Role of protein kinase C (PKC) in agonist-induced mu-opioid receptor down-regulation: II. Activation and involvement of the alpha, epsilon, and zeta isoforms of PKC

J Neurochem. 1999 Feb;72(2):594-604. doi: 10.1046/j.1471-4159.1999.0720594.x.


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 (mu, delta, and kappa), which contain consensus sites for phosphorylation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphorylation of mu- and delta-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 mu-opioids produce PKC translocation in SH-SY5Y neuroblastoma cells. [D-Ala2,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 mu-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-alpha) and novel (PKC-epsilon) isoforms, SH-SY5Y cells also contain a DAG- and Ca2+-independent, atypical PKC isozyme (PKC-zeta), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC-zeta is similarly sensitive to activation by mu-opioids. PKC-zeta translocates from the cytosol to the membrane with kinetics similar to those of PKC-alpha and epsilon in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by mu-opioid agonists is involved in the processes that result in mu-receptor down-regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Analgesics / pharmacology
  • Analgesics, Opioid / pharmacology
  • Biological Transport / drug effects
  • Carcinogens / pharmacology
  • Diprenorphine / pharmacology
  • Down-Regulation / drug effects
  • Down-Regulation / physiology*
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
  • Enkephalin, Leucine / analogs & derivatives
  • Enkephalin, Leucine / pharmacology
  • Enkephalins / pharmacology
  • Enzyme Activation / drug effects
  • Enzyme Activation / physiology
  • Humans
  • Immunoblotting
  • Isoenzymes / analysis
  • Isoenzymes / metabolism
  • Narcotic Antagonists / pharmacology
  • Neuroblastoma
  • Phorbol 12,13-Dibutyrate / pharmacology
  • Phorbol Esters / pharmacology
  • Protein Kinase C / analysis
  • Protein Kinase C / metabolism*
  • Protein Kinase C-alpha
  • Protein Kinase C-epsilon
  • Receptors, Opioid, mu / agonists*
  • Receptors, Opioid, mu / antagonists & inhibitors
  • Receptors, Opioid, mu / metabolism*
  • Second Messenger Systems / physiology
  • Tritium
  • Tumor Cells, Cultured


  • Analgesics
  • Analgesics, Opioid
  • Carcinogens
  • Enkephalins
  • Isoenzymes
  • Narcotic Antagonists
  • Phorbol Esters
  • Receptors, Opioid, mu
  • Tritium
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
  • Diprenorphine
  • Phorbol 12,13-Dibutyrate
  • Enkephalin, Leucine
  • enkephalin, Ser(2), Leu(5), Thr(6)-
  • protein kinase C zeta
  • PRKCA protein, human
  • PRKCE protein, human
  • Protein Kinase C
  • Protein Kinase C-alpha
  • Protein Kinase C-epsilon