Figure 5

Effect of M3R activation-evoked PM PIP2 depletion and PI4P resynthesis on β2AR internalization. (A) PIP2 depletion efficiency was measured by BRET as detailed earlier in Fig. 1B, with the difference that cells expressed type-3 muscarinic acetylcholine receptors (M3R), and lipid depletion was triggered by stimulation with the M3R agonist carbachol (Cch, concentrations as indicated) at time point 0 after pretreatment for 10 min with either vehicle (left graph) or A1 (10 nM, right graph). The signal change was normalized to the last data point before CCh stimulation (100%). Black curves show unstimulated samples. Data show the means ± SEM of 3 independent experiments, each performed in triplicate. (B) Internalization of β2AR was measured by BRET between β2AR-Sluc and Venus-Rab5 as detailed in Fig. 2A, with the distinction that M3R was expressed in all cells instead of AT1R-FRB. Cells were pretreated with either vehicle (left panel) or A1 (10 nM, right panel) for 10 min. M3R was stimulated at time point 0 with CCh (concentrations as indicated on the graph) for approximately 5 min, followed by β2AR-Sluc stimulation with isoprenaline (1 μM). The BRET ratio change was calculated by subtracting baselines not stimulated with isoprenaline. Data show the mean ± SEM of 3 independent experiments, each performed in triplicate. (C) The bar graph on the left shows the rate of β2AR-Sluc internalization 10–15 min after isoprenaline (1 μM) treatment under the marked circumstances, normalized to the nonpretreated and non-PIP2-depleted sample (clear gray bar). Data are means (± SEM) of the last 5 measurement points of the curves from panel B, using the same color code. The graph on the right shows the rate of PIP2 depletion under the same conditions. Bars show the means (± SEM) of the last 5 measurement points of the curves from panel A, using the same color code. Data were analyzed using two-way ANOVA, which showed a significant difference (p < 0.01) between the groups. The Holm‒Sidak post hoc test was used for comparisons within groups, and the results are indicated over the bars (NS—not significant, ***p < 0.001). n = 3.