title: Heterologous regulation of agonist-independent μ-opioid receptor phosphorylation by protein kinase C.
abstract: OBJECTIVE
  Homologous agonist-induced phosphorylation of the μ-opioid receptor (MOR) is initiated at the carboxyl-terminal S375, followed by phosphorylation of T370, T376 and T379. In HEK293 cells, this sequential and hierarchical multi-site phosphorylation is specifically mediated by G-protein coupled receptor kinases 2 and 3. In the present study, we provide evidence for a selective and dose-dependent phosphorylation of T370 after activation of PKC by phorbol esters.
  
  
  METHODS
  We used a combination of phospho site-specific antibodies, kinase inhibitors and siRNA knockdown screening to identify kinases that mediate agonist-independent phosphorylation of the MOR in HEK293 cells. In addition, we show with phospho site-specific antibodies were also used to study constitutive phosphorylation at S363 of MORs in mouse brain in vivo.
  
  
  RESULTS
  Activation of PKC by phorbol esters or heterologous activation of substance P receptors co-expressed with MORs in the same cell induced a selective and dose-dependent phosphorylation of T370 that specifically requires the PKCα isoform. Inhibition of PKC activity did not compromise homologous agonist-driven T370 phosphorylation. In addition, S363 was constitutively phosphorylated in both HEK293 cells and mouse brain in vivo. Constitutive S363 phosphorylation required ongoing PKC activity. When basal PKC activity was decreased, S363 was also a substrate for homologous agonist-stimulated phosphorylation.
  
  
  CONCLUSIONS
  Our results have disclosed novel mechanisms of heterologous regulation of MOR phosphorylation by PKC. These findings represent a useful starting point for definitive experiments elucidating the exact contribution of PKC-driven MOR phosphorylation to diminished MOR responsiveness in morphine tolerance and pathological pain.
authors.affiliation: Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany.
authors.forename: Susann
authors.initials: S
authors.lastname: Illing
authors.forename: Anika
authors.initials: A
authors.lastname: Mann
authors.forename: Stefan
authors.initials: S
authors.lastname: Schulz
mesh_terms: D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000701:Analgesics, Opioid; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate
keywords: MOR; PKC; analgesia; morphine; opioid receptor; phosphorylation; tolerance