Localization of the sites for Ca2+-binding proteins on G protein-coupled receptor kinases.
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.
Mesh Terms:
Amino Acid Sequence, Animals, Binding Sites, Binding, Competitive, Calcium-Binding Proteins, Calmodulin, Cattle, Cyclic AMP-Dependent Protein Kinases, Eye Proteins, G-Protein-Coupled Receptor Kinase 1, G-Protein-Coupled Receptor Kinase 5, GTP-Binding Proteins, Hippocalcin, Humans, Lipoproteins, Molecular Sequence Data, Nerve Tissue Proteins, Peptide Fragments, Protein Binding, Protein Kinase Inhibitors, Protein Kinases, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Receptor Protein-Tyrosine Kinases, Recoverin, beta-Adrenergic Receptor Kinases
Amino Acid Sequence, Animals, Binding Sites, Binding, Competitive, Calcium-Binding Proteins, Calmodulin, Cattle, Cyclic AMP-Dependent Protein Kinases, Eye Proteins, G-Protein-Coupled Receptor Kinase 1, G-Protein-Coupled Receptor Kinase 5, GTP-Binding Proteins, Hippocalcin, Humans, Lipoproteins, Molecular Sequence Data, Nerve Tissue Proteins, Peptide Fragments, Protein Binding, Protein Kinase Inhibitors, Protein Kinases, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Receptor Protein-Tyrosine Kinases, Recoverin, beta-Adrenergic Receptor Kinases
Biochemistry
Date: Sep. 29, 1998
PubMed ID: 9753452
View in: Pubmed Google Scholar
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