Heterologous Activation of Protein Kinase C Stimulates Phosphorylation of δ-Opioid Receptor at Serine 344, Resulting in β-Arrestin- and Clathrin-mediated Receptor Internalization

Bin Xiang,Guo-Hua Yu,Jun Guo,Li Chen,Wei Hu,Gang Pei,Lan Ma

doi:10.1074/jbc.m006187200

Abstract

The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor phosphorylation, indicating that PKC-mediated phosphorylation occurs at Ser-344. PKC-mediated Ser-344 phosphorylation was also induced by activation of G(q)-coupled alpha(1A)-adrenergic receptor or increase in intracellular Ca(2+) concentration. Activation of PKC by PMA, alpha(1A)-adrenergic receptor agonist, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism. Further study demonstrated that agonist-dependent G protein-coupled receptor kinase (GRK) phosphorylation sites in DOR are not targets of PKC. Agonist-dependent, GRK-mediated receptor phosphorylation and agonist-independent, PKC-mediated DOR phosphorylation were additive, but agonist-induced receptor phosphorylation could inhibit PKC-catalyzed heterologous DOR phosphorylation and subsequent internalization. These data demonstrate that the responsiveness of opioid receptor is regulated by both PKC and GRK through agonist-dependent and agonist-independent mechanisms and PKC-mediated receptor phosphorylation is an important molecular mechanism of heterologous regulation of opioid receptor functions.

Highlights

The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms
The PKA activator forskolin did not stimulate DOR phosphorylation (Fig. 2). These data indicate that phorbol 12-myristate 13acetate (PMA)-stimulated DOR phosphorylation is mediated by PKC, whereas other protein kinases such as PKA and MAPK are not critically involved
The results show that incubation of PMA followed by DPDPE treatment increased DOR phosphorylation to ϳ2.5fold of phosphorylation stimulated by DPDPE or PMA alone (ϳ10-fold of the basal level), whereas receptor phosphorylation in response to incubation with DPDPE followed by PMA stimulation was not significantly different from that stimulated by DPDPE or PMA alone (Fig. 8D)

Summary

Introduction

The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Agonist-dependent, GRK-mediated receptor phosphorylation and agonist-independent, PKC-mediated DOR phosphorylation were additive, but agonist-induced receptor phosphorylation could inhibit PKC-catalyzed heterologous DOR phosphorylation and subsequent internalization. Experimental results indicate that GPCR kinase (GRK), not PKC and PKA, is the primary protein kinase involved in homologous phosphorylation of opioid receptors stimulated by opioid agonist and plays an important role in agonist-induced homologous desensitization of opioid receptors [1, 6, 9, 10]. It has been observed that chronic opiate treatment strongly increases PKC activity in specific brain regions, and inhibition of PKC activity attenuates the development of opioid tolerance and dependence [22, 23] These data strongly suggest that opioid signaling is regulated heterologously by agonist-independent pathways in vivo and PKC is likely an important mediator

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