title: Protein kinase C-mediated phosphorylation of the μ-opioid receptor and its effects on receptor signaling.
abstract: Phosphorylation of the μ opioid receptor (MOPr), mediated by several protein kinases, is a critical process in the regulation of MOPr signaling. Although G protein-coupled receptor kinases are known to play an essential role in the agonist-induced phosphorylation and desensitization of MOPr, evidence suggests that other protein kinases, especially protein kinase C (PKC), also participate in the regulation of MOPr signaling. In this study, we investigated the biochemical nature and downstream effects of PKC-mediated MOPr phosphorylation. We observed in vitro phosphorylation of the MOPr C terminus by purified PKC. Protein mass spectrometry and site-directed mutagenesis implicated Ser363 of MOPr as the primary substrate for PKC, and this was confirmed in Chinese hamster ovary cells stably expressing full-length MOPr using an antibody that specifically recognizes phosphorylated Ser363. Alanine mutation of Ser363 did not affect the affinity of MOPr-ligand binding and the efficiency of receptor G-protein coupling. However, the S363A mutation attenuated the desensitization of receptor G-protein coupling induced by phorbol 12-myristate. Our research thus has identified a specific PKC phosphorylation site in MOPr and demonstrated that PKC-mediated phosphorylation of MOPr induces receptor desensitization at the G protein coupling level.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Feng
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Li
authors.forename: Jia Bei
authors.initials: JB
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D007527:Isoenzymes / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology*; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D007527:Isoenzymes; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D011493:Protein Kinase C