title: Regulation of G protein-coupled receptor kinases by calmodulin and localization of the calmodulin binding domain.
abstract: G protein-coupled receptor kinases (GRKs) specifically phosphorylate and regulate the activated form of multiple G protein-coupled receptors. Recent studies have revealed that GRKs are also subject to regulation. In this regard, GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Here we demonstrate that calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5 (IC50 approximately 50 nM) > GRK6 >> GRK2 (IC50 approximately 2 microM) >> GRK1. Calmodulin inhibition of GRK5 is mediated via a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Moreover, calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor even in the absence of calmodulin. Using glutathione S-transferase-GRK5 fusion proteins either to inhibit calmodulin-stimulated autophosphorylation or to bind directly to calmodulin, we determined that an amino-terminal domain of GRK5 (amino acids 20-39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues, characteristics typical of calmodulin binding sites, and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity.
authors.affiliation: Department of Biochemistry, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: A N
authors.initials: AN
authors.lastname: Pronin
authors.forename: D K
authors.initials: DK
authors.lastname: Satpaev
authors.forename: V Z
authors.initials: VZ
authors.lastname: Slepak
authors.forename: J L
authors.initials: JL
authors.lastname: Benovic
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D002147:Calmodulin; C092794:GPR3 protein, human; D008081:Liposomes; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D012243:Rhodopsin; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C517898:GRK5 protein, human; D019204:GTP-Binding Proteins