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PMID: 15843435 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganization of the actin cytoskeleton.

Molecular biology of the cell ·Vol. 16 ·No. 7 ·2005-07-00 ·Pages 3088-99

Cant SH, Pitcher JA

Abstract

G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes activated G protein-coupled receptors (GPCRs). Here, we identify ezrin as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates glutathione S-transferase (GST)-ezrin, but not an ezrin fusion protein lacking threonine 567 (T567), in vitro. These results suggest that T567, the regulatory phosphorylation site responsible for maintaining ezrin in its active conformation, represents the principle site of GRK2-mediated phosphorylation. Two lines of evidence indicate that GRK2-mediated ezrin-radixinmoesin (ERM) phosphorylation serves to link GPCR activation to cytoskeletal reorganization. First, in Hep2 cells muscarinic M1 receptor (M1MR) activation causes membrane ruffling. This ruffling response is ERM dependent and is accompanied by ERM phosphorylation. Inhibition of GRK2, but not rho kinase or protein kinase C, prevents ERM phosphorylation and membrane ruffling. Second, agonist-induced internalization of the beta2-adrenergic receptor (beta2AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized beta2AR and phosphorylated ERM is not dependent on Na+/H+ exchanger regulatory factor binding to the beta2AR. Inhibition of ezrin function impedes beta2AR internalization, further linking GPCR activation, GRK activity, and ezrin function. Overall, our results suggest that GRK2 serves not only to attenuate but also to transduce GPCR-mediated signals.

MeSH Terms
Actins/chemistry Animals Binding Sites Cell Line Cell Membrane/metabolism Cytoskeletal Proteins Cytoskeleton/metabolism DNA, Complementary/metabolism Endocytosis Flow Cytometry G-Protein-Coupled Receptor Kinase 2 Glutathione Transferase/metabolism Humans Microscopy, Fluorescence Phosphoproteins/chemistry,metabolism Phosphorylation Protein Binding Receptor, Muscarinic M1/metabolism Receptors, G-Protein-Coupled/metabolism Signal Transduction Time Factors Transfection beta-Adrenergic Receptor Kinases/metabolism,physiology
Chemicals
Actins Cytoskeletal Proteins DNA, Complementary Phosphoproteins Receptor, Muscarinic M1 Receptors, G-Protein-Coupled ezrin Glutathione Transferase GRK2 protein, human beta-Adrenergic Receptor Kinases G-Protein-Coupled Receptor Kinase 2
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Cant Sarah H
MRC Laboratory for Molecular and Cellular Biology and Department of Pharmacology, University College London, London, WC1E 6BT United Kingdom.
Pitcher Julie A
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.e04-10-0877
Published
2005-07-00
Epub
2005-00-20
Pages
3088-99
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC1165394
Subset
IM
Grants
Wellcome Trust · United Kingdom
External Links
PubMed source DOI: 10.1091/mbc.e04-10-0877 PMC Full Text View journal details
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