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

Underwood, Owen; Fritzwanker, Sebastian; Glenn, Jaqueline; Blum, Nina Kathleen; Batista-Gondin, Arisbel; Drube, Julia; Hoffmann, Carsten; Briddon, Stephen J.; Schulz, Stefan; Canals, Meritxell

Abstract

Desensitisation 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, phosphorylation is important for β-arrestin recruitment to the receptor, and ligands of different efficacies induce distinct phosphorylation barcodes. The C-tail 370TREHPSTANT379 motif harbours Ser/Thr residues important for these regulatory functions. 375Ser is the primary phosphorylation site of a ligand-dependent, hierarchical, and sequential process, whereby flanking 370Thr, 376Thr and 379Thr get subsequently and rapidly phosphorylated. Here we used GRK KO cells, phosphosite specific antibodies and site-directed mutagenesis to evaluate the contribution of the different GRK subfamilies to ligand-induced phosphorylation barcodes and β-arrestin2 recruitment. We show that both GRK2/3 and GRK5/6 subfamilies promote phosphorylation of 370Thr and 375Ser. Importantly, only GRK2/3 induce phosphorylation of 376Thr and 379Thr, and we identify these residues as key sites to promote robust β-arrestin recruitment to the MOR. These data provide insight into the mechanisms of MOR regulation and suggest that the cellular complement of GRK subfamilies plays an important role in determining the tissue responses of opioid agonists.

Citation

Underwood, O., Fritzwanker, S., Glenn, J., Blum, N. K., Batista-Gondin, A., Drube, J., Hoffmann, C., Briddon, S. J., Schulz, S., & Canals, M. (2024). Key phosphorylation sites for robust β-arrestin2 binding at the MOR revisited. Communications Biology, 7, Article 933. https://doi.org/10.1038/s42003-024-06571-1

• Journal Article Type: Article
• Acceptance Date: Jul 9, 2024
• Online Publication Date: Aug 2, 2024
• Publication Date: Aug 2, 2024
• Deposit Date: Aug 1, 2024
• Publicly Available Date: Aug 5, 2024
• Journal: Communications Biology
• Electronic ISSN: 2399-3642
• Publisher: Nature Publishing Group
• Peer Reviewed: Peer Reviewed
• Volume: 7
• Article Number: 933
• DOI: https://doi.org/10.1038/s42003-024-06571-1
• Public URL: https://nottingham-repository.worktribe.com/output/37887073
• Publisher URL: https://www.nature.com/articles/s42003-024-06571-1