Structure, function and regulation of muscarinic acetylcholine receptors

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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