovirus", J.BIOL.CHEM., vol. 278, no. 27, 4 July 2003 (2003-07-04), pages 24552 - 24562, XP002267779 *
YOU, H. ET AL.: "Expression of dopamine D1 receptor in Sf9 insect cells and agonism of 1-12-chloroscoulerine on recombinant D1 receptor", ACTA PHARMACOLOGICA SINICA, vol. 24, no. 3, March 2003 (2003-03-01), pages 225 - 229, XP002983183 *

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