Platelet P2Y1 receptor exhibits constitutive G protein signaling and β-arrestin 2 recruitment
BMC Biology volume 21, Article number: 14 (2023)
Purinergic P2Y1 and P2Y12 receptors (P2Y1-R and P2Y12-R) are G protein-coupled receptors (GPCR) activated by adenosine diphosphate (ADP) to mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y12-R is the major target of antiplatelet drugs, no P2Y1-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y1-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding. In this context, an accurate characterization of P2Y1-R antagonists constitutes an important preliminary step.
Here, we investigated the pharmacology of P2Y1-R signaling through Gq and β-arrestin pathways in HEK293T cells and in mouse and human platelets using highly sensitive resonance energy transfer-based technologies (BRET/HTRF). We demonstrated that at basal state, in the absence of agonist ligand, P2Y1-R activates Gq protein signaling in HEK293T cells and in mouse and human platelets, indicating that P2Y1-R is constitutively active in physiological conditions. We showed that P2Y1-R also promotes constitutive recruitment of β-arrestin 2 in HEK293T cells. Moreover, the P2Y1-R antagonists MRS2179, MRS2279 and MRS2500 abolished the receptor dependent-constitutive activation, thus behaving as inverse agonists.
This study sheds new light on P2Y1-R pharmacology, highlighting for the first time the existence of a constitutively active P2Y1-R population in human platelets. Given the recent interest of P2Y12-R constitutive activity in patients with diabetes, this study suggests that modification of constitutive P2Y1-R signaling might be involved in pathological conditions, including bleeding syndrome or high susceptibility to thrombotic risk. Thus, targeting platelet P2Y1-R constitutive activation might be a promising and powerful strategy for future antiplatelet therapy.
Co-activation of both P2Y1-R and P2Y12-R is necessary for full platelet aggregation by ADP . P2Y1-R is responsible for platelet shape change and initiates small and reversible ADP-induced platelet aggregation by triggering Gq-dependent phospholipase C (PLC) activation leading to inositol triphosphate (IP3) production and subsequent calcium release into the cytoplasm . P2Y12-R activation results in amplification and stabilization of the aggregation response, through Gi-dependent adenylyl cyclase inhibition and subsequent cyclic adenosine monophosphate (cAMP) decrease . Antiplatelet drugs that target P2Y12-R activation have been extensively developed and are widely used in the treatment and prevention of arterial thrombosis [4, 5], but, by contrast, no selective P2Y1-R antagonist have yet been developed for clinical use. One reason for this is that while P2Y12-R expression is mostly restricted to platelets, P2Y1-R exhibits a broader expression pattern, thereby raising concerns about P2Y1-R inhibition and possible unforeseen outcomes. However, P2Y1-R-null mice are viable with no apparent abnormalities, thus suggesting that P2Y1-R might be a promising target for the development of antiplatelet drugs. Indeed, platelets from P2Y1-R-deficient mice exhibited impaired platelet aggregation in response to ADP and a strong resistance to thrombosis [6, 7]. Moreover, selective inhibition of P2Y1-R in rats using P2Y1-R antagonists reduced both venous and arterial thrombosis . Importantly, P2Y1-R inhibition resulted in only moderate prolongation of the bleeding time [9, 10], making it a good candidate for inhibiting platelet activation with presumably less bleeding outcome while bleeding risk is the major drawback of anti-P2Y12-R therapies. Indeed, P2Y12-R blockers face some limitations as irreversible platelet inhibition achieved by thienopyridines (clopidogrel, prasugrel) displayed a delayed onset of action and increased the risk of bleeding , while reversible binding drugs such as ticagrelor and cangrelor have been recently associated with adverse effects [12, 13].
Recently, the diadenosine tetraphosphate derivative GLS-409 designed to achieve dual inhibition of both P2Y1-R and P2Y12-R was shown to promote potent inhibition of canine coronary artery thrombosis and reversible human platelet inhibition [14, 15]. Thus, developing selective P2Y1-R-targeting drugs might be a novel and promising antithrombotic strategy to ensure efficient inhibition of platelet aggregation with a minimal effect on bleeding.
