Interaction between the conserved region in the C-terminal domain of GRK2 and rhodopsin is necessary for GRK2 to catalyze receptor phosphorylation

J Biol Chem. 2000 Mar 24;275(12):8469-74. doi: 10.1074/jbc.275.12.8469.

Abstract

The C-terminal domain of G protein-coupled receptor kinases (GRKs) consists of a conserved region and a variable region, and the variable region has been shown to direct the membrane translocation of cytosolic enzymes. The present work has revealed that the C-terminal domain may also be involved in kinase-receptor interaction that is primarily mediated by the conserved region. Truncation of the C-terminal domain or deletion of the conserved region in this domain of GRK2 resulted in a complete loss of its ability to phosphorylate rhodopsin and in an obvious decrease in its sensitivity to receptor-mediated phosphorylation of a peptide substrate. On the contrary, deletion of the betagamma subunit binding region in the C-terminal domain of GRK2 did not significantly alter the ability of the enzyme to phosphorylate rhodopsin. In addition, the recombinant proteins that represent the C-terminal domain and the conserved region of GRK2 could inhibit GRK2-mediated phosphorylation of rhodopsin and receptor-mediated activation of GRK2 but not GRK2-mediated phosphorylation of the peptide substrate. Furthermore, the conserved region as well as the C-terminal domain could directly bind rhodopsin in vitro. These results indicate that the C-terminal domain, or more precisely, the conserved region of this domain, is important for enzyme-receptor interaction and that this interaction is required for GRK2 to catalyze receptor phosphorylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adrenergic beta-Antagonists
  • Amino Acid Sequence
  • Animals
  • Cattle
  • Conserved Sequence*
  • Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Escherichia coli / genetics
  • G-Protein-Coupled Receptor Kinase 2
  • Mutagenesis
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Phosphorylation
  • Protein Binding
  • Receptors, Adrenergic, beta / genetics
  • Receptors, Adrenergic, beta / metabolism*
  • Recombinant Proteins / metabolism
  • Rhodopsin / metabolism*
  • Sequence Deletion
  • beta-Adrenergic Receptor Kinases
  • rho GTP-Binding Proteins / metabolism

Substances

  • Adrenergic beta-Antagonists
  • Peptide Fragments
  • Receptors, Adrenergic, beta
  • Recombinant Proteins
  • Rhodopsin
  • Cyclic AMP-Dependent Protein Kinases
  • GRK2 protein, human
  • beta-Adrenergic Receptor Kinases
  • G-Protein-Coupled Receptor Kinase 2
  • rho GTP-Binding Proteins