Structure, function and regulation of muscarinic acetylcholine receptors

从猪大脑或心房中纯化毒蕈碱类乙酰胆碱受体，用从猪大脑中纯化的G蛋白进行重组，研究毒蕈碱类乙酰胆碱受体的结构、功能和调控。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f内糖苷酶去除糖的影响。(2)[^3H]丙基苄胆碱芥菜的结合位点位于包括第二和第三推测的跨膜段和蛋白激酶C在C端12-14 kDa肽上的磷酸化位点。(3)第二和第三胞外环之间存在s - s键，s - s键的减少导致对毒菌碱配体的亲和力降低。与DTNB或NEM的烷基化导致对毒蕈碱激动剂的亲和力增加。(4)在特异性脂质存在的情况下，可以获得更多的对吡仑西平具有不同亲和力的信息。受体和G蛋白之间的相互作用：大脑和心房纯化受体与三种纯化的G蛋白（Gi， Go和Gn）中的一种相互作用，在重构的脂质囊泡中具有相似的效力。使用心房膜作为受体来源而不是纯化受体，也获得了类似的结果。毒蕈碱受体的磷酸化：脑毒蕈碱受体可被蛋白激酶c磷酸化，而心房毒蕈碱受体未被蛋白激酶c磷酸化。心房受体是camp依赖性蛋白激酶较好的底物。一种依赖于激动剂存在而使毒蕈碱受体磷酸化的激酶从猪大脑中部分纯化。脑和心房受体均被该激酶磷酸化。用G蛋白重组毒蕈碱受体后磷酸化抑制，加入GTP或GDP后抑制恢复。少

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