Interestingly, we and others recently demonstrated that P2Y12-R exhibited constitutive activity on Gi/o proteins and downstream adenylyl cyclase inhibition in human resting platelets [16, 17]. At resting state, this constitutive P2Y12-R signaling might be essential for platelets to respond rapidly to a vessel injury, by lowering cAMP levels and sensitizing platelets prior to activation. Importantly, P2Y12-R constitutive signaling needs to be fine-tuned to ensure proper control of hemostasis since loss of P2Y12-R constitutive activity was associated with bleeding syndrome  while enhanced P2Y12-R constitutive signaling was correlated with platelet hyperactivity in diabetes . Similarly, transgenic mice expressing constitutively active P2Y12-R chimera exhibited increased platelet activation and thrombosis , highlighting the pro-thrombotic role of the P2Y12-R constitutive activity. Therefore, monitoring platelet P2Y12-R constitutive activity might be a powerful readout to evaluate the thrombotic status of patients and thus adjust the antiplatelet therapy by balancing the antithrombotic beneficial effects with the bleeding risk. In this context, the identification of inverse agonists might be a promising avenue for the development of new therapeutic molecules able to precisely modulate the constitutive activity of P2Y12-R and ultimately prevent thrombosis. Interestingly, many P2Y12-R antagonists (AR-C78511, cangrelor, ticagrelor, selatogrel) were indeed described as inverse agonists at P2Y12-R constitutive signaling [16,17,18,19,20], suggesting that the clinical benefits of antiplatelet drugs might be directly related to inverse agonism at P2Y12-R.
Since P2Y1-R is a good candidate for inhibiting platelet activation with presumably less bleeding outcome, here we characterized the pharmacological properties of P2Y1-R regarding the receptor constitutive activity. We demonstrated that much like P2Y12-R on Gi/o protein signaling, P2Y1-R exhibited constitutive activity leading to Gq protein activation and downstream PLC/IP3 signaling both in HEK293T cells and in mouse and human resting platelets. In addition to G protein-dependent signaling, we also investigated β-arrestin 2 recruitment as β-arrestin 2 plays a prominent role in platelet GPCR signaling. In marked contrast with P2Y12-R, we showed that P2Y1-R also displayed constitutive association with β-arrestin 2 in HEK293T cells, highlighting a constitutive recruitment of β-arrestin 2. Interestingly, the P2Y1-R antagonists, MRS2179, MRS2279, and MRS2500, acted as an inverse agonist at P2Y1-R by counteracting both constitutive G protein signaling and β-arrestin 2 recruitment to the receptor.
Altogether, our data provide new critical insights toward P2Y1-R pharmacological characterization, highlighting for the first time the constitutive activity of this receptor and inverse agonism in resting human platelets. Importantly, the level of agonist-independent P2Y1-R and P2Y12-R basal signaling might be directly correlated with the platelet responsiveness and therefore represent a promising readout to evaluate the thrombotic risk.
P2Y1-R exhibits constitutive G𝛼q-dependent signaling in HEK293T cells
Since P2Y12-R was recently shown to exhibit constitutive activity on Gαi/o protein signaling in human resting platelets [16,17,18], we investigated whether P2Y1-R would display ligand-independent activation as well. As previously described [17, 21, 22], using a BRET2 (bioluminescence resonance energy transfer)-based assay, we directly monitored the basal Gαq protein activation by measuring the interaction between Gαq and Gγ2 subunits of the Gαqβ1γ2 heterotrimer complex. Indeed, this assay is based on the non-radiative transfer between the Renilla reniformis luciferase (RLuc8) energy donor fused to the Gαq protein subunit and the fluorescent GFP2 energy acceptor fused to Gγ2 protein subunit (Fig. 1a). At basal state, in the absence of agonist, the preassembled inactive Gαqβ1γ2 heterotrimeric complex will favor the detection of a basal BRET signal due to the close proximity between Gαq and Gγ2 protein subunits. By contrast, ligand-independent constitutive receptor activation