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

Muscarinic 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 ), 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.