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Vol. 19, Issue 7, 2973-2983, July 2008

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EGF Transregulates Opioid Receptors through EGFR-mediated GRK2 Phosphorylation and Activation

Pharmacology Research Center and State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai 200032, China

Submitted October 22, 2007; Revised March 26, 2008; Accepted April 28, 2008
Monitoring Editor: Richard Assoian

G protein–coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR function. Here we demonstrate that activation of epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase family, stimulates GRK2 activity and transregulates the function of G protein–coupled opioid receptors. Our data showed that EGF treatment promoted DOR internalization induced by DOR agonist and this required the intactness of GRK2-phosphorylation sites in DOR. EGF stimulation induced the association of GRK2 with the activated EGFR and the translocation of GRK2 to the plasma membrane. After EGF treatment, GRK2 was phosphorylated at tyrosyl residues. Mutational analysis indicated that EGFR-mediated phosphorylation occurred at GRK2 N-terminal tyrosyl residues previously shown as c-Src phosphorylation sites. However, c-Src activity was not required for EGFR-mediated phosphorylation of GRK2. In vitro assays indicated that GRK2 was a direct interactor and a substrate of EGFR. EGF treatment remarkably elevated DOR phosphorylation in cells expressing the wild-type GRK2 in an EGFR tyrosine kinase activity–dependent manner, whereas EGF-stimulated DOR phosphorylation was greatly decreased in cells expressing mutant GRK2 lacking EGFR tyrosine kinase sites. We further showed that EGF also stimulated internalization of µ-opioid receptor, and this effect was inhibited by GRK2 siRNA. These data indicate that EGF transregulates opioid receptors through EGFR-mediated tyrosyl phosphorylation and activation of GRK2 and propose GRK2 as a mediator of cross-talk from RTK to GPCR signaling pathway.

* These authors contributed equally to this work.

Address correspondence to: Lan Ma (lanma{at}shmu.edu.cn)

Abbreviations used: DPDPE, D-Pen2, D-Pen5 enkephalin; GRKs, G protein–coupled receptor kinases; DOR, -opioid receptor; M4/5/6, a DOR mutant lacks GRK2 phosphorylation sites; MOR, µ-opioid receptor; EGFR, epidermal growth factor receptor; GST-EGFR, GST-tagged EGFR kinase domain; HEK293, human embryonic kidney 293 cells.

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