Phosphorylation of a phosphoinositidase C-linked muscarinic receptor by a novel kinase distinct from β-adrenergic receptor kinase

AbstractMuscarinic receptor kinase activity previously described in intact CHO cells transfected with human m3-muscarinic receptor cDNA (CHO-m3 cells) [Tobin, A.B and Nahorski, S.R (1993) J. Biol. Chem. 268,9817\3-9823] was found to be associated, at least in part, with a crude membrane fraction of CHO-m3 cell lysates. Phosphorylation of the m3-muscarinic receptor was agonist dependent, reaching a maximum after 10 min exposure to carbachol (1 mM) and was completely blocked by atropine (10μM). m3-Muscarinic receptor phosphorylation was insensitive to Zn2+ (0.1 mM) and heparin (1 μgml), concentrations that inhibit endogenous β-adrenergic receptor kinase activity present in CHO-m3 cells strongly suggesting that the m3-muscarinic receptor kinase is distinct fromβ-adrenergic receptor kinase. A role for protein kinase C can also be eliminated on the basis that the potent protein kinase C inhibitor, Ro-318220 (1μM), had no effect on agonist-mediated m3-muscarinic receptor phosphorylation. Further, the inability of calcium (300μM), cAMP (0.2 mM) and cGMP (0.2 mM) to elevate the basal phosphorylation state of m3-muscarinic receptors eliminates a role for protein kinases regulated by these second messengers. Finally, agonist mediated phosphorylation appears to be independent of G-protein activation as both GDP-β-S (500μM) and GTP-γ-S (100μM) did not influence m3-muscarinic receptor phosphorylation