or agonist-induced receptor activation will promote Gαq/Gγ2 subunit dissociation, thereby leading to a decrease of the basal BRET signal (Fig. 1a). At basal state, a high basal BRET signal was detected in control cells (pcDNA3.1), indicative of Gαq/Gγ2 proximity and Gαqβ1γ2 inactive complex (Fig. 1b). By contrast, at comparable Gαq protein BRET probe expression (Additional File 1: Fig. S1a), expression of P2Y1-R promoted a strong and significant decrease of the basal BRET signal, suggesting that P2Y1-R constitutively activated Gαq protein (Fig. 1b). To preclude any P2Y1-R activation by a passive release of ADP by HEK293T cells, we performed similar experiments in the presence of high concentration of apyrase (0.2U/mL) to degrade any trace of ADP in the medium (Additional File 2: Fig. S2a). Under these conditions, P2Y1-R expression still induces a marked and significant decrease of the basal BRET signal compared to control cells (pcDNA3.1), thereby supporting a P2Y1-R-dependent constitutive activation of Gαq protein. Surprisingly, ADP only triggered a significant but moderate decrease of the BRET signal (Fig. 1c), indicating a weak Gαq protein activation following P2Y1-R stimulation. These results suggested that, in these experimental conditions, P2Y1-R exhibited a strong constitutive activity on Gαq protein signaling, thereby narrowing ADP-dependent receptor activation, as previously described for P2Y12-R on Gαi/o protein signaling . Interestingly, the constitutive activity is a specific feature of P2Y1-R since the basal BRET signal was gradually decreased with increasing cell surface expression of P2Y1-R (Additional File 3: Fig. S3a, left panel) while, at similar Gαq protein BRET probe expression levels (Additional File 1: Fig. S1b), it was not significantly impacted after increasing plasma membrane expression of another Gαq protein-coupled receptor, the angiotensin II type 1 receptor (AT1-R) (Fig. 1d and Additional File 3: Fig. S3a, right panel). Of note, because P2Y1-R and AT1-R were respectively Myc- and HA-tagged and were thus detected using different primary antibodies, their expression at the cell surface cannot be compared between each other’s. These results demonstrate that, contrary to AT1-R, P2Y1-R displays constitutive activation of Gαq protein in HEK293T cells and that this ligand-independent basal activation correlates with the receptor expression level, in agreement with the well-known receptor expression dependency of GPCR constitutive activity .
Since P2Y12-R antagonists such as ticagrelor and cangrelor were shown to target the receptor constitutive activity, thereby acting as inverse agonists at P2Y12-R [16, 17], we then investigated the pharmacological properties of a P2Y1-R antagonist, MRS2179 , regarding the constitutive activity of P2Y1-R on Gαq protein activation. MRS2179 stimulation induced an increase of the BRET signal of G protein biosensors in cells expressing P2Y1-R (Fig. 1e), indicating that MRS2179 counteracted the constitutive activation of P2Y1-R, thus behaving as an inverse agonist. Indeed, inverse agonists inhibit the constitutive activity of a receptor, by switching the receptor from an active R* to an inactive R conformational state. Consequently, inverse agonists should be prone to increase the BRET signal between Gαq and Gγ2 BRET biosensors, reflecting Gαq/Gγ2 reassembly (Fig. 1a) [17, 25]. This inhibition on P2Y1-R constitutive activity was dependent on receptor amount since MRS2179-modulated BRET signal increased with cell surface P2Y1-R expression level (Fig. 1e and Additional File 3: Fig. S3a, left panel), while Gαq protein BRET probe expression remained constant (Additional File 1: Fig. S1c). Noticeably, MRS2179 exhibited significant inverse agonist efficacy at the highest P2Y1-R expression levels (Fig. 1e), in agreement with the Gαq protein constitutive activation detected at similar receptor expression levels (Fig. 1d). To gain more insight into the inverse agonist efficacy of MRS2179 at P2Y1-R, we performed a concentration-response curve on Gαq protein activity and observed that MRS2179 exhibited a relatively low potency (EC50 = 11.08 μM) (Fig. 1f).
