Fig. 4.

PAR2 activates PIP5K. (A) Cartoon of voltage-sensitive 5-phosphatase (VSP) used to deplete PM PI(4,5)P₂. (B) Differential interference contrast (DIC) and confocal imaging combined with patch clamp. PAR2-CFP, GFP-VSP and PH-RFP were expressed at the same time. (C) Reaction scheme for PM PI(4,5)P₂ depletion by VSP and resynthesis by PIP5K. (D) Time course of cytoplasmic PH-RFP during and after one brief activation of VSP by a voltage jump. A representative trace. (E) Red symbols: Catalytic rate constant of PIP5K determined from the exponential recovery of PI(4,5)P₂ after repeated VSP pulses applied before and during treatment with AP (100 μM). Mean of five independent experiments. Black symbols: mean time course of PH-RFP probe translocation during the activation of PAR2 by AP. (F and G) Average intensity of clustered β-arrestin-2-mRFP and of GFP-PIP5K-Iγ recorded in conventional TIRF microscopy while AP was applied to PAR2-expressing cells. In the analysis, regions of interest (ROIs) containing individual β-arrestin-2-mRFP clusters (magenta) were selected to measure intensity changes of GFP-PIP5K-Iγ (green), reflecting recruitment of the kinase into β-arrestin 2 clusters. (F) One subset of data with monotonic PIP5K-Iγ recruitment into β-arrestin 2 clusters (group 1, 14 ROIs). (G) The second, remaining subset of data with biphasic PIP5K-Iγ signals at β-arrestin 2 clusters (group 2, 13 ROIs). The gray line was copied from the β-arrestin 2 clustering in F for comparison. The gray line and magenta symbols are significantly different (P < 0.001). (H) FRET between CFP-tagged cytoplasmic PIP5K-Iγ and YFP-tagged β-arrestin 2 (“+ cytoplasmic PIP5K,” n = 9 cells). For the control experiments (“Control,” n = 8 cells), cells were transfected with CFP instead of CFP-PIP5K-Iγ together with β-arrestin-2-YFP and dark PAR2. AP (100 µM) was used to activate PAR2 receptors. *P < 0.05.