Agonist Binding and Desensitization of the μ-Opioid Receptor Is Modulated by Phosphorylation of the C-Terminal Tail Domain

Abstract

Sustained activation of G protein-coupled receptors can lead to a rapid decline in signaling through acute receptor desensitization. In the case of the m-opioid receptor (MOPr), this desensitization may play a role in the development of analgesic tolerance. It is understood that phosphorylation of MOPr promotes association with b-arrestin proteins, which then facilitates desensitization and receptor internalization. Agonists that induce acute desensitization have been shown to induce a noncanonical high-affinity agonist binding state in MOPr, conferring a persistent memory of prior receptor activation. In the current study, live-cell confocal imaging was used to investigate the role of receptor phosphorylation in agonist binding to MOPr. A phosphorylation cluster in the C-terminal tail of MOPr was identified as a mediator of agonist-induced affinity changes in MOPr. This site is unique from the primary phosphorylation cluster responsible for b-arrestin binding and internalization. Electrophysiologic measurements of receptor function suggest that both phosphorylation clusters may play a parallel role during acute receptor desensitization. Desensitization was unaffected by alanine mutation of either phosphorylation cluster, but was largely eliminated when both clusters were mutated. Overall, this work suggests that there are multiple effects of MOPr phosphorylation that appear to regulate MOPr function: one affecting b-arrestin binding and a second affecting agonist binding.

The Complex Roles of μ-Opioid Receptor Phosphorylation: A Key Determinant in Receptor Signaling and Regulation

“…An important observation is that the rates of MOPr desensitization do not correlate with the rates of receptor phosphorylation. In agreement with the study by Birdsong et al (2015), Yousuf and colleagues show that Met-enkephalin-induced desensitization is only severely compromised when the 11 Ser/Thr residues of MOPr C-tail are mutated to Ala. In contrast, morphine-induced desensitization is unaffected by these mutations but compromised by protein kinase C inhibition, both at the wild-type receptor and more so at the receptor mutant lacking all phosphorylation sites.…”

“…However, the impact of the different receptor phosphorylation profiles in the regulation of other aspects of MOPr signaling remains uncertain. In this issue, two independent studies from different groups tackle this important question by using "phosphosite" receptor mutants (Birdsong et al, 2015 andYousuf et al, 2015). Together these studies highlight that agonist-dependent phosphorylation of specific C-terminal motifs participates in the regulation of MOPr signaling via multiple mechanisms that extend beyond b-arrestin recruitment.…”

“…Importantly, both studies highlight that 1) desensitization occurs even when the residues required for b-arrestin recruitment are mutated, and 2) the multiple mechanisms participating in MOPr desensitization are ligand dependent and, in some cases, can occur independently of Ser/Thr phosphorylation. Although the contribution of the TSST motif was not directly assessed in the study by Yousuf et al (2015), it is likely that, as suggested by Birdsong et al (2015), the motif participates in MOPr desensitization by stabilizing an uncoupled conformation of the receptor.…”

Phosphoproteomic analysis of the mouse brain mu‐opioid (MOP) receptor

“…Though differences in detection sensitivity between native preparation and overexpressed receptors cannot be excluded, this suggests that the 354 TSST 357 motif is not a primary phosphorylation cluster in vivo. In this sense, alanine mutations within this motif show unconsistent results in the literature: no effect on MOP receptor phosphorylation level [11,31], desensitization [24,31] and internalization [16]; decreased phosphorylation and desensitization in the case of the rat S355A/T357A receptor mutant [32]; impact on agonist dissociation kinetics [24].…”

Role of Phosphorylation Sites in Desensitization ofµ-Opioid Receptor

“…Significant differences between wild-type MOPr and 11S/T-A were seen at **P , 0.01 and ***P , 0.001 (two-way analysis of variance, Bonferroni post-tests) (n = 4). phosphorylation of sites additional to those required for arrestin recruitment (lost in the 6S/T-A mutant) are necessary (Birdsong et al, 2015;Canals, 2015). T394 might not be crucial because Lau et al (2011) were unable to detect agonist-induced phosphorylation of this site.…”