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.
Levay
K
K
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33136, USA.
Satpaev
D K
DK
Pronin
A N
AN
Benovic
J L
JL
Slepak
V Z
VZ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D054774:G-Protein-Coupled Receptor Kinase 5; D019204:GTP-Binding Proteins / Q000378:metabolism*; D051597:Hippocalcin; D006801:Humans; D008074:Lipoproteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051603:Recoverin; D051552:beta-Adrenergic Receptor Kinases
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; C496731:RCVRN protein, human; D051603:Recoverin; D051597:Hippocalcin; D011494:Protein Kinases; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; C496764:GRK1 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human; D019204:GTP-Binding Proteins