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Development of procedures to knock out the function of intracellular proteins by expression of cDNAs encoding specific antibodies.

Research Project

Project/Area Number 02557013
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field General medical chemistry
Research InstitutionTokyo University

Principal Investigator

NUKADA Toshihide  Tokyo University, Faculty of Medicine, Assistant Professor, 医学部(医), 講師 (80189349)

Co-Investigator(Kenkyū-buntansha) TANAKA Masato  Mitsubisi Kasei Institute of Life Science, Department of Biomolecular Research,, 生体分子科学研究部, 主任研究員
SATO Showbu  Mitsubisi Kasei Institute of Life Science, Department of Biomolecular Research,, 生体分子科学研究部, 主任研究員
HAGA Tatsuya  Tokyo University, Faculty of Medicine, Professor, 医学部, 教授 (30011646)
Project Period (FY) 1990 – 1992
Project Status Completed (Fiscal Year 1992)
Budget Amount *help
¥14,200,000 (Direct Cost: ¥14,200,000)
Fiscal Year 1992: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1991: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥11,000,000 (Direct Cost: ¥11,000,000)
KeywordsGTP-binding protein / Monoclonal Antibody / cDNA Cloning / Immunoglobulin / Phospholipase C / Pertussis Toxin / Baculovirus / Muscarinic Acetylcholine Receptor / 免疫ブロッティング / 単クロ-ン抗体 / cDNAクロ-ニング / ホスホリパ-ゼC / ウェスタンブロッティング / ムスカリン性アセチルコリン受容体
Research Abstract

1. cDNA cloning of two novel GTP-binding protein alpha-subunits (Galphas) from a bovine liver cDNA library. These alpha-subunits (designated as GL1alpha and GL2alpha) lack apparent ADP-ribosylation sites for pertussis toxin. 2. Expression of exogenous Galphas in neuronal cells and Xenopus oocytes. Exogenous Gi3alpha mediated muscarinic receptor- (MAchR) dependent inhibition of high-threshold, voltage-dependent Ca^<2+> currents in NG108-15 cells Exogenous GL1alpha and GL2alpha that coupled to both the m1 subtype of mAchR and metabotropic glutamate receptor 1 mediated inhibition and stimulation of phospholipase C activity in Xenopus oocytes, respectively. 3. preparation of monoclonal antibodies specific for GL1alpha, GL2alpha, Gi1alpha, Goalpha using cell fusion method. 4. cDNA cloning of the specific monoclonal antibodies for each Galpha. The primary structures of the variable regions of the immunoglobulin heavy (gamma1 and mu) and light (kappa) chains were determined from the respectiv … More e cDNAs. 5. Establishment of an E. coli system for the simultaneous expression of mu and kappa cDNAs for anti-GL1alpha and Gi1alpha monoclonal antibodies. mu and kappa proteins secreted into the periplasmic space formed disulfide bonds and may be mu_2kappa_2.6. Mass production of Galpha proteins using a baculovirus expression system. solubilization of GL1alpha and GL2alpha proteins from membrane fractions by Lubrol PX or sodium cholate was made possible by the c-expression of G-protein beta_1gamma_2-subunits. 7. Insertion of immunoglobulin heavy and light chain cDNAs into expression vectors that contain an inducible promoter derived from mouse mammary tumor virus or Drosophila heat-shock protein 70 gene. We are planning to co-transfect vectors expressing the heavy and light chains of monoclonal antibodies for specific Galphas into cultured cells. We will then study the intracellular changes that result from the inhibition of specific subtypes of Galphas following glucocorticoid- or heat- induction of monoclonal antibody synthesis. Less

Report

(4 results)
  • 1992 Annual Research Report   Final Research Report Summary
  • 1991 Annual Research Report
  • 1990 Annual Research Report
  • Research Products

    (25 results)

All Other

All Publications (25 results)

