Platelet P2Y1 receptor exhibits constitutive G protein signaling and β-arrestin 2 recruitment

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…

Fig. 2. P2Y1-R exhibits constitutive signaling in resting mouse and human platelets. a, b Washed murine (a) or human (b) platelets were incubated in the absence (basal, dashed line) 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.d. of 13 mice (a) or 16 healthy donors (b) and are expressed as the percentage of matched basal values. The statistical comparison between untreated (basal or vehicle) and treated (ADP, MRS2179 or Gq inhibitor respectively) platelets was assessed using Kruskal-Wallis test followed by Dunn’s post-tests (*p < 0.05; **p < 0.01; ***p < 0.001)

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…

Fig. 4. P2Y1-R antagonists behave as inverse agonists. a 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, MRS2279, or MRS2500 for 1 min. Results are expressed as the difference in the BRET signal measured in the presence and in the absence of ligand and are normalized to the mean value of maximal MRS2179 response. Data represent the mean ± s.e.m. of six independent experiments. Statistical significance between unstimulated and stimulated cells was assessed by one-way ANOVA followed by Sidak’s post-tests (**p < 0.01; ***p < 0.001; ****p < 0.0001). b 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, MRS2279, or MRS2500 for 15 min. Results are expressed as the difference in the BRET signal measured in the presence and in the absence of ligand and are normalized to the mean value of maximal MRS2179 response. Data represent the mean ± s.e.m. of five independent experiments. Statistical significance between unstimulated and stimulated cells was assessed by…

Fig. 5. P2Y1-R constitutive activity depends on receptor expression level. Increasing P2Y1-R expression switched the receptor equilibrium from an inactive R conformation to an active R* conformation, thereby promoting a constitutive activation of Gq protein signaling as well as constitutive recruitment of β-arrestin 2, and precluding ADP-induced signaling. By contrast, decreasing P2Y1-R expression had an opposite effect, balancing the receptor equilibrium towards an inactive R state responding to agonist stimulation