This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged ␦ and receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulsechase experiments using [35 S]methionine metabolic labeling indicated that the turnover rate of ␦ receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G i and G o proteins by pertussis toxin-attenuated down-regulation of the opioid receptor, while down-regulation of the ␦ opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on and ␦ opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced and ␦ receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steadystate and ␦ opioid receptor levels. Immunoprecipitation of and ␦ 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.The pharmacological effects of opioid drugs and the physiological effects of endogenous opioid peptides are initiated through the binding and activation of opioid receptors (1), which are members of the G protein-coupled receptor (GPCR) 1 family (2). GPCRs comprise a diverse superfamily of integral membrane proteins that mediate signal transduction across the plasma membrane. All GPCRs are postulated to have amino termini located on the extracellular side of the plasma membrane that are linked to seven-transmembrane helices connected by relatively short intracellular and extracellular loops, and contain carboxyl termini that face the interior of the cell. Ligands approach and engage GPCRs from the extracellular space, and receptor activation results in coupling to heterotrimeric G proteins on the intracellular face of membrane. The amino termini of nearly all G protein-coupled receptors contain consensus amino acid sequences for asparagine-linked glycosylation; two sites for N-linked glycosylation are present in ␦ and receptors, while five are found in the receptor (3).Three types of opioid receptor, ␦, , and , have been cloned and characterized extensively (4 -7). Opioid receptors have unique ligand specificities, anatomical distributions, and physiological functions (8 -15). Opioid receptors exhibit ϳ60% identity in their amino acid sequences, however, marked differences in sequence conservation are evident within receptor subdomains. The amino acid sequences of putative transmembrane spanning segments and the three intracellular loops are highly conserved among opioid receptor types, whereas sequences in the extracellular amino termini, second and third extracellular...

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