Abstract
This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta 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 mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta 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.
Highlights
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]
The major conclusions to be drawn from this study are as follows: 1) proteolysis of and ␦ opioid receptor protein is evident 2– 4 h following DADL treatment and there is a near complete loss of receptor protein following 18 h of exposure. 2)
3) Functional pertussis toxin-sensitive G proteins are required for agonistinduced down-regulation of the opioid receptor but not for ␦ opioid receptor down-regulation
Summary
Cell Culture and Transfection—Human embryonic kidney (HEK) 293 cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin sulfate. Membranes were blocked for 1 h in 2.3% dried milk, 0.5% bovine serum albumin, 0.1% Nonidet P-40, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20, followed by overnight incubation at 4 °C with mouse anti-FLAG M1 monoclonal antibody (Sigma). Membranes were blocked for 1 h in 2.3% dried milk, 0.5% bovine serum albumin, 0.1% Nonidet P-40, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween 20, followed by overnight incubation with mouse monoclonal anti-phospho-MAPK antibody or rabbit anti-MAP kinase antibody (New England Biolabs, Beverly, MA). HEK 293 cells expressing FLAG-tagged ␦ or opioid receptors were preincubated at 37 °C for 3 h in serum-free Dulbecco’s modified Eagle’s medium with or without 100 ng/ml pertussis toxin (Ptx), incubated for 18 h in the absence and presence of 1 M DAMGO ( receptors) or DADL (␦ receptors). Data represent mean Ϯ S.E. of three to four experiments conducted in duplicate
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