Abstract
The trafficking of the μ-opioid receptor (MOR), a member of the rhodopsin G protein-coupled receptor (GPCR) family, can be regulated by interaction with multiple cellular proteins. To determine the proteins involved in receptor trafficking, using the targeted proteomic approach and mass spectrometry analysis, we have identified that Ribophorin I (RPNI), a component of the oligosaccharide transferase complex, could directly interact with MOR. RPNI can be shown to participate in MOR export by the intracellular retention of the receptor after small interfering RNA knockdown of endogenous RPNI. Overexpression of RPNI rescued the surface expression of the MOR 344KFCTR348 deletion mutant independent of calnexin. Furthermore, RPNI regulation of MOR trafficking is dependent on the glycosylation state of the receptor, as reflected by the inability of overexpression of RPNI to affect the trafficking of the N-glycosylation-deficient mutants, or GPCRs that have minimal glycosylation sites. Hence, this novel RPNI chaperone activity is a consequence of N-glycosylation-dependent direct interaction with MOR.
Footnotes
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This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA007339, DA016674, DA000564, DA011806].
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ABBREVIATIONS: GPCR, G protein-coupled receptor; ER, endoplasmic reticulum; OST, oligosaccharide transferase; KOR, κ-opioid receptor; N2A, neuro2A neuroblastoma cell; RPNI, ribophorin I; MOR, μ-opioid receptor; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; siRNA, small interfering RNA; GFP, green fluorescent protein; AR, adrenergic receptor; HA, hemagglutinin; FACS, fluorescence activated cell sorting; LC MS/MS, liquid chromatography-tandem mass spectrometry; EndoH, endoglycosidase H; PNGase F, peptide-N--(N-acetyl-β-glucosaminyl)asparagine amidase; IP, immunoprecipitation; co-IP, coimmunoprecipitation; AR, adrenergic receptor; MOR5ND, MOR with Asn9, Asn31, Asn38, Asn46, and Asn53 residues at the N terminus all mutated to Asp; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; DOR, δ-opioid receptor; BiP, ER luminal binding protein; C2, MOR with the 344KFCTR348 sequence deleted.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received December 7, 2008.
- Accepted March 16, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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