PHOSPHORYLATION OF A PHOSPHOINOSITIDASE C-LINKED MUSCARINIC RECEPTOR BY A NOVEL KINASE DISTINCT FROM BETA-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, 9817-9823] 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 (10 mu M). m3-Muscarinic receptor phosphorylation was insensitive to Zn2+ (0.1 mM) and heparin (1 mu g/ml), concentrations that inhibit endogenous beta-adrenergic receptor kinase activity present in CHO-m3 cells strongly suggesting that the m3-muscarinic receptor kinase is distinct from beta-adrenergic receptor kinase. A role for protein kinase C can also be eliminated on the basis that the potent protein kinase C inhibitor, Re-318220 (1 mu M), had no effect on agonist-mediated m3-muscarinic receptor phosphorylation. Further, the inability of calcium (300 mu M), 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-beta-S (500 mu M) and GTP-gamma-S (100 mu M) did not influence m3-muscarinic receptor phosphorylation.