Different mechanisms of homologous and heterologous μ‐opioid receptor phosphorylation

Abstract

The efficiency of μ‐opioid receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite‐specific μ‐opioid receptor antibodies, which have proved to be excellent tools for monitoring the spatial and temporal dynamics of receptor phosphorylation and dephosphorylation. Agonist‐induced phosphorylation of μ‐opioid receptors occurs at a conserved 10 residue sequence 370TREHPSTANT379 in the receptor's carboxyl‐terminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at S375, present in the middle of this sequence, but only high‐efficacy opioids have the ability to drive higher order phosphorylation on flanking residues (T370, T376 and T379). S375 is the initiating residue in a hierarchical phosphorylation cascade. In contrast, agonist‐independent heterologous μ‐opioid receptor phosphorylation occurs primarily at T370. The combination of phosphosite‐specific antibodies and siRNA knockdown screening also facilitated the identification of relevant kinases and phosphatases. In fact, morphine induces a selective S375 phosphorylation that is predominantly catalysed by GPCR kinase 5 (GRK5), whereas multisite phosphorylation induced by high‐efficacy opioids specifically requires GRK2/3. By contrast, T370 phosphorylation stimulated by phorbol esters or heterologous activation of Gq‐coupled receptors is mediated by PKCα. Rapid μ‐opioid receptor dephosphorylation occurs at or near the plasma membrane and is catalysed by protein phosphatase 1γ (PP1γ). These findings suggest that there are distinct phosphorylation motifs for homologous and heterologous regulation of μ‐opioid receptor phosphorylation. However, it remains to be seen to what extent different μ‐opioid receptor phosphorylation patterns contribute to the development of tolerance and dependence in vivo. Linked Articles This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2

Role of Phosphorylation Sites in Desensitization ofµ-Opioid Receptor

“…The present results suggest that PKC-mediated phosphorylation of S363 and T370 (Doll et al, 2011;Feng et al, 2011;Illing et al, 2014;Mann et al, 2015) is not necessary because morphine-induced desensitization persisted when all S/T sites were mutated. Chu et al (2010) reported that morphine-induced, PKC-mediated desensitization is mediated partly by phosphorylation of Ga i2 subunits, which should be independent of C-terminal phosphorylation.…”

“…Desensitization by ME and morphine were both unaffected in 3S/T-A that removes S375, which is efficiently phosphorylated by GRK2/3, as well as two proximal residues, T370, that is quite efficiently phosphorylated (Doll et al, 2011), and S363 which is constitutively phosphorylated and a substrate for PKC-phosphorylation (Illing et al, 2014;Mann et al, 2015). This finding suggests other S/T residues may be phosphorylated to contribute to desensitization and internalization.…”

“…Phosphorylation and Desensitization of m-Opioid Receptors (S363, T370; Feng et al, 2011;Illing et al, 2014;Mann et al, 2015).…”

Endogenous Analgesia, Dependence, and Latent Pain Sensitization

“…The CA state of MOR is thought to depend on receptor modifications, including receptor interactions with accessory proteins or phosphorylation at numerous sites on MOR (Sadee et al, 2005, Connor and Traynor, 2010, Mann et al, 2014), including constitutive phosphorylation at S363 in vivo (Illing et al, 2014). With the emergence of phosphosite-specific MOR antibodies and other pharmacological tools (Mann et al, 2014), future studies could determine whether CFA increases MOR phosphorylation or dephosphorylation that then contributes to MOR CA .…”

“…With the emergence of phosphosite-specific MOR antibodies and other pharmacological tools (Mann et al, 2014), future studies could determine whether CFA increases MOR phosphorylation or dephosphorylation that then contributes to MOR CA .…”