title: The amino-terminal domain of G-protein-coupled receptor kinase 2 is a regulatory Gbeta gamma binding site.
abstract: G-protein-coupled receptor kinase 2 (GRK2) is activated by free Gbetagamma subunits. A Gbetagamma binding site of GRK2 is localized in the carboxyl-terminal pleckstrin homology domain. This Gbetagamma binding site of GRK2 also regulates Gbetagamma-stimulated signaling by sequestering free Gbetagamma subunits. We report here that truncation of the carboxyl-terminal Gbetagamma binding site of GRK2 did not abolish the Gbetagamma regulatory activity of GRK2 as determined by the inhibition of a Gbetagamma-stimulated increase in inositol phosphates in cells. This finding suggested the presence of a second Gbetagamma binding site in GRK2. And indeed, the amino terminus of GRK2 (GRK2(1-185)) inhibited a Gbetagamma-stimulated inositol phosphate signal in cells, purified GRK2(1-185) suppressed the Gbetagamma-stimulated phosphorylation of rhodopsin, and GRK2(1-185) bound directly to purified Gbetagamma subunits. The amino-terminal Gbetagamma regulatory site does not overlap with the RGS domain of GRK-2 because GRK2(1-53) with truncated RGS domain inhibited Gbetagamma-mediated signaling with similar potency and efficacy as did GRK2(1-185). In addition to the Gbetagamma regulatory activity, the amino-terminal Gbetagamma binding site of GRK2 affects the kinase activity of GRK2 because antibodies specifically cross-reacting with the amino terminus of GRK2 suppressed the GRK2-dependent phosphorylation of rhodopsin. The antibody-mediated inhibition was released by purified Gbetagamma subunits, strongly suggesting that Gbetagamma binding to the amino terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a previously unrecognized Gbetagamma binding site that regulates GRK2-mediated receptor phosphorylation and inhibits Gbetagamma-stimulated signaling.
authors.affiliation: Institut für Pharmakologie und Toxikologie, Versbacher Strasse 9, D-97078 Würzburg, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Eichmann
authors.forename: Kristina
authors.initials: K
authors.lastname: Lorenz
authors.forename: Michaela
authors.initials: M
authors.lastname: Hoffmann
authors.forename: Jörg
authors.initials: J
authors.lastname: Brockmann
authors.forename: Cornelius
authors.initials: C
authors.lastname: Krasel
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
authors.forename: Ursula
authors.initials: U
authors.lastname: Quitterer
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins