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Structure, function and regulation of muscarinic acetylcholine receptors

Research Project

Project/Area Number 62490008
Research Category

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

Allocation TypeSingle-year Grants
Research Field 広領域
Research InstitutionThe University of Tokyo (1988-1989)
Hamamatsu University School of Medicine (1987)

Principal Investigator

HAGA Tatsuya  Institute of Brain Research, Professor, 医学部(医), 教授 (30011646)

Co-Investigator(Kenkyū-buntansha) ICHIYAMA Arata  Hamamatsu University, Professor, 医学部, 教授 (90025601)
Project Period (FY) 1987 – 1989
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 1989: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1988: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1987: ¥3,400,000 (Direct Cost: ¥3,400,000)
KeywordsMuscarinic receptor / Acetylcholine receptor / GTP binding protein / Pirenzepine / Reconstitution / Receptor / Phosphorylation / Protein kinase C / ムスカリン性アセチルコリン受容体 / GTP結合蛋白質 / レセプター
Research Abstract

structure, function and regulation of muscarinic acetylcholine receptors were examined on the receptors purified from porcine cerebrum or atrium and then reconstituted with G proteins, which were purified form porcine cerebrum.
1. Structure and ligand binding properties of muscarinic receptors: (1) The interaction of receptors with muscarinic ligands and G proteins was not affected by removal of sugars with endoglycosidase F. (2) The binding site of [^3H]propylbenzy1choline mustard was located on the region including the second and the third putative transmembrane segments and the phosphorylation sites by protein kinase C on the 12-14 kDa peptide of the C-terminus. (3) The presence of S-S bond between the second and the third extracellular loops was indicated, and the reduction of S-S bond(s),was shown to result in the decrease in the affinity for muscarinic ligands. Alkylation with DTNB or NEM resulted in the increase in the affinity for muscarinic agonists. (4) The subtype-specific co … More nformations with different affinities for pirenzepine were indicated to be taken in the presence Qf specific lipids.
2. Interaction between receptors and G proteins: Both cerebral and atrial purified receptors interacted with one of purified three different G proteins (Gi, Go and Gn) with similar potencies in reconstituted lipid vesicles. Similar results were obtained by using atrial membranes as a source of receptors instead of purifiedreceptors.
3. Phosphorylation of muscarinic receptors: Cerebral but not atrial muscarinic receptors were phosphorylated by protein kinase C. Atrial receptors were better substrates of cAMP-dependent protein kinase than cerebral receptors. A kinase which phosphorylates muscarinic receptors depending on the presence of agonists was partially purified from porcine cerebrum. Both cerebral and atrial receptors were phosphorylated by the kinase. The phosphorylation was inhibited when muscarinic receptors were reconstituted with G proteins and then subjected to phosphorylation, and the inhibition was restored by addition of GTP or GDP. Less

Report

(4 results)
  • 1989 Annual Research Report   Final Research Report Summary
  • 1988 Annual Research Report
  • 1987 Annual Research Report
  • Research Products

    (48 results)

All Other

All Publications (48 results)

