title: Phosphorylation of a phosphoinositidase C-linked muscarinic receptor by a novel kinase distinct from beta-adrenergic receptor kinase.
abstract: 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 microM). m3-Muscarinic receptor phosphorylation was insensitive to Zn2+ (0.1 mM) and heparin (1 microgram/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, Ro-318220 (1 microM), had no effect on agonist-mediated m3-muscarinic receptor phosphorylation. Further, the inability of calcium (300 microM), 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 microM) and GTP-gamma-S (100 microM) did not influence m3-muscarinic receptor phosphorylation.
authors.affiliation: Leiscester University, Department of Cell Physiology and Pharmacology, UK.
authors.forename: A B
authors.initials: AB
authors.lastname: Tobin
authors.forename: B
authors.initials: B
authors.lastname: Keys
authors.forename: S R
authors.initials: SR
authors.lastname: Nahorski
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015290:Second Messenger Systems; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D011976:Receptors, Muscarinic; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051552:beta-Adrenergic Receptor Kinases; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase