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Protein kinase C-mediated phosphorylation of the μ-opioid receptor and its effects on receptor signaling

Bo Feng1, Zhihua Li, Jia Bei Wang

  • 1Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA.

Molecular Pharmacology
|January 8, 2011
PubMed

Insights

Protein kinase C (PKC) phosphorylates the μ opioid receptor (MOPr) at Ser363, a key step in regulating MOPr signaling and desensitization. This specific phosphorylation site impacts receptor desensitization at the G protein coupling level.

Area of Science:

  • Biochemistry
  • Molecular Pharmacology
  • Cell Signaling

Background:

  • Opioid receptor signaling is crucial for pain management and other physiological processes.
  • G protein-coupled receptor kinases (GRKs) are known regulators of μ opioid receptor (MOPr) phosphorylation and desensitization.
  • The role of other kinases, such as protein kinase C (PKC), in MOPr regulation remains less understood.

Purpose of the Study:

  • To investigate the specific mechanisms and consequences of PKC-mediated phosphorylation of the MOPr.
  • To identify the precise site of PKC phosphorylation on the MOPr.
  • To determine the functional impact of this phosphorylation on MOPr signaling and desensitization.

Main Methods:

  • In vitro kinase assays using purified PKC and MOPr C-terminal peptides.
  • Protein mass spectrometry to identify phosphorylation sites.
  • Site-directed mutagenesis to create specific MOPr mutants (e.g., S363A).
  • Cell-based assays in Chinese hamster ovary (CHO) cells expressing MOPr, including ligand binding, G-protein coupling assays, and Western blotting with phospho-specific antibodies.

Main Results:

  • PKC directly phosphorylates the MOPr C terminus in vitro.
  • Serine 363 (Ser363) was identified as the primary PKC phosphorylation site on MOPr.
  • Mutation of Ser363 to alanine (S363A) did not alter MOPr ligand binding affinity or basal G protein coupling efficiency.
  • The S363A mutation significantly attenuated phorbol 12-myristate-induced desensitization of MOPr G protein coupling.

Conclusions:

  • PKC phosphorylates MOPr at Ser363, representing a novel regulatory pathway.
  • PKC-mediated phosphorylation of MOPr at Ser363 is critical for agonist-induced desensitization of G protein coupling.
  • This finding elucidates a specific molecular mechanism by which PKC modulates opioid receptor function.

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