RAPID AGONIST-MEDIATED PHOSPHORYLATION OF M3-MUSCARINIC-RECEPTORS REVEALED BY IMMUNOPRECIPITATION

A specific antiserum against the human m3-muscarinic receptor subtype was made by subcloning a variant region of the third intracellular loo p of the m3-receptor (Ser345-Leu463) into a bacterial expression plasm id that produced a fusion protein with glutathione S-transferase. In i mmunoblot studies this antiserum identified the human m3-receptor expr essed in transfected Chinese hamster ovary (CHO) cells (CHO-m3 cells, 1343 fmol/mg protein) as a diffuse band at approximately 97-110 kDa. I n vivo labeling of the ATP pool in CHO-m3 cells with [P-32]orthophosph ate followed by immunoprecipitation of solubilized m3-receptors reveal ed that the unstimulated receptor existed in a phosphorylated form. In cubation of CHO-m3 cells with the cholinergic agonist carbachol (1 mM) increased the phosphorylated state of the receptor dramatically, prim arily at serine. The time course for agonist-dependent phosphorylation was very rapid occurring within seconds of agonist addition and was m aintained for at least 30 min. The muscarinic antagonist atropine (10 muM) inhibited agonist-stimulated phosphorylation. Neither forskolin ( 10 muM) nor the calcium ionophore, ionomycin (1 muM), had any effect o n the state of phosphorylation of the m3-receptor, eliminating a role for cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protei n kinase in the agonist-dependent phosphorylation of m3-receptors. 4Be ta-phorbol 12beta-myristate 13alpha-acetate (100 nM) did increase m3-r eceptor phosphorylation, an effect that was inhibited by the selective protein kinase C inhibitor RO-318220 (10 muM). However, agonist-stimu lated m3-receptor phosphorylation was not inhibited by RO-318220 indic ating that protein kinase C was not involved in agonist-induced m3-rec eptor phosphorylation. In conclusion the phosphorylation of m3-recepto rs, in vivo, was increased following the application of muscarinic ago nist or PMA. The response to agonist was mediated via a kinase distinc t from protein kinase C, protein kinase A and Ca2+/calmodulin dependen t protein kinase, whereas the effect of 4beta-phorbol 12beta-myristate 13alpha-acetate was mediated by protein kinase C.