Fig. 1

P2Y₁-R constitutively activates Gq protein-dependent signaling in HEK293T cells. a Schematic representation depicting the BRET signal measured at basal state, reflecting the inactive Gαqβ1γ2 complex, and resulting from an energy transfer between the energy donor RLuc8 fused to Gαq protein and the energy acceptor GFP2 fused to Gγ2 protein. Agonist-induced or constitutive receptor activation will promote G protein activation and dissociation that is reflected by a decrease of the BRET signal. b Basal Gαq protein activation was evaluated by measuring basal BRET signal in HEK293T cells co-expressing Gαq-RLuc8, GFP2-Gγ2, and Gβ1 in the absence (pcDNA3.1) or in the presence of P2Y₁-R. Data represent the mean ± s.e.m. of six independent experiments and statistical significance between cells expressing P2Y₁-R or not was assessed using an unpaired t-test (***p < 0.001). c Gαq protein activation was evaluated by measuring BRET signal in HEK293T cells co-expressing Gαq-RLuc8, GFP2-Gγ2, and Gβ1 in the absence (pcDNA3.1) or in the presence of P2Y₁-R, after stimulation or not with ADP (10 μM) for 1 min. Results are expressed as the difference in the BRET signal measured in the presence and in the absence of ADP. Data represent the mean ± s.e.m. of six independent experiments and statistical significance of ADP-induced BRET modulation between cells expressing P2Y₁-R or not was assessed using an unpaired t-test (*p < 0.05). d Basal Gαq protein activation was evaluated by measuring basal BRET signal in HEK293T cells co-expressing Gαq-RLuc8, GFP2-Gγ2, and Gβ1 in the absence (0 μg) or in the presence of increasing amounts of vectors (ranging from 0.001 to 4 μg/dish) encoding AT1-R or P2Y₁-R. Data represent the mean ± s.e.m. of five independent experiments and statistical significance between cells expressing receptors or not (0 μg) was assessed using two-way ANOVA followed by Sidak’s post-tests (****p < 0.0001; ns, not statistically significant). e Gαq protein activation was evaluated by measuring BRET signal in HEK293T cells co-expressing Gαq-RLuc8, GFP2-Gγ2, and Gβ1 in the absence (0 μg) or in the presence of increasing amounts of vectors encoding P2Y₁-R (ranging from 0.001 to 4 μg/dish), after stimulation or not with MRS2179 (10 μM) for 1 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.e.m. of seven independent experiments and statistical significance of MRS2179-induced BRET modulation between cells expressing P2Y₁-R or not (0 μg) was assessed using one-way ANOVA followed by Holm-Sidak’s post-tests. (**p < 0.01; ****p < 0.0001; ns, not statistically significant). f Dose-response curve was performed in HEK293T cells co-expressing Gαq-RLuc8, GFP2-Gγ2, Gβ1, and P2Y₁-R after stimulation or not with increasing concentrations of MRS2179 for 1 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.e.m. of five independent experiments. Statistical significance between unstimulated and stimulated cells was assessed by one-way ANOVA followed by Sidak’s post-tests (*p < 0.05; ***p < 0.001; ****p < 0.0001). Maximal efficacy (Emax ± s.e.m.) and potency (EC50 and pEC50 ± s.e.m.) of MRS2179 are indicated in the inset. g HEK293T cells expressing P2Y₁-R, P2Y₁₂-R, or not (pcDNA3.1) were incubated in the absence (basal) or in the presence of ADP (100 μM), MRS2179 (10 μM), vehicle (DMSO) ,or the Gq inhibitor (100 nM) for 2 h and IP1 accumulation was quantified. Data represent the mean ± s.e.m. of five independent experiments and are expressed as IP1 concentration (nM). The statistical comparison between unstimulated (basal or vehicule (DMSO)) and stimulated (ADP, MRS2179, or Gq inhibitor) cells or between cells expressing the different receptors was assessed using two-way ANOVA followed by Bonferroni’s or Dunnett’s post-tests respectively (*p < 0.05; **** or ^####p < 0.0001; ns, not statistically significant)