| Project/Area Number |
06558107
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 試験 |
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| Research Field |
Neuroscience in general
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| Research Institution | University of Tokyo |
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Principal Investigator |
HAGA Tatsuya Tokyo Univ., Fac. Medicine Professor, 医学部(医), 教授 (30011646)
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| 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)
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| Project Period (FY) |
1994 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥17,700,000 (Direct Cost: ¥17,700,000)
Fiscal Year 1996: ¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 1995: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1994: ¥9,300,000 (Direct Cost: ¥9,300,000)
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| Keywords | Receptor / Conformation / Large-Scale Purification / Transmitter / TRNOE / G Protein / Acetylcholine / Muscarine / レセプター / 伝達物質 |
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| 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.
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