Fig. 3

P2Y₁-R triggers constitutive recruitment of β-arrestin 2. a Schematic representation depicting the BRET signal measured between β-arrestin 2 fused to the energy donor RLuc and the receptor fused to the energy acceptor Venus. The recruitment of β-arrestin 2 to the receptor in the presence of an agonist will promote an increase of the BRET signal compared to the basal state as the proximity between the β-arrestin 2 energy donor and the receptor energy acceptor increased. b Basal BRET signal was evaluated in HEK293T cells expressing β-arrestin 2-RLuc alone (pcDNA3.1) or in the presence of P2Y₁-R-Venus, or P2Y₁₂-R-Venus. Data represent the mean ± s.e.m. of five independent experiments and statistical significance between cells expressing receptors or not was assessed using one-way ANOVA followed by Dunnett’s post-tests (***p < 0.001; ns, not statistically significant). c Basal BRET signal was measured in HEK293T cells co-expressing a fixed amount of β-arrestin 2-RLuc and increasing amounts of P2Y₁-R-Venus, P2Y₁₂-R-Venus, or Venus. Results are expressed as the Net BRET and were analyzed by nonlinear regression on a pooled data set from five independent experiments assuming a model with one-site binding. d–f β-arrestin 2 recruitment was evaluated by monitoring BRET signal in HEK293T cells co-expressing β-arrestin 2-RLuc and P2Y₁-R-Venus, P2Y₁₂-R-Venus, or AT1-R-Venus, after stimulation or not with ADP (10 μM) (d), AngII (10 μM) (e), or MRS2179 (10 μM) (f) for 5 min. The 2 first minutes represent the BRET signal at basal state, before injecting the ligand. Results are expressed as the Net BRET and data represent the mean ± s.e.m. of five independent experiments. g Dose-response curve was performed in HEK293T cells co-expressing β-arrestin 2-RLuc and P2Y₁-R-Venus after stimulation or not with increasing concentrations of MRS2179 for 15 min. Results are expressed as the difference in the BRET signal measured in the presence and in the absence of MRS2179. Data represent the mean ± s.d. of six independent experiments. Statistical significance between unstimulated and stimulated cells was assessed by Friedman test followed by Dunn’s post-tests (**p < 0.01; ***p < 0.001; ****p < 0.0001). Maximal efficacy (Emax) and potency (EC50 and pEC50 ± s.e.m.) of MRS2179 are indicated in the inset. h β-arrestin 2 recruitment was evaluated by monitoring BRET signal in HEK293T cells co-expressing β-arrestin 2-RLuc and decreasing amounts of vectors encoding P2Y₁-R-Venus after stimulation or not with ADP (10 μM) or MRS2179 (10 μM) for 15 min. Results are expressed as the difference in the BRET signal measured in the presence and in the absence of ligand. Data represent the mean ± s.e.m. of five independent experiments. Statistical significance between unstimulated and stimulated cells was assessed using a paired t-test (**p < 0.01; ***p < 0.001; ns, not statistically significant)