We then further explored the P2Y1-R constitutive activity by monitoring downstream Gαq-dependent signaling in P2Y1-R-expressing HEK293T cells (Fig. 1g). Since Gαq protein activation is known to induce PLC activation leading to IP3 and subsequent cytosolic calcium release, we analyzed the constitutive Gαq-dependent P2Y1-R signaling by quantifying intracellular inositol monophosphate (IP1) using a monoclonal antibody-based competitive ELISA. IP1 is a downstream metabolite of IP3 that accumulates in cells following Gαq protein-coupled receptor activation, making it an ideal readout for Gαq protein-dependent signaling pathways. Interestingly, at basal state, while more expressed than P2Y1-R at the plasma membrane (Additional File 3: Fig. S3b), P2Y12-R did not impact IP1 levels compared to control cells (pcDNA3.1) while P2Y1-R markedly increased IP1 production (Fig. 1g), suggesting a specific constitutive activity of P2Y1-R on PLC/IP3 pathway, in agreement with the constitutive activity detected on the Gαq protein (Fig. 1b). Once again, this basal P2Y1-R-dependent IP1 production cannot be due to the presence of ADP in the medium since it was still detected in P2Y1-R expressing cells in the presence of high apyrase concentration (0.2U/mL), even after 30-min accumulation (Additional File 2: Fig. S2b). Interestingly, IP1 production was slightly but significantly potentiated upon ADP stimulation in cells expressing P2Y1-R, suggesting that under these conditions, a part of P2Y1-R was still in an inactive R conformation that is sensitive to ADP stimulation, in agreement with the detection of ADP-promoted Gαq protein activation (Fig. 1c). P2Y1-R-mediated constitutive IP1 production was strongly inhibited following MRS2179 treatment, thus demonstrating that MRS2179 behaved as an inverse agonist on constitutive Gq/PLC/IP3 signaling (Fig. 1g). Consistently, the plant-derived Gαq inhibitor (FR900359)  also significantly reduced the constitutive P2Y1-R-dependent IP1 production (Fig. 1g).
Altogether, these results strongly support that P2Y1-R constitutively activates the Gq/PLC-dependent signaling in HEK293T cells and that MRS2179 acts as an inverse agonist on this P2Y1-R constitutive signaling (Fig. 1).
Platelet P2Y1-R constitutive signaling is counteracted by MRS2179 inverse agonist
Since our data demonstrated that P2Y1-R exhibits constitutive ADP-independent signaling in HEK3293T cells (Fig. 1), we investigated the relevance of such P2Y1-R constitutive activity and inverse agonist efficacy of MRS2179 in both murine (Fig. 2a) and human (Fig. 2b) washed platelets, in the presence of apyrase (0.02 U/mL) to degrade any trace of ADP and indomethacin to prevent thromboxane A2 production. As performed in HEK293T cells, we thus quantified intracellular IP1 levels to monitor Gαq protein-dependent signaling pathways. We observed that MRS2179 decreased IP1 levels, thus behaving as an inverse agonist and thereby demonstrating a basal IP1 production in the absence of agonist. These results indicate the existence of a P2Y1-R constitutive activation of Gαq signaling in murine (Fig. 2a) and human (Fig. 2b) resting platelets. P2Y1-R constitutive activity was still detected in human platelets incubated with higher apyrase concentration (0.2 U/mL), indicating that the basal IP1 production did not result from platelet activation and ADP release (Additional File 4: Fig. S4a). Accordingly, we demonstrated that dense granule exocytosis and ADP release did not occur in washed human platelets since when assessing surface expression of platelet CD63 activation marker in resting and activated platelets, we showed that stimulation with thrombin receptor activating peptide (TRAP) was able to trigger a strong exposure of CD63 at the platelet surface (Additional File 4: Fig. S4b). Consistently with what we observed in P2Y1-R expressing HEK293T cells (Fig. 1g), the basal IP1 production was also inhibited by the Gαq inhibitor in both mouse (Fig. 2a) and human (Fig. 2b) washed platelets. Notably, IP1 production was significantly increased upon ADP stimulation in murine and human platelets, much like in P2Y1-R expressing HEK293T cells, suggesting the existence of two P2Y1-R populations at resting state: one “pre-active” ADP-insensitive receptor population exhibiting constitutive activity and another “inactive” receptor population that is responsive to ADP.
Altogether, these data highlighted the constitutive activity at P2Y1-R/Gαq pathways in mouse and human resting platelets and demonstrated that MRS2179 displayed inverse agonist efficacy by counteracting P2Y1-R-dependent signaling.
