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