Fig. 6: PIP₃ controls βarr2 recruitment at the plasma membrane (part 2).

A Principle of the experiment to recruit PTEN selectively to the plasma membrane through rapamycin-induced heterodimerization between FKBP (FK506 binding protein 12) and FRB (FKBP12 rapamycin binding) fused to mcherry-tagged PTEN (PTEN^mcherry) and Lyn₁₁, respectively; PIP₃ was detected using PH-Akt^venus. B Time-course recording βarr-2^YFP recruitment to the plasma membrane under TIRF illumination in response to PTH (10 nM) ± rapamycin in HEK293 cells co-expressing PTH₁R, Lyn₁₁-FRB and FKBP-tagged PTEN^mCherry. Means ± SEM of N = 6–9 experiments. C Representative trajectories of βarr2^mNG molecules in HEK293 cells expressing PTH₁R in the absence (basal), and the presence of PTH without (PTH) or with TGX221, (PTH + TGX221). Scale bar: 5 µm. D, E Average number of βarr2^mNG molecules normalized by time (s) and the area of the cell in the presence of PTH alone or together with TGX-221 (D) and corresponding survival functions (1-CDF) for the duration of βarr2^mNG trajectories (E, left). Curves were fit using a double exponential decay function (right). Mean ± 95% confidence intervals (C.I.) of n = 14/2744 (basal), n = 27/9099 (PTH), n = 24/6984 (PTH + TGX-221) and n = 14/5228 (fixed) (D) cells/average trajectories per cell of N = 3 experiments. *P = 0.0311 by unpaired t-test (D). ****P < 0.0001 and ns, not significant (P = 0.3358) by one-way ANOVA with a Dunnett multiple comparison with respect to fixed cells expressing PTH₁R^mNG (E). Schematic in panel (A) created in BioRender. Vilardaga, J.P. (2025) https://BioRender.com/e81d105.