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 cell s) [Tobin,A.B and Nahorski,S.R (1993) J. Biol. Chem. 268, 9817-9823] w as found to be associated, at least in part, with a crude membrane fra ction of CHO-m3 cell lysates. Phosphorylation of the m3-muscarinic rec eptor 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 bet a-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 rece ptor phosphorylation. Further, the inability of calcium (300 mu M), cA MP (0.2 mM) and cGMP (0.2 mM) to elevate the basal phosphorylation sta te of m3-muscarinic receptors eliminates a role for protein kinases re gulated by these second messengers. Finally, agonist mediated phosphor ylation 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-musc arinic receptor phosphorylation.