Fig. 5: PIP3 controls βarr2 recruitment at the plasma membrane (part 1). | Nature Communications

Fig. 5: PIP3 controls βarr2 recruitment at the plasma membrane (part 1).

From: Fast-diffusing receptor collisions with slow-diffusing peptide ligand assemble the ternary parathyroid hormone–GPCR–arrestin complex

Fig. 5

A Principle of the experiments using TGX-221. PTH induces Gq activation (1); Gβγ-dependent recruitment and activation of PI3Kβ at the plasma membrane (2), which in turn catalyzes the formation of PIP3 from PIP2 (3); recruitment of βarr2 (4); and its interaction with PTH1R (5). TGX-221 is a potent PI3Kβ inhibitor. B–D Time-course recording under TIRF illumination of PIP3 levels (B), FRET between βarr-2YFP and PTH1RCFP (C), and βarr-2YFP recruitment to the plasma membrane (D) in response to 10 nM PTH ± TGX-221 in HEK293 cells. Mean ± SEM of N = 3–6 experiments. E Time courses of internalization and recycling of the PTH1R tagged with superecliptic pHluorin (PTHRSEP) that exhibits fluorescence intensity reduction in acidic environments in response to PTH in HEK293 cells preincubated with DMSO (control, black) or 100 nM TGX-221 (pink) for 30 min before live cell imaging control and in βarr2-KO HEK293 cells, measured by time-lapse confocal microscopy. Cells were perfused with a 100 nM PTH (horizontal bar) and then washed out. Mean ± SD from N = 2–3 experiments and n ≥ 100 cells per condition. Expression of β-arrestins in parental and βarrs-KO HEK-293 cells using an anti-βarr1/2 antibody (right panel). Schematic in (A, E) created in BioRender. Vilardaga, J.P. (2025) https://BioRender.com/e81d105 and https://bioRender.com/o82q948, respectively.

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