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, |iS/ 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 r ,n 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 EC50 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 μ8/Τ[ί3+ΟεΓ]Α 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.

Chronic morphine treatment up-regulates mu opioid receptor binding in cells lacking filamin A

“…In addition, it was shown (Capeyrou et al, 1997) that when all serine and threonine residues within the third intracellular loop and carboxyl terminal tail of MOP were mutated to alanine, an increase in MOP binding sites was seen after chronic agonist treatment. As suggested by the authors, a receptor conformation involved in MOP internalization, degradation and recycling pathways could be disrupted in the mutant receptor, resulting in a lower rate of receptor degradation than receptor synthesis, leading to receptor up-regulation.…”

“…As suggested by the authors, a receptor conformation involved in MOP internalization, degradation and recycling pathways could be disrupted in the mutant receptor, resulting in a lower rate of receptor degradation than receptor synthesis, leading to receptor up-regulation. It is unclear whether the mutant receptor has impaired ability to internalize into endosomes or has an increased rate of recycling, due to the unphosphorylated state of the receptor (Capeyrou et al, 1997).…”

“…The importance of the carboxyl tail in the down-regulation and trafficking of the opioid receptors is well established (Burd et al, 1998;Capeyrou et al, 1997;Chaipatikul et al, 2003;Finn and Whistler, 2001;Trapaidze et al, 1996). There are many examples of association of the GPCRs, including MOP with other proteins, such as beta-arrestins, (Lefkowitz, 1998), GRKs (Pitcher et al, 1998) and small G proteins (Mitchell et al, 1998).…”

Phosphorylation of Ser363, Thr370, and Ser375 Residues within the Carboxyl Tail Differentially Regulates μ-Opioid Receptor Internalization

“…Interestingly, in addition to the subtype-specific regulation of opioid receptors (9 -12), individual opioid receptors are differentially regulated by distinct opioid agonists (5-8, 13, 14). In the case of MOR, opioid agonists demonstrating equivalent ability to activate receptor signaling exhibit remarkable differences in their abilities to functionally desensitize (5,6) and induce internalization of the receptor in both transfected cells and neurons (7,8,(13)(14)(15). However, the detailed molecular events underlying this differential regulation of MOR by distinct agonists remain unclear.…”

U50,488H-induced Internalization of the Human κ Opioid Receptor Involves a β-Arrestin- and Dynamin-dependent Mechanism

“…Agonist-induced internalization of and ␦ opioid receptors has been extensively studied (20,(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). Etorphine and various peptide agonists promote internalization of both and ␦ opioid receptors to transferrin-containing endosomes, while morphine and levorphanol do not (32, 33, 37, 39 -42).…”