Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid μ-opioid receptor desensitization

Abstract

G protein receptor kinases (GRKs) and β-arrestins are key regulators of μ-opioid receptor (MOR) signaling and trafficking. We have previously shown that high-efficacy opioids such as DAMGO stimulate a GRK2/3-mediated multisite phosphorylation of conserved C-terminal tail serine and threonine residues, which facilitates internalization of the receptor. In contrast, morphine-induced phosphorylation of MOR is limited to Ser and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and β-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was β-arrestin to a DAMGO-activated MOR. β-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on the phosphorylation sites in the C-terminal tail, specifically four serine and threonine residues within the TREHPSTANT motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and β-arrestins. We identified two components of high-efficacy agonist desensitization of MOR: a sustained component, which required GRK2-mediated phosphorylation and a potential soluble factor, and a rapid component, which was likely mediated by GRK2 but independent of receptor phosphorylation. Elucidating these complex receptor-effector interactions represents an important step toward a mechanistic understanding of MOR desensitization that leads to the development of tolerance and dependence.

A cellular perspective of bias at G protein‐coupled receptors

“…In a separate study, GRK/β‐arrestin function was disrupted by mutation of S375 in MOR 105 . S375 is the initiating site for MOR multiphosphorylation and mutation of this site to alanine reduced overall MOR phosphorylation, β‐arrestin recruitment, and agonist‐dependent MOR endocytosis 79,82,84 . In a S375A‐MOR knock‐in mouse, development of tolerance to high efficacy opioids was reduced while development of tolerance to morphine was retained in the electrical tail root assay for analgesia 105 .…”

“…The mu opioid receptor (MOR) provides a contrasting example of the cellular response to partial/biased agonism. In cultured cell models, the full agonist DAMGO was shown to cause GRK2/3‐mediated phosphorylation of MOR phosphorylation sites: S356, T357, T370, S375, T376, and T379 79–84 . This result was compared with the lower efficacy agonist morphine, which is typically considered a partial agonist in vitro although with systems‐dependent efficacy 44,85–88 .…”

“…This result was compared with the lower efficacy agonist morphine, which is typically considered a partial agonist in vitro although with systems‐dependent efficacy 44,85–88 . Morphine caused phosphorylation of S375 in MOR, produced minimal phosphorylation of T370, T376, and T379, and thus drove 5‐ to 15‐fold‐less multiphosphorylation of MOR 79–84 . Unexpectedly, and in contrast to β2AR, morphine caused a switch to GRK5 (part of the GRK4/5/6 subfamily) as the kinase primarily required for phosphorylation of MOR in cultured cells 80,82 .…”

Recent Progress in Opioid Research from an Electrophysiological Perspective

“…Much of this has come from mutations of phosphorylation sites in MOR (Chen et al, 2013;Doll et al, 2011Doll et al, , 2012Just et al, 2013;Lau et al, 2011;Mies et al, 2018;Moulledous et al, 2015;Wang et al, 2002) and the development of a selective and potent G protein-coupled receptor kinase (GRK) inhibitor, Compound101 (Thal et al, 2011;Lowe et al, 2015). Electrophysiological studies have been made in cell lines, neurons in brain slices with expressed receptors and in brain slices from knockin animals expressing MORs lacking regulatory phosphorylation sites in the C-terminal tail of the receptor (Yousuf et al, 2015;Miess et al, 2018;Kliewer et al, 2019;Birdsong 2013Birdsong , 2015Arttamangkul et al, 2018Arttamangkul et al, , 2019b. Both acute agonist-dependent desensitization and measures of long-term tolerance are reduced or eliminated in cells expressing phosphorylation-deficient mutant receptors.…”

Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells

“…However, this binding involves multiple biochemical steps and, in particular, it typically requires the receptor to undergo prior agonist-induced phosphorylation [23][24][25] . This has been clearly established for opioid receptors 13,26 , for which strong interaction with beta-arrestin requires the receptor to be phosphorylation at multiple sites in the cytoplasmic tail through a defined sequence of agonist-dependent reactions which are catalyzed by distinct GPCR kinase (GRK) isoforms 10,[27][28][29] . Accordingly, beta-arrestin recruitment measured in such assays clearly reflects a process that is considerably more complex than allosteric selection by the receptor.…”