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Medientyp:
E-Artikel
Titel:
Heterologous regulation of agonist‐independent μ‐opioid receptor phosphorylation by protein kinase C
Beteiligte:
Illing, Susann;
Mann, Anika;
Schulz, Stefan
Erschienen:
Wiley, 2014
Erschienen in:
British Journal of Pharmacology, 171 (2014) 5, Seite 1330-1340
Sprache:
Englisch
DOI:
10.1111/bph.12546
ISSN:
0007-1188;
1476-5381
Entstehung:
Anmerkungen:
Beschreibung:
Background and PurposeHomologous 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.Experimental ApproachWe 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.Key ResultsActivation 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 and ImplicationsOur 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.