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1996 Fiscal Year Final Research Report Summary

Conformation of the Muscarinic Acetylcholine Receptor

Research Project

Project/Area Number 06558107
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section試験
Research Field Neuroscience in general
Research InstitutionUniversity of Tokyo

Principal Investigator

HAGA Tatsuya  Tokyo Univ., Fac. Medicine Professor, 医学部(医), 教授 (30011646)

Co-Investigator(Kenkyū-buntansha) YOKOYAMA Shigeyuki  Tokyo Univ., Fac. Science Professor, 理学系研究科, 教授 (00159229)
TOYOSHIMA Chikasi  Tokyo Univ., Inst. Mol. Cell. Biol. Professor, 分子細胞生物学研究所, 教授 (70172210)
FUJISAWA Tadami  Tsumura Co. Ltd., Devel. Res. Section Chief, 企画開発本部, 課長
SASAKI Hiromi  Tsumura Co. Ltd., Devel. Res. Dep. Head, 企画開発本部, 部長
KAMEYAMA Kimihiko  Tokyo Univ., Fac. Medicine, Associate, 医学部, 助手 (50224697)
Project Period (FY) 1994 – 1996
KeywordsReceptor / Conformation / Large-Scale Purification / Transmitter / TRNOE / G Protein / Acetylcholine / Muscarine
Research Abstract

We have developed a large-scale expression system of a m2 muscarinic receptor mutant using the baculovirus-Sf9 cells and a simple purification system of the m2 receptor using Co^<2+>-chelating column. We have made different kinds of m2 receptor mutants and checked for their expression level, stability and ability to interact with G proteins. We chose a mutant with (1) substitution of Asn by Asp in order to avoid glycosylation, (2) deletion of the central part of the third intracellular loop to avoid proteolysis, and (3) addition of six histidine tag at the carboxy terminus in order to be used for purification with Co^<2+>-column. We could obtain approximately 1 mg of the purified m2 receptor mutant every month. The receptor was used for two-dimensional crystallization and for measurements of Transfer Nuclear Overhauser Effect of methacholine (S (+)-O-acetyl-beta-methylcholine) bound to the m2 receptor. We could observe a putative one-dimensional array of the receptor but have not yet succeeded in obtaining two-dimensional crystal. We could obtain evidence that the C_<alpha>-C_<beta> bond of methacholine bound to the m2 receptor takes the trans conformation, whereas the bond of free methacholine does the gauche conformation.
In the course of this project, we have found that (1) the five subtypes of muscarinic receptors (m1-m5) could be expressed in the baculovirus-Sf9 system, enabling us to examine their ligand binding properties and capability to be solubilized in active forms, (2) most of competitive ligands bind to five subtypes with similar affinities, whereas allosteric ligands, including a newly found alpha-lapachone, bind in a subtype-specific manner, (3) a palmitoyl residue is present in the carboxyl terminus of the m2 receptor, and depalmitoylation attenuates the ability of the m2 receptor to activate G proteins.

  • Research Products

    (16 results)

All Other

All Publications (16 results)

  • [Publications] Rinken,A.: "Solubilization of muscarinic receptor subtypes from baculovirus infected Sf9 insect cells." Biochem.Pharmacol.48. 1245-1251 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Guo,Z.D.: "Ligand binding properties of muscarinic acetylcholine receptor subtypes (m1-m5) expressed in baculovirus-infected insect cells." J.Pharmacol.Exp.Then.274. 378-384 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Guo,Z.D.: "Allosteric binding of 9-methoxy-alpha-lapachone and alcuronium to the muscarinic acetylcholine receptor m2 subtype." Biomed.Res,. 16. 327-335 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nakamura,F.: "Characterization of Gq family G proteins GL1alpha (G14alpha),GL2alpha (G11alpah),and Gqalpha expressed in the baculovirus-insect cell system." J,Biol.Chem.270. 6246-6253 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hayashi,M.K.: "Purification and functional reconstitution with GTP-binding regulatory prote-ins of hexahistidine-tagged muscarinic acetylcholine receptors (m2 subtype)." J.Biochem.120. 1232-1238 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hayashi,M.K.: "Palmitoylation of muscarinic acetylcholine receptor m2 subtypes:" Archiv.Biochem.Biophys.(in press). (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Haga,T.: "Phosphorylation and sequestration of muscarinic receptors. In : Molecular mechanisms of muscarinic acetylcholine receptor function" edited by Wess,J.Austin : CRC Press,1995,p.227-245., 19 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Haga,T.: "Receptor Biochemistry. In : Encyclopedia of Molecular Biology and Molecular Medicine,Volume 5" edited by Meyers,R.A.Weiheim,New York : VCH,1997,p.229-243., 15 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Rinken, A., K.Kameyama, T.Haga and L.Engstrom.: "Solubilization of muscarinic receptor subtypes from baculovirus infected Sf9 insect cells." Biochem. Pharmacol.48. 1245-1251 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Guo, Z.D., K.Kameyama, A.Rinken and T.Haga.: "Ligand binding properties of muscarinic acetylcholine receptor subtypes (m1-m5) expressed in baculovirus-infected insect cells." J.Pharmacol. Exp. Ther.274. 378-384 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Guo, Z.D., Haga, H.Itokawa and F.Mizobe.: "Allosteric binding of 9-methoxy-alpha-lapachone and alcuronium to the muscarinic acetylcholine receptor m2 subtype." Biomed. Res.16. 327-335 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nakamura, F., M.Kato, K.Kameyama, T.Nukaba, T.Haga, H.Kato, T.Takenawa and U.Kikkawa.: "Characterization of Gq family G proteins GL 1alpha (G14alpha), GL2alpha (G11alpah), and Gqalpha expressed in the baculovirus-insect cell system." J.Biol. Chem.270. 6246-6253 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hayashi, M.K.and T.Haga.: "Purification and functional reconstitution with GTP-binding regulatory prote-ins of hexahistidine-tagged muscarinic acetylcholine receptors (m2 subtype)." J.Biochem.120. 1232-1238 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hayashi, M.K.and T.Haga.: "Palmitoylation of muscarinic acetylcholine receptor m2 subtypes : Reduction in their ability to activate G proteins by mutation of a putative palmitoylation site, cysteine 457, in the carboxyl-terminal tail." Arch. Biochem. Biophys.(in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Haga, T., Haga, K., Kameyama, K.and Tsuga, H.: "Phosphorylation and sequestration of muscarinic receptors." In : Molecular mechanisms of muscarinic acetylcholine receptor function edited by Wess, J.Austin : CRC Press. 227-245 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Haga, T.: "Receptor Biochemistry." In : Encyclopedia of Molecular Biology and Molecular Medicine, Volume 5 edited by Meyers, R.A.Weiheim, New York : VCH. 229-243 (1997)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-03-09  

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