  • [Publications] Fumio NAKAMURA: "Identification of Two Novel GTP-binding Protein α-Subunits that Lack Apparent ADP-ribosylation Sites for Pertussis Toxin" Journal of Biological Chemistry. 266. 12676-12681 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Kazuko HAGA: "Activation by G protein βγ-Subunits of Agonistor Light-Dependent Phosphorylation of Muscarinic Acetylcholine Receptors and Rhodopsin" Journal of Biological Chemistry. 267. 2222-2227 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Kazumasa SHIOZAKI: "Effects of Magnesium Ion on the Interaction of Atrial Muscarinic Acetylcholine Receptors and GTP-Binding Regulatory Proteins" Biochemistry. 31. 10634-10642 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Tatsuya HAGA: "Phosphorylation of Muscarinic Receptors:Regulation by G Proteins" Life Sciences. 52. 421-428 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Ago Rinken: "Solubilization and Characterization of Atrial Muscarinic Acetylcholine Receptors in Sucrose Monolaurate" Archives of Biochemistry and Biophysics. 301. (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] D.A.Brown: "Coupling of Muscarinic Receptor Subtypes to Ion Channels:Experiments on Neuroblastoma Hybrid Cells" Annu.New York Acad.Sci.(1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] T.Haga: "Reconstitution of muscarinic acetylcholine receptors and G proteins.In "Receptor-Effector Coupling:A practical approach",ed.by E.C.Hulme." Oxford University Press,Oxford, 83-98 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Toshihide Nukada: "Expression of G-protein α-Subunit cDNA.In "Neuroreceptor Mechanisms In Brain"ed.by S.Kito,T.Segawa and R.W.Olsen" Plenum Press,New York, 141-149 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] F. Nakamura: "Identification of Two Novel GTP-binding Protein alpha-Subunits that Lack Apparent ADP-ribosylation Sites for pertussis Toxin." Journal of Biological Chemistry. 266. 12676-12681 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] K. Haga: "Activation by G protein betagamma-Subunits of Agonist- or Light-Dependent Phosphorylation of Muscarinic Acetylcholine Receptors and Phodopsin." Journal of Biological Chemistry. 267. 2222-2227 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] K. Shiozaki: "Effects of Magnesium Ion on the Interaction of Atrial Muscarinic Acetylcholine Receptors and GTP-Binding Regulatory Proteins." Biochemistry. 31. 10634-10642 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] T. Haga: "Phosphorylation of Muscarinic Receptor: Regulation by G Proteins." Life Sciences. 52. 421-428 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] A. Rinken: "Solubilization and Characterization of Atrial Muscarinic Acetylcholine Receptors in Sucrose Monolaurate." Archives of Biochemistry and Biophysics. 301. (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] D. A. Brown: "Coupling of Muscarinic Receptor Subtypes to Ion Channels: Experiments on Neuro-blastoma Hybrid Cells." Annu. New York Acad. Sci.(1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] T. Haga: "Reconstitution of muscarinic acetylcholine receptors and G proteins. In "Receptor-Effector Coupling: A practical approach", ed. by E. C. Hulme." Oxford University Press, Oxford. 83-98 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] T. Nukada: "Expression of G-protein alpha-Subunit cDNA. In "Neuro-receptor Mechanisms In Brain" ed. by S. Kito, T. Segawa and R. W. Olsen" Plenum Press, New York. 141-149 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Kazumasa Shiozaki: "Effects of Magnesium Ion on the Interaction of Atrial Muscarinic Acetylcholine Receptors and GTP-Binding Regulatory Proteins" Biochemistry. 31. 10634-10642 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Tatsuya Haga: "Phosphorylation of Muscarinic Receptprs:Regulation by G Proteins" Life Sciences. 52. 421-428 (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] Ago Rinken: "Solubilization and Characterization of Atrial Muscarinic Acetylcholine Receptors in Sucrose Monolaurate" Archives of Biochemistry and Biophysics. 301. (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] D.A Brown: "Coupling of Muscarinic Receptor Subtypes to Ion Channels:Experiments on Neuroblastoma Hybrid Cells" Annu.New York Acad.Sci.(1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] Fumio Nakamura: "Identification of Two Novel GTP-binding Protein α-Subunits that Lack Apparent ADP-ribosylation Sites for Pertussis Toxin" Journal of Biological Chemistry. 266. 12676-12681 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Kazuko Haga: "Activation by G protein βγ-Subunits of Agonist- or Light- Dependent Phosphorylation of Muscarinic Acetylcholine Receptors and Rhodopsin" Journal of Biological Chemistry. 267. 2222-2227 (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] Fumio Nakamura: "Identification of Two Novel GTPーbinding Protein αーSubunits that Lack Apparent ADPーribosylation Sites for Pertussis Toxin" Journal of Biological Chemistry.

    • Related Report
      1990 Annual Research Report
  • [Publications] Toshihide Nukada: "Expression of GーProtein αーSubunit cDNA" Neureceptor Mechanisms In Brain. 141-149 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] T.Haga: "Reconstitution of muscarinic acetylcholine receptors and G proteins.In"ReceprorーEffector Coupling:A practical approach",ed.by E.C.Hulme." Oxford University Press,Oxford, 83-98 (1990)

    • Related Report
      1990 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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