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bioRxiv · 10.64898/2026.10.05.756893

Phosphorylation of β-arrestins impacts recruitment to Parathyroid Hormone Receptor 1 (PTH1R) at the cell surface and endosomes

Carruthers, E. R.·Oyagawa, C. R.·Poulsen, R. C.·Grimsey, N. L.
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Abstract

{beta}-arrestins 1 and 2 are intracellular adaptor proteins that regulate the subcellular localisation and signalling of GPCRs including Parathyroid Hormone Receptor 1 (PTH1R), which produces {beta}-arrestin-dependent signalling that may selectively stimulate bone anabolism. {beta}-arrestins can themselves be regulated by post-translational modifications including phosphorylation, which dictates the translocation and signalling of the few GPCRs studied in this context to date. We aimed to investigate whether phosphorylation of {beta}-arrestins modulates their subcellular translocation in response to PTH1R activation. Translocation of {beta}-arrestins to the plasma membrane and early endosomes was quantified using BRET biosensors and immunocytochemistry with high content analysis. Phosphoknockout or phosphomimetic mutations were introduced to previously demonstrated phosphorylation sites in {beta}-arrestins 1 and 2, initially in bulk to assess overall effects. Plasma membrane translocation was altered only subtly, whereas early endosome translocation was markedly reduced for both mutations but to a greater degree for the phosphomimetic. Contributions of individual or paired sites were assessed by restoration to wildtype from the bulk phosphomimetic mutants. Dual restoration of S13 and T275 in {beta}-arrestin 1, or the analogous sites in {beta}-arrestin 2, restored the greatest degrees of early endosomal translocation, with complete restoration for {beta}-arrestin 1 and partial for {beta}-arrestin 2. Restoration of other sites had minimal or no effect. Dephosphorylation therefore appears to be required for PTH1R-stimulated {beta}-arrestin early endosomal translocation, with different phosphorylation sites implicated than some other GPCRs. These findings raise the possibility of differential {beta}-arrestin regulation by distinct stimuli which may have implications in therapeutic targeting of {beta}-arrestin-dependent pathways.

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Carruthers, E. R., Oyagawa, C. R., Poulsen, R. C., Grimsey, N. L.. 2026-10-12. Phosphorylation of β-arrestins impacts recruitment to Parathyroid Hormone Receptor 1 (PTH1R) at the cell surface and endosomes. https://doi.org/10.64898/2026.10.05.756893

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