Structure, function and regulation of muscarinic acetylcholine receptors

从猪脑或心房中提纯的乙酰胆碱受体与从猪大脑中提纯的G蛋白重组后，对M胆碱受体的结构、功能和调节进行了研究。M受体的结构和配基结合特性：(1)受体与M配体和G蛋白的相互作用不受内切糖苷酶F脱糖的影响。(2)[~(3 H)]丙基苄基胆碱芥末的结合部位位于第二和第三跨膜片段以及C末端12-14 kDa多肽上的蛋白激酶C的磷酸化部位。(3)细胞外第二环和第三环之间存在S-S键，而S-S键(S)的减少导致与M配体的亲和力降低。与DTNB或NEM的烷基化反应增加了对M受体激动剂的亲和力。(4)亚型特异性co…在存在QF特异性脂类的情况下，哌仑西平具有不同亲和力的信息更多。受体与G蛋白的相互作用：在重组脂泡中，脑型和心房型纯化的受体都能与纯化的三种不同G蛋白(Gi、Go和Gn)中的一种相互作用。用心房膜代替纯化的受体作为受体的来源也得到了类似的结果。M受体的磷酸化：脑型而不是心房型M受体被蛋白激酶C磷酸化，心房受体是cAMP依赖的蛋白激酶的更好底物。一种依赖于激动剂存在而使M受体磷酸化的激酶是从猪大脑中部分纯化出来的。脑型和心房型受体均被该激酶磷酸化。M受体与G蛋白重组后磷酸化被抑制，加入GTP或GDP可恢复这种抑制作用。较少

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