PHOSPHORYLATION OF BRAIN MUSCARINIC ACETYLCHOLINE-RECEPTORS AND G-PROTEINS - EFFECTS OF A COPURIFIED AGONIST-DEPENDENT MEMBRANE-PROTEIN KINASE AND PROTEIN-KINASE-C

Muscarinic acetylcholine receptors(mAChRs) and guanine nucleotide binding proteins(G proteins) were partially purified from plasma membranes and subcellular fractions of control and carbachol treated rat brain by affinity chromatography with ABT and heptylamine, respectively. A membrane associated protein kinase was co-purified with the mAChRs. Phosphorylation of mAChRs by this kinase is both time and concentration dependent, stimulated by mAChR agonists acetylcholine and carbachol and low concentrations of G(O), and inhibited by mAChR antagonist atropine and tau-S-GTP. Both mAChRs and G proteins were phosphorylated with protein kinase C. The membrane protein kinase and protein kinase C also phosphorylated a 110 kDa polypeptide co-purified with G proteins by heptylamine chromatography. The 110 kDa polypeptide was dually labelled with [H-3]-PrBCM, a mAChR-binding ligand, and [tau-S-35]-GTP, a G protein-binding ligand, and was further identified by photoaffinity labelling with [tau-P-32]-8-azido-GTP, Treatment with carbachol increased the distribution of mAChRs and G(alpha) in light membrane fractions, indicating translocation of both of these proteins.