  • [Publications] T.Haga: "Interaction of the muscarinic acetylcholine receptor and GTP-binding proteins." Biomedical Research. 8. 149-156 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] G.Berstein: "Agonist and antagonist binding of muscarinic acetylcholine recepotors purified from porcine brain:Interconversion of high and low affinity sites by sulfhydryl reagents." Journal of Neurochemistry. 50. 1687-1694 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T.Haga: "Molecular properties of muscarinic receptors." Trends in Pharmacological Sciences Supplement III. 12-18 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T.Haga: "Biochemical studies on the muscarinic acetylcholine receptor." In Neuroreceptors and signal transduction,ed.by S.Kito,T.Segawa,K.Kuriyama,M.T'hyama,and R.W.Olsen,Plenum Publishing Company,New York,London and washington D.C.239-254 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K.Haga: "Cerebral muscarinic acetylcholine receptors interact with three kinds of GTP-binding proteins in a reconstitution system of purified components." Molecular Pharmacology. 35. 286-294 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T.Nishiyama: "Comparison between purified cerebral and atrial muscarinic acetylcholine receptors.Pirenzepine binding and the effect of sulf-hydryl reagents." Biomedical Research. 10. 251-260 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] G.Berstein: "Effect of the lipid environment on the differential affinity of purified cerebral and atrial muscarinic acetylcholine receptors for pirenzepine." Molecular Phyrmacology. 36. 601-607 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K.Haga: "Agonist-dependent phosphorylation of cerebral and atrial muscarinic receptors:Blockade of the phosphorylation by GTP-binding regulatory proteins and its reversal by guanine nucleotides." Biomedical Research. 10. 293-299 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K.Haga: "Phosphorylation by protein kinase C of the muscarinic acetycholine receptor." J.Neurochemisty(in press). (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] H.Uchiyama: "Location in muscarinic acetylchoine receptors of sites for(^3H)propylbenzylcholine mustard binding and for phosphorylation with protein kinase C." Journal Neurochemstry(in press). (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T.Ikegaya: "Interaction of atrial muscarinic receptors with three kinds of GTP-binding proteins." Journal Molecular Cellular Cardiology(in press). (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T.Haga: "Muscarinic acetylcholine receptors in porcine atrial membranes interact with three kinds of G proteins." Japanese Circulation Journal(in press). (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T.Haga: "Reconstitution of muscarinic cholinoceptors and GTP-binding proteins from porcine brain.In Cellualr and molecular basis of cholinergic function,ed.by M.J.Dowdall and J.N.Hawthorne pp 104-115." Ellis Horwood Ltd.,Chichester, (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T.Haga: "The structure and function of muscarinic acetylcholine receptors.In Biosignal transduction mechanisms,ed.by M.Kasai,T.Yishioka and H.Suzuki,pp.115-138." Japan Sci.Soc.Press,Tokyo Springer Verlag,Berlin, (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] G.Berstein: "Molecular aspects of muscarinic receptors.In Current Aspects in the Neurosciences,vol.1,ed.by N.Osborne,pp245-284." MacMillan Press,London, (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T.Haga: "Molecular characterization of muscarinic acetylcholine receptors.In Physiology and pharmacology of trensmembrane signalling,ed.by T.Segawa,M.Endoh,M.Ui and K.Kurihara,pp105-113" Elsevier Science Publishers B.V., (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Haga and K. Haga: "Interaction of the muscarinic acetylcholine receptor and GTP-binding proteins." Biomed. Res. 8, 149-156 (1987).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] G. Berstein, K. Haga, T. Haga and A. Ichiyama: "Agonist and antagonist binding of muscarinic acetylcholine receptors purified from porcine brain: Interconversion of high and low affinity sites by sulfhydryl reagents." J. Neurochem. 50, 1687-1694 (1988).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Haga, K. Haga, G. Berstein, T. Nishiyama, H. Uchiyama and A. Ichiyama: "Molecular properties of muscarinic receptors." Trends in Pharmac. Sci. Supplement III. p12-18 (1988).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Haga, G. Berstein, T. Nishiyama, H. Uchiyama, K. Ohara and K. Haga: "Biochemical studies on the muscarinic acetylcholine receptor." "Neuroreceptors and signal transduction", ed. by S. Kito, T. Segawa, K. Kuriyama, M. Tohyama, and R.W. Olsen, Plenum Publishing Company, New York, London and Washington D.C. pp. 239-254 (1988).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K. Haga, H. Uchiyama, T. Haga, A. Ichiyama, K. Kangawa, and H. Matsuo: "Cerebral muscarinic acetylcholine receptors interact with three kinds of GTP-binding proteins in a reconstitution system of purified components." Mol. Pharmac. 35, 286-294 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Nishiyama, G. Berstein, T. Ikegaya, T. Haga, A. Ichiyama, A. Kobayashi and N. Yamazaki: "Comparison between purified cerebral and atrial muscarinic acetylcholine receptors: pirenzepine binding and the effect of sulfhydryl reagents." Biomed. Res. 10, 251-260 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] G. Berstein, T. Haga and A. Ichiyama: "Effect of the lipid environment on the differential affinity of purified cerebral and atrial muscarinic acetylcholine receptors for pirenzepine." Mol. Pharmac. 