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.
Footnotes
This work was supported by the National Institutes of Health National Institutes of Drug Abuse [Grant DA11925].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/mol.110.069096.
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ABBREVIATIONS:
- GPCR
- G protein-coupled receptor
- CHO
- Chinese hamster ovary
- CT
- carboxyl terminal
- DAMGO
- [d-Ala2-N-Me-Phe4,Gly5-ol]-enkephalin
- EGFP
- enhanced green fluorescence protein
- GRK
- G protein-coupled receptor kinase
- GST
- glutathione transferase
- MALDI-TOF-MS
- matrix-assisted laser desorption ionization-time of flight-mass spectrometry
- MOPr
- μ opioid receptor
- PMA
- phorbol 12-myristate 12-acetate
- PKC
- protein kinase C
- WGA
- wheat germ agglutinin
- WT
- wild type
- AC
- adenylate cyclase
- MS
- mass spectrometry
- PCR
- polymerase chain reaction
- PAGE
- polyacrylamide gel electrophoresis
- TBS-T
- Tris-buffered saline/Tween 20
- ANOVA
- analysis of variance
- DMSO
- dimethyl sulfoxide
- PBS
- phosphate-buffered saline
- [35S]GTPγS
- guanosine 5′-O-(3-[35S]thio)triphosphate.
- Received October 4, 2010.
- Accepted January 5, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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