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Heterologous regulation of agonist-independent ?-opioid receptor phosphorylation by protein kinase C.

ABSTRACT: BACKGROUND AND PURPOSE: Homologous agonist-induced phosphorylation of the ?-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 APPROACH: 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. KEY 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 PKC? 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 IMPLICATIONS: 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.

SUBMITTER: Illing S

PROVIDER: S-EPMC3952808 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Heterologous regulation of agonist-independent μ-opioid receptor phosphorylation by protein kinase C.

Illing Susann S Mann Anika A Schulz Stefan S

British journal of pharmacology 20140301 5

<h4>Background and purpose</h4>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.<h4>Exp ...[more]

PMID: 24308893

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Project description:Background and purposeMorphine activates the µ-opioid receptor without causing its rapid endocytosis. In contrast, full agonists such as [d-Ala(2) -MePhe(4) -Gly-ol]enkephalin (DAMGO) or etonitazene stimulate a rapid and profound internalization. However, the detailed molecular events underlying the differential regulation of receptor trafficking by distinct opioid agonists remain incompletely understood.Experimental approachHere, we have generated phosphosite-specific antibodies for the carboxyl-terminal residues serine 363 (Ser363), threonine 370 (Thr370) and serine 375 (Ser375), which enabled us to selectively detect either the Ser363-, Thr370- or Ser375-phosphorylated form of the receptor.Key resultsWe showed that agonist-induced phosphorylation occurs at Thr370 and Ser375, whereas Ser363 is constitutively phosphorylated in the absence of agonist. We further demonstated that DAMGO and etonitazene stimulated the phosphorylation of both Thr370 and Ser375. In contrast, morphine promoted the phosphorylation of Ser375, but failed to stimulate Thr370 phosphorylation. In the presence of DAMGO, Ser375 phosphorylation occurred at a faster rate than phosphorylation of Thr370, indicating that Ser375 is the primary site of agonist-dependent phosphorylation. Activation of PKC by phorbol 12-myristate 13-acetate increased receptor phosphorylation only on Thr370, but not on Ser375, indicating that Thr370 can also undergo heterologous PKC-mediated phosphorylation. We also showed that µ receptor dephosphorylation can occur within minutes at or near the plasma membrane, and that agonist removal is a major prerequisite for Thr370 and Ser375 dephosphorylation.Conclusions and implicationsTogether, we showed for the first time that distinct agonists stimulate site-specific patterns of phosphorylation, which are intimately related to their ability to elicit µ-opioid receptor sequestration.Linked articleThis article is commented on by Kelly, pp. 294-297 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01387.x.

Dataset Information

Heterologous regulation of agonist-independent ?-opioid receptor phosphorylation by protein kinase C.

Publications

Heterologous regulation of agonist-independent μ-opioid receptor phosphorylation by protein kinase C.

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