Localization of the sites for Ca2+-binding proteins on G protein-coupled receptor kinases

Biochemistry. 1998 Sep 29;37(39):13650-9. doi: 10.1021/bi980998z.

Abstract

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-binding proteins has recently emerged as a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca2+-binding protein recoverin, other GRKs can be inhibited by Ca2+-calmodulin. To dissect the mechanism of this inhibition at the molecular level, we localized the GRK domains involved in Ca2+-binding protein interaction using a series of GST-GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites were localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin had no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin-GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5-calmodulin interaction. While specific mutation within the N-terminal site had no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuated the effect of calmodulin on GRK5, and the simultaneous mutation of both sites rendered the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca2+-dependent regulation of GRKs.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Binding, Competitive / drug effects
  • Calcium-Binding Proteins / antagonists & inhibitors
  • Calcium-Binding Proteins / metabolism
  • Calmodulin / metabolism*
  • Calmodulin / pharmacology
  • Cattle
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Eye Proteins*
  • G-Protein-Coupled Receptor Kinase 1
  • G-Protein-Coupled Receptor Kinase 5
  • GTP-Binding Proteins / metabolism*
  • Hippocalcin
  • Humans
  • Lipoproteins*
  • Molecular Sequence Data
  • Nerve Tissue Proteins*
  • Peptide Fragments / metabolism
  • Protein Binding / drug effects
  • Protein Kinase Inhibitors
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases*
  • Receptor Protein-Tyrosine Kinases / antagonists & inhibitors
  • Receptor Protein-Tyrosine Kinases / genetics
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Recoverin
  • beta-Adrenergic Receptor Kinases

Substances

  • Calcium-Binding Proteins
  • Calmodulin
  • Eye Proteins
  • Lipoproteins
  • Nerve Tissue Proteins
  • Peptide Fragments
  • Protein Kinase Inhibitors
  • RCVRN protein, human
  • Recoverin
  • Hippocalcin
  • Protein Kinases
  • Receptor Protein-Tyrosine Kinases
  • Protein-Serine-Threonine Kinases
  • Cyclic AMP-Dependent Protein Kinases
  • G-Protein-Coupled Receptor Kinase 1
  • GRK1 protein, human
  • beta-Adrenergic Receptor Kinases
  • G-Protein-Coupled Receptor Kinase 5
  • GRK5 protein, human
  • GTP-Binding Proteins