The amino-terminal domain of G-protein-coupled receptor kinase 2 is a regulatory Gbeta gamma binding site

J Biol Chem. 2003 Mar 7;278(10):8052-7. doi: 10.1074/jbc.M204795200. Epub 2002 Dec 16.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cell Line
  • Cyclic AMP-Dependent Protein Kinases / chemistry
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • GTP-Binding Proteins / metabolism*
  • Humans
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • Transfection
  • beta-Adrenergic Receptor Kinases

Substances

  • Recombinant Proteins
  • Cyclic AMP-Dependent Protein Kinases
  • beta-Adrenergic Receptor Kinases
  • GTP-Binding Proteins