Role of Protein Kinase C (PKC) in Agonist‐Induced α‐Opioid Receptor Down‐Regulation

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Abstract
: Phosphorylation of specific amino acid residues is believed to be crucial for the agonist-induced regulation of several G protein-coupled receptors. This is especially true for the three types of opioid receptors (μ, δ, and α), which contain consensus sites for phosphorylation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphorylation of μ- and δ-opioid receptors and to potentiate agonist-induced receptor desensitization. In this series of experiments, we continue our investigation of how opioid-activated PKC contributes to homologous receptor down-regulation and then expand our focus to include the exploration of the mechanism(s) by which μ-opioids produce PKC translocation in SH-SY5Y neuroblastoma cells. [dAla2,N-Me-Phe4,Gly-ol]enkephalin (DAMGO)-induced PKC translocation follows a time-dependent and biphasic pattern beginning 2 h after opioid addition, when a pronounced translocation of PKC to the plasma membrane occurs. When opioid exposure is lengthened to >12 h, both cytosolic and particulate PKC levels drop significantly below those of control-treated cells in a process we termed “reverse translocation.” The opioid receptor antagonist naloxone, the PKC inhibitor chelerythrine, and the L-type calcium channel antagonist nimodipine attenuated opioid-mediated effects on PKC and μ-receptor down-regulation, suggesting that this is a process partially regulated by Ca2+-dependent PKC isoforms. However, chronic exposure to phorbol ester, which depletes the cells of diacylglycerol (DAG) and Ca2+-sensitive PKC isoforms, before DAMGO exposure, had no effect on opioid receptor down-regulation. In addition to expressing conventional (PKC-α) and novel (PKC-ε) isoforms, SH-SY5Y cells also contain a DAG-and Ca2+-independent, atypical PKC isozyme (PKC-ξ), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC-ξ is similarly sensitive to activation by μ-opioids. PKC-ξ translocates from the cytosol to the membrane with kinetics similar to those of PKC-α and ε in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by μ-opioid agonists is involved in the processes that result in μ-receptor down-regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved.