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

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 d opioid receptor (DOR) containing six Ser/Thr residues abolished both DPDPEand phorbol 12-myristate 13acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wildtype DOR, whereas S344G substitution blocked PMAinduced receptor phosphorylation, indicating that PKCmediated phosphorylation occurs at Ser-344. PKCmediated Ser-344 phosphorylation was also induced by activation of Gq-coupled a1A-adrenergic receptor or increase in intracellular Ca concentration. Activation of PKC by PMA, a1A-adrenergic receptor agonist, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative b-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a b-arrestinand 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. Opioid receptors are G protein-coupled receptors (GPCR) and include d, k, and m subtypes. Interaction of opioid receptors on the surface of neurons in the central nervous system with endogenous opioid peptides and synthetic alkaloids produces strong analgesic effect, but chronic use of opioid drug results in drug tolerance and dependence. The molecular mechanisms of regulation of the receptor responsiveness and opioid tolerance and dependence are not well understood, although desensitization of opioid receptor has been implicated as one of the major mechanisms. The responsiveness of opioid receptor reduces upon exposure to opioid agonist, and this agonist-dependent desensitization is defined as homologous desensitization of the opioid receptor. Several mechanisms contribute to desensitization of opioid receptors. It has been demonstrated that, following stimulation of opioid agonist, the opioid receptor becomes phosphorylated rapidly (1–4), the phosphorylated receptor uncouples from G proteins and binds to b-arrestins (5, 6), the receptor is subsequently sequestered in an intracellular compartment (7), and even the expression of the opioid receptor is down-regulated (8). Phosphorylation of opioid receptors is the initial step in opioid receptor desensitization, and phosphorylation of d, k, and m subtypes of opioid receptors in response to agonist stimulation has been demonstrated by other laboratories as well as our own (1–4, 9). 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). In addition to agonist-specific receptor desensitization, functions of GPCRs can be regulated by agonist-independent mechanisms, namely, heterologous desensitization. Second messenger-dependent protein kinases such as PKA and PKC mediate receptor phosphorylation, and this has been implicated in heterologous regulation of activities of a number of GPCRs recently (11, 12). Accumulating evidence suggest that the sensitivity of opioid receptor in response to neural signals is