P2Y1-R is constitutively associated with β-arrestin 2
In addition to G protein-dependent signaling, GPCRs elicit β-arrestin-dependent signaling pathways. Since β-arrestin 2 was involved in platelet GPCR desensitization, we then investigated β-arrestin 2 recruitment following P2Y1-R activation.
We assessed β-arrestin 2 recruitment to the receptor using BRET1 assay. The assay measures the interaction between β-arrestin 2 fused to the Renilla reniformis luciferase (RLuc) energy donor and the receptor fused to fluorescent Venus energy acceptor (Fig. 3a). The recruitment of cytosolic β-arrestin 2 to the receptor at the plasma membrane in the presence of an agonist will promote a significant increase of the BRET signal compared to the basal state, reflecting the close proximity between the β-arrestin 2 energy donor and the receptor energy acceptor .
Interestingly, at basal state, we detected a significant BRET signal between P2Y1-R-Venus and β-arrestin 2-RLuc, indicating that P2Y1-R and β-arrestin 2 are already associated prior any agonist stimulation (Fig. 3b), thus demonstrating the constitutive activity of P2Y1-R on β-arrestin 2 pathway. Conversely, in similar conditions, P2Y12-R did not exhibit such a constitutive interaction with β-arrestin 2. As observed for P2Y1-R-dependent constitutive Gq protein activation (Additional File 2: Fig. S2a), the basal interaction between P2Y1-R—but not P2Y12-R—and β-arrestin 2 was also detected in the presence of high concentration of apyrase (Additional File 5: Fig. S5), thereby precluding any ADP-dependent β-arrestin 2 recruitment at P2Y1-R due to ADP release in the medium. To further confirm the specificity of P2Y1-R/β-arrestin 2 constitutive interaction, we performed BRET saturation curves in HEK293T cells co-expressing a fixed amount of the energy donor β-arrestin 2-RLuc and an increasing amount of the energy acceptor P2Y1-R-Venus. We detected a basal BRET signal between P2Y1-R-Venus and β-arrestin 2-RLuc that increased hyperbolically and saturated at high P2Y1-R-Venus concentration, thus demonstrating the specificity of the BRET signal (Fig. 3c). By contrast, a very weak BRET signal was detected when experiments were performed with P2Y12-R-Venus or soluble Venus as a negative control. This BRET signal is independent of the Venus expression level leading to linear curves, most likely reflecting bystander, nonspecific BRET signal (random collision) (Fig. 3c).
Surprisingly, in cells expressing P2Y1-R or P2Y12-R, ADP stimulation did not trigger β-arrestin 2 recruitment (Fig. 3d). By contrast and as expected, in AT1-R-expressing cells, Angiotensin II (AngII) promoted a time-dependent β-arrestin 2 recruitment (Fig. 3e). Importantly, MRS2179 significantly decreased the BRET signal between P2Y1-R-Venus and β-arrestin 2-RLuc, but not with P2Y12-R, reflecting a dissociation between P2Y1-R and β-arrestin 2 and thus demonstrating that MRS2179 behaved as a specific inverse agonist at P2Y1-R on the β-arrestin 2 recruitment (Fig. 3f). To further characterize the inverse agonist potency of MRS2179 at P2Y1-R, we performed a concentration-response curve on β-arrestin 2 recruitment and observed that much like on Gαq signaling (Fig. 1f), MRS2179 exhibited a low potency in the micromolar range (EC50 = 4.561 μM) (Fig. 3g).
Since GPCR constitutive activity is known to correlate with receptor expression level (Fig. 1d–e), we then monitored β-arrestin 2 recruitment in cells expressing decreasing amount of P2Y1-R. We found that decreasing receptor expression unveiled a significant agonist efficacy of ADP on β-arrestin 2 recruitment, with a concomitant loss of the inverse agonist efficacy of MRS2179 (Fig. 3h). Thus, decreasing receptor expression level—while keeping constant β-arrestin 2-RLuc probe expression (Additional File 6: Fig. S6a)—switched P2Y1-R from a constitutive active R* state targeted by MRS2179 to an inactive R and agonist-sensitive state, as reflected by the decrease of the basal BRET signal between P2Y1-R-Venus and β-arrestin 2-RLuc (Additional File 6: Fig. S6b). Accordingly, and in agreement with what we observed with ADP stimulation (Fig. 3h), the potent purinergic agonist 2-methylthioadenosine diphosphate (2MeSADP) only promoted β-arrestin 2 recruitment in cells expressing low levels of P2Y1-R (Additional File 6: Fig. S6c).
