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MBC in Press, published online ahead of print April 20, 2005
Mol. Biol. Cell 10.1091/mbc.E04-10-0877

A more recent version of this article appeared on July 1, 2005
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Submitted on October 7, 2004
Revised on March 30, 2005
Accepted on April 8, 2005

G Protein-coupled Receptor Kinase 2-mediated Phosphorylation of Ezrin Is Required for G Protein-coupled Receptor-dependent Reorganization of the Actin Cytoskeleton

Sarah H. Cant and Julie A. Pitcher

MRC Laboratory for Molecular and Cellular Biology and Department of Pharmacology, University College London, London, WC1E 6BT United Kingdom

Monitoring Editor: Anthony Bretscher

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 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 ERM phosphorylation serves to link GPCR activation to cytoskeletal reorganisation. 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 (PKC), prevents ERM phosphorylation and membrane ruffling. Second, agonist-induced internalisation of the {beta}2-adrenergic receptor ({beta}2AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized {beta}2AR and phosphorylated ERM is not dependent on Na+/H+ exchanger regulatory factor (NHERF)-binding to the {beta}2AR. Inhibition of ezrin function impedes {beta}2AR internalisation, 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.

Address correspondence to: Julie A. Pitcher (julie.pitcher{at}ucl.ac.uk)






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