title: Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases.
abstract: The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.
authors.affiliation: Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Daaka
authors.forename: J A
authors.initials: JA
authors.lastname: Pitcher
authors.forename: M
authors.initials: M
authors.lastname: Richardson
authors.forename: R H
authors.initials: RH
authors.lastname: Stoffel
authors.forename: J D
authors.initials: JD
authors.lastname: Robishaw
authors.forename: R J
authors.initials: RJ
authors.lastname: Lefkowitz
mesh_terms: D000818:Animals; D019556:COS Cells; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D054770:G-Protein-Coupled Receptor Kinase 3; D054774:G-Protein-Coupled Receptor Kinase 5; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008246:Lysophospholipids / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D020794:Receptor Protein-Tyrosine Kinases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D008246:Lysophospholipids; D010446:Peptide Fragments; D018179:Receptors, Thrombin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C082835:thrombin receptor peptide (42-47); D005982:Glutathione Transferase; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; D051552:beta-Adrenergic Receptor Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; D019204:GTP-Binding Proteins; D007545:Isoproterenol