Regulation of G protein-coupled Receptor Kinase 5 (GRK5) by Actin

doi:10.1074/jbc.273.32.20653

Regulation of G protein-coupled Receptor Kinase 5 (GRK5) by Actin*

From the ^§Howard Hughes Medical Institute and Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and ^¶The Salk Institute, La Jolla, California 92037

Abstract

G protein-coupled receptor kinases (GRKs) initiate pathways leading to the desensitization of agonist-occupied G-protein-coupled receptors (GPCRs). Here we report that the cytoskeletal protein actin binds and inhibits GRK5. Actin inhibits the kinase activity directly, reducing GRK5-mediated phosphorylation of both membrane-bound GPCRs and soluble substrates. GRK5 binds actin monomers with a K _d of 0.6 μm and actin filaments with a K _d of 0.2 μm. Mutation of 6 amino acids near the amino terminus of GRK5 eliminates actin-mediated inhibition of GRK5. Calmodulin has previously been shown to bind to the amino terminus of GRK5 (Pronin, A. N., and Benovic, J. L. (1997) J. Biol. Chem. 272, 3806–3812) and here we show calmodulin displaces GRK5 from actin. Calmodulin inhibits GRK5-mediated phosphorylation of GPCRs, but not soluble substrates such as casein. Thus in the presence of actin, calmodulin determines the substrate specificity of GRK5 by preferentially allowing phosphorylation of soluble substrates over membrane-bound substrates.

Footnotes

↵* This work was supported by National Institutes of Health Grants HL 16737 (to R. J. L.) and GM26338 (to T. D. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ These authors contributed equally to the results of this work.
↵‖ To whom correspondence should be addressed: Howard Hughes Medical Institute and Depts. of Medicine and Biochemistry, Duke University Medical Center, Box 3821, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875; E-mail: lefko001{at}mc.duke.edu.
Abbreviations:

GRK

G protein-coupled receptor kinase

GPCR

G protein-coupled receptor

FI

fluorescence intensity

NTPB

NH₂-terminal polybasic

CTPB

COOH-terminal polybasic

MARCKS

myristoylated alanine-rich PKC substrate

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis.
- Received February 24, 1998.
- Revision received June 2, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.