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

: Phosphorylation of specific amino acid residues isbelieved to be crucial for the agonist‐induced regulation of several Gprotein‐coupled receptors. This is especially true for the three types ofopioid receptors (μ, δ, and α), which contain consensus sitesfor phosphorylation by numerous protein kinases. Protein kinase C (PKC) hasbeen shown to catalyze the in vitro phosphorylation of μ‐ andδ‐opioid receptors and to potentiate agonist‐induced receptordesensitization. In this series of experiments, we continue our investigationof how opioid‐activated PKC contributes to homologous receptor down‐regulationand then expand our focus to include the exploration of the mechanism(s) bywhich μ‐opioids produce PKC translocation in SH‐SY5Y neuroblastoma cells.[ d Ala 2 , N ‐Me‐Phe 4 ,Gly‐ol]enkephalin(DAMGO)‐induced PKC translocation follows a time‐dependent and biphasicpattern beginning 2 h after opioid addition, when a pronounced translocationof PKC to the plasma membrane occurs. When opioid exposure is lengthened to>12 h, both cytosolic and particulate PKC levels drop significantly belowthose of control‐treated cells in a process we termed “reversetranslocation.” The opioid receptor antagonist naloxone, the PKCinhibitor chelerythrine, and the L‐type calcium channel antagonist nimodipineattenuated opioid‐mediated effects on PKC and μ‐receptor down‐regulation,suggesting that this is a process partially regulated byCa 2+ ‐dependent PKC isoforms. However, chronic exposure to phorbolester, which depletes the cells of diacylglycerol (DAG) andCa 2+ ‐sensitive PKC isoforms, before DAMGO exposure, had no effecton opioid receptor down‐regulation. In addition to expressing conventional(PKC‐α) and novel (PKC‐ε) isoforms, SH‐SY5Y cells also contain aDAG‐and Ca 2+ ‐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.