Arrestin‐centred interactions at the membrane and their conformational determinants

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.

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Relating GPCR domains with functionality: receptor helix-bundle and C-terminus differentially influence GRK-specific functions and β-arrestin-mediated regulation

Matthees,

Haider,

Klement

et al. 2024

Preprint

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G protein-coupled receptors (GPCRs) orchestrate diverse physiological responses via intracellular signaling through G proteins, GPCR kinases (GRKs), and arrestins. While the role of G proteins in receptor signaling is well-established, the contributions of GRKs and arrestins remain incompletely understood. Here, we investigate the influence of arrestin-interacting GPCR domains (helix-bundle/C-terminus) on β-arrestin conformations and functions using refined biosensors and advanced cellular knockout systems. By focusing on prototypical class A (b2AR) and B (V2R) receptors and their chimeras (b2V2/V2b2), we can now characterize differential β-arrestin conformational changes as primarily mediated by the receptor C-terminus or helix-bundle. Moreover, we demonstrate that some β-arrestin-supported processes are governed by distinct receptor domains, such as ERK1/2 activation (helix-bundle) and arrestin co-internalization (C-terminus), while others depend on the overall GPCR configuration, such as receptor internalization. Our findings elucidate how individual GPCR domains dictate downstream signaling events, shedding light on the structural basis of receptor-specific signaling and regulation.

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Arrestin‐centred interactions at the membrane and their conformational determinants

Cited by 4 publications

References 119 publications

GPCR-dependent and -independent arrestin signaling

GPCR-dependent and -independent arrestin signaling

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

Relating GPCR domains with functionality: receptor helix-bundle and C-terminus differentially influence GRK-specific functions and β-arrestin-mediated regulation

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