Agonist‐induced signaling and trafficking of the μ‐opioid receptor: role of serine and threonine residues in the third cytoplasmic loop and C‐terminal domain

Abstract The human μ-opioid receptor and a mutant form, μS/T[i3+Cter]A, in which all Ser and Thr residues from the third cytoplasmic loop and C-terminal domain were changed to Ala, were studied after expression in CHO-K1 cells. Although the mutant receptors had similar affinities for agonists and EC 50 values for inhibition of adenylyl cyclase as compared to wild-type receptors, the E max were almost 2-fold decreased, suggesting a role of the mutated residues in G-protein coupling. After chronic morphine or etorphine, the EC 50 values of the agonists were about 5-fold increased at both receptors but the E max values were not altered; upon agonist withdrawal forskolin-stimulated cAMP levels were increased to almost 200% of control levels. Sequestration and rapid down-regulation of the μ-opioid receptor were induced by DAGO and etorphine but not morphine. In contrast, the μS/T[i3+Cter]A receptor was not sequestered and was up-regulated (150–380%) after treatment with agonists. The results indicate that the Ser and Thr residues in the third cytoplasmic loop and C-terminus of the μ-opioid receptor are not involved in the limited desensitization or in the adenylyl cyclase superactivation promoted by agonists but that their integrity and/or their phosphorylation is required in the intricate and coordinately regulated pathways involved in receptor signaling and trafficking.