Altogether, these results strongly supported that at basal state, and at high expression level, P2Y1-R displayed a constitutive activation of β-arrestin 2 signaling and that MRS2179 behaved as an inverse agonist to counteract receptor-dependent constitutive β-arrestin 2 recruitment.
P2Y1-R antagonists behaved as inverse agonists
We further deeply depicted the pharmacological signature of P2Y1-R by testing two other receptor antagonists. Thus, we explored the pharmacological properties of MRS2279 and MRS2500 and compared to MRS2179 by performing new concentration-response curve experiments on both Gαq protein activity and β-arrestin 2 recruitment in HEK293T cells. We observed that like MRS2179, the two P2Y1-R antagonists MRS2279 and MRS2500 behaved as inverse agonists on both P2Y1-R-dependent constitutive Gαq protein (Fig. 4a) and β-arrestin 2 (Fig. 4b) signaling.
In particular, we observed that although the three compounds displayed comparable maximal efficacy (Emax) on both constitutive Gαq protein activation and β-arrestin 2 recruitment, MRS2500 exhibited a greater potency than MRS2179 and MRS2279 (Table 1). Noteworthy, while MRS2179 had comparable potencies on both Gαq protein and β-arrestin 2 signaling, MRS2500 appeared to be more potent on β-arrestin 2 signaling than on Gαq protein signaling (Table 1), indicating that MRS2500 behaved as a biased inverse agonist compared to MRS2179.
Altogether, these results demonstrated the different pharmacological signatures of the three molecules at P2Y1-R constitutive activity.
Importantly, we also validated the physiological relevance of those results in human washed platelets and showed that as observed for MRS2179, MRS2279, and MRS2500 also exhibited inverse agonist efficacy on P2Y1-R-dependent constitutive Gαq signaling in human platelets with physiological expression levels of both endogenous P2Y1-R and G proteins (Fig. 4c).
In this study, we demonstrated for the first time the constitutive P2Y1-R signaling on PLC/IP3 pathway and the inverse agonist efficacy of MRS2179 in both HEK293T cells and mouse and human resting platelets. Interestingly, P2Y12-R, another ADP receptor playing a key role in platelet aggregation, also displayed constitutive signaling in human platelets [16, 17], emphasizing the importance of basal activation of ADP receptors in platelet physiology. As already suggested for constitutive P2Y12-R/Gi/o activation, we can speculate that at resting state, platelets already exhibit basal “low noise” P2Y1-R/Gq signaling that could not initiate per se platelet activation but that would ensure a rapid response to vascular injury.
Moreover, we showed that P2Y1-R—but not P2Y12-R—also exhibited constitutive interaction with β-arrestin 2. β-arrestins are key players of GPCR function and were primarily proposed to turn off GPCR signaling by triggering receptor desensitization and internalization . Further studies then reported that β-arrestins also acted as signal transducers by scaffolding signaling complexes leading to the activation of signaling pathways, including MAPK signaling . Therefore, constitutive basal P2Y1-R/β-arrestin 2 interaction could ultimately regulate receptor internalization and therefore affect receptor intracellular location and signaling capabilities. While the role of P2Y1-R/Gq signaling was well described in platelet aggregation and closely related to shape change through an increase in intracellular calcium triggered by Gq/PLC activation, the β-arrestin 2 signaling was contrastingly poorly documented. Recently, β-arrestins were described as negative regulators of GPCR signaling, acting like a brake on platelet aggregation . In the literature, conflicting reports described either β-arrestin-dependent or β-arrestin-independent P2Y-R internalization and/or desensitization. Thus, Mundell and colleagues failed to observe β-arrestin 2 recruitment in P2Y1-R-expressing cells following ADP stimulation but showed an ADP-induced β-arrestin 2 translocation at P2Y12-R [30, 31]. In marked contrast, P2Y1-R was shown to promote ADP-induced β-arrestin 2 recruitment to the plasma membrane while P2Y12-R did not . These discrepancies between different studies might be directly related to P2Y1-R expression level. Indeed, as already reported in the literature , GPCR constitutive activity is known to rise as a function of increasing receptor expression. Accordingly, we demonstrated here that increasing P2Y1-R expression triggered a constitutive association of the receptor with β-arrestin 2, precluding ADP-induced β-arrestin 2 recruitment while decreasing P2Y1-R expression had an opposite effect, balancing the receptor equilibrium towards an inactive population responding to agonist stimulation and promoting β-arrestin 2 recruitment (Figs. 3h and 5 and Additional File 6: Fig. S6c).