36, 601-607 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K. Haga and T. Haga: "Agonist-dependent phosphorylation of cerebral and atrial muscarinic receptors: Blockade of the phosphorylation by GTP-binding regulatory proteins and its reversal by guanine nucleotides." Biomed. Res. 10, 293-299 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K. Haga, T. Haga and A. Ichiyama: "Phosphorylation by protein kinase C of the muscarinic acetylcholine receptor." J. Neurochem.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] H. Uchiyama, K. Ohara, K. Haga, T. Haga and A. Ichiyama: "Location in muscarinic acetylcholine receptors of sites for [^3H]propylbenzylcholine mustard binding and for phosphorylation with protein kinase C." J. Neurochem.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Ikegaya, T. Nishiyama, K. Haga, T. Haga, A. Ichiyama, A. Kobayashi and N. Yamazaki: "Interaction of atrial muscarinic receptors with three kinds of GTP-binding proteins." J. Mol. Cell. Cardiol.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Haga, T. Ikegaya and K. Haga: "Muscarinic acetylchoine receptors in porcine atrial membranes interact with three kinds of G proteins." Jap. Circulation J.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Haga and K. Haga: "Reconstitution of muscarinic cholinoceptors and GTP-binding proteins from porcine brain." "Cellular and molecular basis of cholinergic function", ed. by M.J. Dowdall and J.N. Hawthorne, Ellis Horwood Ltd., Chichester, pp 104-115 (1987).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Haga: "The structure and function of muscarinic acetylcholine receptors." "Biosignal Transduction Mechanisms", ed. by M. Kasai, T. Yoshioka and H. Suzuki, Japan Sci. Soc. Press, Tokyo/Springer Verlag, Berlin. pp. 115-138 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] G. Berstein and T. Haga: "Molecular aspects of muscarinic receptors." "Current Aspects in the Neurosciences, Vol.1", ed. by N. Osborne, Macmillan Press, London. pp 245-284 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Haga, K. Haga, G. Berstein, H. Uchiyama, T. Nishiyama, and T. Ikegaya: "Molecular characterization of muscarinic acetylcholine receptors." "Physiology and pharmacology of transmembrane signalling", ed. by T. Segawa, M. Endoh, M. Ui and K. Kurihara, Elsevier Science Publishers B. V. pp. 105-113 (1989).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Haga, K. Haga and E.C. Hulme: "Solubilization, purification and molecular characterization of receptors: principles and strategy." "Receptor Biochemistry: A practical approach", ed. by E.C. Hulme, Oxford University Press, Oxford.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Haga, K. Haga and E.C. Hulme: "Purification and molecular characterization of muscarinic acetylcholine receptors." "Receptor Biochemistry: A practical approach", ed. by E.C. Hulme, Oxford University Press, Oxford.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Haga and K. Haga: "Reconstitution of muscarinic acetylcholine receptors and G proteins." "Receptor-Effector Coupling: A practical approach", ed. by E.C. Hulme, Oxford University Press, Oxford.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] G.Berstein: "Effect of the lipid environment on the differentioal affinity of purified cerebral and atrial muscarinic acetylcholine receptors for pirenzepine." Molecular Pharmacology. 36. 601-607 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Haga: "Agonist-dependent phosphorylation of cerebral and atrial muscarinic receptors:Blockade of the phosphorylation by GTP-binding regulatory proteins and its reversal by guanine nucleotides." Biomedical Research. 10. 293-299 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Haga: "Phosphorylation by protein kinase C of the muscarinic acetylcholine receptor." J.Neurochemisty. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] H.Uchiyama: "Location in muscarinic acetylchoine receptors of sites for(3H)propylbenzylcholine mustard binding and for phosphorylation with protein kinase C." Journal Neurochemistry. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] T.Ikegaya: "Interaction of atrial muscarinic receptors with three kinds of GTP-binding proteins." Journal Molecular Cellular Cardiology. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] T.Haga: "Muscarinic acetylcholine receptors in porcine atrial membranes interact with three kinds of G proteins." Japanese Circulation Journal. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] G.Berstein: "Molecular aspects of muscarinic receptors.In Current Aspects in the Neurosciences,vol.1,ed.by N.Osborne,pp 245-284." MacMillan Press,London, (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] T.Haga: "Molecular characterization of muscarinic acetylcholine receptors.In Physiology and pharmacology of transmembrane signalling ed by T.Segawa,M.Endoh,M.Ui and K.Kurihara,pp,105-113" Elsevier Science Publishers B.V., (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] T.Haga: Trends in Pharmac Sci.Supplement III. 12-18 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] G.Berstein: J.Neurochem. 50. 1687-1694 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] K.Haga: Mol.Pharmac.(1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] K.Haga: J.Biol.Chem.in press.

    • Related Report
      1987 Annual Research Report
  • [Publications] T.Haga;eds.S.Kito et al.: in "Neuroreceptor and signal transduction"(eds.S.Kito et al.)Plenum Publishing Co.in press.

    • Related Report
      1987 Annual Research Report

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Published: 1987-04-01   Modified: 2016-04-21  

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