title: Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin.
abstract: Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.
authors.affiliation: Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Japan. haga@m.u-tokyo.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Haga
authors.forename: H
authors.initials: H
authors.lastname: Tsuga
authors.forename: T
authors.initials: T
authors.lastname: Haga
mesh_terms: D000818:Animals; D001285:Atropine / Q000494:pharmacology; D016466:CHO Cells; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology*; D002217:Carbachol / Q000494:pharmacology; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D011487:Protein Conformation; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D013552:Swine; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D002147:Calmodulin; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D000001:Calcimycin; D001285:Atropine; D002217:Carbachol; D012243:Rhodopsin; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D002118:Calcium