So far, whether the constitutive P2Y1-R activation on both G protein and β-arrestin 2 pathways relies solely on a unique receptor population or two separate ones still remains an open question. Thus, understanding the molecular basis of constitutively active P2Y1-R appears as a tremendous challenge to decipher the role of basal P2Y1-R signaling in platelet activation. One may envision that constitutive P2Y-R activity was associated with some receptor populations physically restricted to special membrane domains. GPCR organization at the cell surface relies on a dynamic equilibrium between monomers, dimers, and high-order oligomers . Over the past years, a growing body of evidence reported that oligomerization could influence membrane expression, trafficking, and functional activity of GPCR, sometimes even generating novel pharmacological and signaling properties . More recently, CXCR4 dimerization was closely linked to its basal activity . A CXCR4 mutant displaying no basal activity was monomeric, supporting a positive correlation between CXCR4 basal signaling and dimeric organization. Interestingly, inverse agonists efficiently reduced CXCR4 basal activity but also abolished receptor dimerization .
In living cells, P2Y1-R and P2Y12-R exist as different oligomeric states and are capable to form not only homo-oligomers but also hetero-oligomers [37, 38]. Since previous studies demonstrated reciprocal cross-talk between P2Y1-R and P2Y12-R signaling , one can suggest that P2Y1-R/P2Y12-R dimers or oligomers could regulate basal constitutive activation of both receptors to fine-tune the purinergic signaling in platelets.
Furthermore, an increasing number of studies suggests that membrane components—i.e. lipids—surrounding GPCR may modulate receptor oligomeric states and thus possibly regulate basal receptor signaling . P2Y1-R and P2Y12-R partitioned, at least in part, into cholesterol-enriched raft domains [37, 41]. Interestingly, in vivo clopidogrel treatment mostly converted P2Y12-R oligomers into dimers that partitioned outside the lipid rafts in freshly isolated platelets . In contrast to cangrelor and ticagrelor, it is not known if clopidogrel behaves as an inverse agonist at P2Y12-R/Gi/o signaling, but it is tempting to speculate that ADP receptors inserted into raft domains undergo conformational constraints favoring oligomerization and constitutive activity. Inverse agonists including antiplatelet drugs could then inhibit basal receptor activation, by releasing GPCR out of these domains, possibly by disrupting oligomerization. In this context, inverse agonists might be an interesting field for future investigations to develop new therapeutic molecules able to modulate selectively the constitutive activity of P2Y receptors.
The physiological relevance of GPCR constitutive activity was unveiled in many biological processes as alterations of agonist-independent receptor signaling were associated with various diseases . Accordingly, P2Y12-R displayed increased expression and constitutive activation in subjects with diabetes mellitus that exhibit platelet hyperactivity and high thrombotic risk . By contrast, the bleeding syndrome-related R122C mutation of P2Y12-R correlated with a loss of constitutive receptor signaling . Hence, monitoring P2Y12-R as well as P2Y1-R constitutive activation might be an early marker of platelet hyper-reactivity—usable in clinical practice—that could be directly associated with the bleeding or thrombotic risks. This should be particularly pertinent for the prediction/management of thrombosis in elderly patients as age-related diseases, including cardiovascular diseases, type 2 diabetes and Alzheimer’s disease, are associated with dysregulated platelet functions, platelet hyperactivity, enhanced aggregation, and/or increased risk of thrombotic events [43,44,45]. In this context, the reclassification of P2Y1-R antagonists into inverse agonists could have important pharmacological and therapeutic applications.
