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Structure, function and regulation of muscarinic acetylcholine receptors

毒蕈碱乙酰胆碱受体的结构、功能和调节

基本信息

批准号：
62490008
负责人：
HAGA Tatsuya
金额：
$ 3.78万
依托单位：
The University of Tokyo (1988-1989)Hamamatsu University School of Medicine (1987)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1989
项目状态：
已结题

项目摘要

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

以猪大脑或心房纯化的毒蕈碱乙酰胆碱受体，并用猪大脑纯化的G蛋白重构受体，研究毒蕈碱乙酰胆碱受体的结构、功能和调控。 1．毒蕈碱受体的结构和配体结合特性：（1）受体与毒蕈碱配体和G蛋白的相互作用不受糖苷内切酶F去除糖的影响。（2）[^3H]丙基苄胆碱芥子的结合位点位于包括第二和第三假定跨膜片段和磷酸化位点的区域。 C 末端 12-14 kDa 肽上的蛋白激酶 C。 (3)第二和第三胞外环之间存在S-S键，并且S-S键的减少导致对毒蕈碱配体的亲和力降低。用 DTNB 或 NEM 烷基化导致对毒蕈碱激动剂的亲和力增加。 (4) 对哌仑西平具有不同亲和力的亚型特异性辅酶信息表明是在 Qf 特异性脂质存在的情况下获取的。2．受体和 G 蛋白之间的相互作用：大脑和心房纯化的受体均与纯化的三种不同 G 蛋白（Gi、Go 和 Gn）之一相互作用，在重构的脂质囊泡中具有相似的效力。使用心房膜代替纯化受体作为受体来源也得到了类似的结果。 3．毒蕈碱受体的磷酸化：脑而非心房毒蕈碱受体被蛋白激酶 C 磷酸化。心房受体是比脑受体更好的 cAMP 依赖性蛋白激酶底物。根据激动剂的存在磷酸化毒蕈碱受体的激酶是从猪大脑中部分纯化的。大脑和心房受体均被激酶磷酸化。当毒蕈碱受体用G蛋白重建然后进行磷酸化时，磷酸化被抑制，并且通过添加GTP或GDP来恢复抑制。较少的