Different mechanisms of homologous and heterologous μ‐opioid receptor phosphorylation

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 sequence370TREHPSTANT379in the receptor's carboxyl‐terminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation atS375, 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 andT379).S375 is the initiating residue in a hierarchical phosphorylation cascade. In contrast, agonist‐independent heterologous μ‐opioid receptor phosphorylation occurs primarily atT370. The combination of phosphosite‐specific antibodies and siRNAknockdown screening also facilitated the identification of relevant kinases and phosphatases. In fact, morphine induces a selectiveS375 phosphorylation that is predominantly catalysed byGPCRkinase 5 (GRK5), whereas multisite phosphorylation induced by high‐efficacy opioids specifically requiresGRK2/3. By contrast,T370 phosphorylation stimulated by phorbol esters or heterologous activation ofGq‐coupled receptors is mediated byPKCα. 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 dependencein vivo.Linked ArticlesThis article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visithttp://dx.doi.org/10.1111/bph.2015.172.issue-2