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Vol. 16, Issue 7, 3088-3099, July 2005

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

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

Submitted October 7, 2004; Revised March 30, 2005; Accepted April 8, 2005
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 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 ₂-adrenergic receptor (₂AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized ₂AR and phosphorylated ERM is not dependent on Na⁺/H⁺ exchanger regulatory factor binding to the ₂AR. Inhibition of ezrin function impedes ₂AR 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.

Abbreviations used: ACh, acetylcholine; ₂AR, ₂-adrenergic receptor; cGMP, cyclic guanosine monophosphate; ERM, ezrin-radixinmoesin; FERM, four-point-one, ezrin-radixin-moesin; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; GST, glutathione S-transferase; Iso, isoproterenol; LDL, low-density lipoprotein; M1MR, M1 muscarinic receptor; M2MR, M2 muscarinic receptor; NHERF, Na⁺/H⁺ exchanger regulatory factor; pERM, threonine-phosphorylated ezrin-radixin-moesin; PH, pleckstrin homology; PIP₂, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; RH, regulator of G protein signaling homology; T567, threonine 567.

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

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