Ca2+-Dependent Inhibition of G Protein-Coupled Receptor Kinase 2 by Calmodulin

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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 βγ subunits and partially inhibited in the presence of βγ subunits. The dose−response curve for stimulation by βγ subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of βγ 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 βγ subunits, but the agonist-dependent stimulation of I3−GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of βγ 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 βγ 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.