Proteasome Involvement in Agonist-induced Down-regulation of μ and δ Opioid Receptors

• Published in: The Journal of biological chemistry 2001-04, Vol.276 (15), p.12345-12355
• Main Authors: Chaturvedi, Kirti, Bandari, Persis, Chinen, Norihiro, Howells, Richard D.
• Format: Article
• Language: English
• Subjects: Cell Line; Cysteine Endopeptidases - metabolism; Down-Regulation; Humans; Kinetics; Multienzyme Complexes - metabolism; Phosphorylation; Proteasome Endopeptidase Complex; Receptors, Opioid, delta - agonists; Receptors, Opioid, delta - metabolism; Receptors, Opioid, mu - agonists; Receptors, Opioid, mu - metabolism; Ubiquitins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M008054200

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged δ and μ receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [35S]methionine metabolic labeling indicated that the turnover rate of δ receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional Gi and Go proteins by pertussis toxin-attenuated down-regulation of the μ opioid receptor, while down-regulation of the δ opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on μ and δ opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced μ and δ receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state μ and δ opioid receptor levels. Immunoprecipitation of μ and δ opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.