Key phosphorylation sites for robust β-arrestin2 binding at the MOR revisited

Abstract

FigureDesensitisation of the mu-opioid receptor (MOR) is proposed to underlie the initiation of opioid analgesic tolerance and previous work has shown that agonist-induced phosphorylation of the MOR C-tail contributes to this desensitisation. Moreover, we and others have shown that phosphorylation is important for β-arrestin recruitment to the receptor, and that ligands of different efficacies induce distinct patterns, or barcodes, of receptor phosphorylation. Within the MOR C-tail, the370TREHPSTANT379motif harbours Ser/Thr residues important for these regulatory functions.375Ser acts as a primary phosphorylation site of a ligand-dependent, hierarchical, and sequential process, whereby flanking370Thr,376Thr and379Thr residues can get subsequently phosphorylated. Here we used HEK293 GRK KO cells, in combination with phosphosite specific antibodies and site-directed mutagenesis of the MOR, to evaluate the contribution of the different GRK subfamilies to ligand-induced phosphorylation barcodes and β-arrestin2 recruitment. We show that both GRK subfamilies (GRK2/3 and GRK5/6) promote phosphorylation of Thr370and Ser375. However, only GRK2/3 induce phosphorylation of Thr376and Thr379, which is required to promote robust β-arrestin recruitment to the receptor. Moreover, while DAMGO and fentanyl can engage all kinases to promote Thr370and Ser375phosphorylation, under endogenous GRK expression conditions, morphine-induced phosphorylation of these residues is specifically mediated by GRK5/6. These data provide insight into the mechanisms of MOR regulation and suggest that the cellular complement of the different GRK subfamilies plays an important role in determining the tissue responses of distinct opioid agonists.