Figure 6

Effect of local PI4P resynthesis on AT1R internalization. (A) Internalization of AT1R was measured by BRET in HEK 293T cells between AT1R-Rluc and three different endosomal marker proteins: Venus-Rab5 (upper row), Venus-FYVE (WDFY2) (middle row) and Venus-FYVE (EEA1) (lower row). Cells were pretreated with either vehicle (black curves) or A1 (10 nM, blue curves) for 10 min. AT1R-Rluc was stimulated with Ang II (1 nM, 10 nM and 100 nM for the left, middle and right columns, respectively) at time point 0. The BRET ratio change was calculated against nonstimulated baselines (not shown) for each curve. Data are the mean ± SEM of 3 independent experiments, each performed in triplicate. (B) Internalization experiments of the upper row in panel (A) were repeated with the G-protein activation deficient mutant of AT1R (DRY/AAY). This time, BRET was measured between AT1R DRY/AAY-Rluc and Venus-Rab5, as detailed in panel (A). (C) The bar graphs show the rate of AT1R-Rluc internalization 15–20 min after AngII treatment as measured by BRET between AT1R and endosomal markers as indicated. Data are means (± SEM) of the last 5 measurement points of the curves from the left and middle columns of panels (A) and (B). Data were analyzed using one-way ANOVA for each group separately, which showed a significant difference between samples within each group (p < 0.001 for all three groups with wild-type AT1R, p = 0.002 for AT1R DRY/AAY). The Holm-Sidak post hoc test was used for pairwise comparisons within groups, and significance is marked between corresponding vehicle- and A1-pretreated samples. **p < 0.01; NS p > 0.05; n = 3.