Abstract
There is increasing evidence that the signal transduction of opioid receptors is modulated by receptor-associated proteins. In the search for proteins regulating μ-opioid receptor (MOPr) endocytosis, synaptophysin was found to bind to the rat μ-opioid receptor in yeast two-hybrid assay. Coimmunoprecipitation experiments and bioluminescence resonance energy transfer assays confirmed that the μ-opioid receptor constitutively interacts with synaptophysin in human embryonic kidney 293 cells overexpressing MOPr and synaptophysin. In this study, we show that overexpression of synaptophysin enhances the μ-opioid receptor endocytosis. One explanation for the observed effects is that synaptophysin recruits dynamin to the plasma membrane, facilitating fission of clathrin-coated vesicles. This suggestion is supported by our finding that overexpression of a synaptophysin truncation mutant, which breaks the interaction between synaptophysin and dynamin, prevents agonist-mediated μ-opioid receptor endocytosis. In addition, the synaptophysin-augmented μ-opioid receptor trafficking leads to attenuated agonist-induced receptor desensitization and faster receptor resensitization. Taken together, our findings strongly suggest that synaptophysin plays an important role in the regulation of μ-opioid receptor trafficking and signaling.
Footnotes
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This work was supported by grants HO 1027/10-1 and SFB 426/A2 from the Deutsche Forschungsgemeinschaft (to V.H.) and Bundesministerium für Bildung und Forschung grant 01ZZ0407 (to T.K.).
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Y.-J.L. and D.-F.W contributed equally to this work.
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ABBREVIATIONS: PCR, polymerase chain reaction; Syp, synaptophysin; MOPr, the μ-opioid receptor; CMV, cytomegalovirus; HEK, human embryonic kidney; HA, influenza hemagglutinin-HA-epitope (YPYDVPDYA); HA-MOPr, HA epitope-tagged MOPr; Myc, epitope tag (MASMQKLI-SEEDL); GST, glutathione transferase; BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; DAMGO, [d-Ala2,Me-Phe4,Glyol5]enkephalin; IP, immunoprecipitation; AP, adaptor protein; 3ILμ, third intracellular loop of the μ-opioid receptor; ANOVA, analysis of variance.
- Received May 2, 2006.
- Accepted September 27, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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