title: Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2.
abstract: G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.
authors.affiliation: Department of Biochemistry, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: C V
authors.initials: CV
authors.lastname: Carman
authors.forename: J L
authors.initials: JL
authors.lastname: Parent
authors.forename: P W
authors.initials: PW
authors.lastname: Day
authors.forename: A N
authors.initials: AN
authors.lastname: Pronin
authors.forename: P M
authors.initials: PM
authors.lastname: Sternweis
authors.forename: P B
authors.initials: PB
authors.lastname: Wedegaertner
authors.forename: A G
authors.initials: AG
authors.lastname: Gilman
authors.forename: J L
authors.initials: JL
authors.lastname: Benovic
authors.forename: T
authors.initials: T
authors.lastname: Kozasa
mesh_terms: D017607:Aluminum Compounds / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology; D054770:G-Protein-Coupled Receptor Kinase 3; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D054799:Phospholipase C beta; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014020:Tissue Extracts / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D017607:Aluminum Compounds; D007527:Isoenzymes; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; D014020:Tissue Extracts; C050992:tetrafluoroaluminate; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human; D051552:beta-Adrenergic Receptor Kinases; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D005459:Fluorides