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

Konstantin Levay, Daulet K. Satpaev, Alexey N. Pronin, Jeffrey L. Benovic, Vladlen Z. Slepak

Research output: Contribution to journalArticle

64 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)13650-13659
Number of pages10
JournalBiochemistry
Volume37
Issue number39
DOIs
StatePublished - Sep 29 1998

ASJC Scopus subject areas

  • Biochemistry

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