Apart from platelets, P2Y1-R also achieved many additional roles in other cell types, and particularly throughout the brain in neurons, astrocytes, and microglia. Interestingly, astrocyte hyperactivity, which is an important contributor to neuronal-glial network dysfunction in Alzheimer’s disease, was driven by enhanced P2Y1-R expression and activity . Importantly, P2Y1-R inhibition with MRS2179 restored network homeostasis and protected from the decline of spatial learning and memory in an Alzheimer’s disease mouse model , thereby highlighting P2Y1-R as a novel target in the treatment of Alzheimer’s disease. Similarly, in epileptic models, brain P2Y1-R expression is increased  and correlated with an abnormal pattern of intracellular calcium oscillations. In this model, P2Y1-R antagonists normalize the duration of astroglial calcium oscillations and protect against seizure-induced cortical damages [48, 49]. Although those defects of intercellular calcium waves were primarily attributed to a release of P2Y1-R agonist both in Alzheimer’s disease and in epilepsy, we cannot exclude that such modifications of P2Y1-R expression levels can directly affect basal signaling of the receptor. Indeed, neuronal dysfunction and astrocyte hyperactivity might be directly linked to a detrimental increased P2Y1-R expression, and consequently enhanced basal P2Y1-R/Gq signaling, that would be responsible, at least in part and independently of agonist stimulation, for the increase in spontaneous astroglial calcium events. In this context, basal P2Y1-R constitutive activity could be an important feature to evaluate in the receptor pharmacological signature as it could unexpectedly contribute to disease pathogenesis.
In recent years, the understanding of GPCR pharmacology and ligand efficacy opened up new avenues for GPCR drug discovery strategies. In particular, accumulating data have provided unequivocal evidences for the physiological relevance of the ligand-independent constitutive activation of GPCR and demonstrated the therapeutic value of modulating the constitutive activity by inverse agonists. In the case of the P2Y12 receptor, its constitutive activation started to gain interest since it was associated with high thrombotic risk when enhanced in diabetes patients or correlated with bleeding syndrome when abolished.
Here, we demonstrated that P2Y1-R also exhibits constitutive signaling in human platelets and that MRS2179, MRS2279 and MRS2500 behave as inverse agonists. Since this ligand-independent P2Y1-R constitutive activity is closely related to receptor expression level, monitoring P2Y1-R expression and constitutive activation could be a promising readout to evaluate the thrombotic risk in platelets and also in other cell types as P2Y1-R functions in a broad range of tissues. In the future, the development of selective inverse agonists of P2Y1-R might be a powerful strategy for antiplatelet therapy as well as for treatments for neurodegenerative disorders.
Human P2Y1-R (NCBI Reference Sequence: NM_002563.2) and P2Y12-R (NCBI Reference Sequence: NM_022788.3) were fused to double Myc epitope and rat AT1-R (NCBI Reference Sequence: NM_030985.4) to HA tag in N-terminus extracellular region. Alternatively, receptors were also fused to Venus tag in C-terminus intracellular region. Plasmids encoding Gαq-RLuc8, GFP2-Gγ2 and untagged Gβ1 were previously described [17, 21, 22]. ADP was purchased from Sigma-Aldrich/Merck (Darmstadt, Germany). MRS2179, MRS2279, MRS2500, and 2MeSADP were purchased from Tocris Bioscience (Bristol, UK) and luciferase substrates (coelenterazine 400a and h) from Interchim (Los Angeles, CA, USA). The Gq inhibitor (FR900359) was kindly supplied by Evi Kostenis (University of Bonn. Germany).
Cell culture and transfection
Human embryonic kidney HEK293T/17 cells (ATCC) were maintained in DMEM AQmedia (Sigma-Aldrich/Merck, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Life technologies) and 100 units/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. Twenty-four hours after cell splitting, transient transfections were performed using polyethylenimine (PEI, Polysciences Inc.) according to the manufacturer’s instructions.
Bioluminescence resonance energy transfer (BRET) measurements
G protein activation and β-arrestin 2 recruitment were performed as previously described [17, 21, 22]. Briefly, vectors encoding receptors, Gαq-RLuc8, GFP2-Gγ2, Gβ1, or β-arrestin 2 were transiently cotransfected into HEK293T/17 cells as indicated in the figure legends. Forty-eight hours after transfection, cells were washed and resuspended in PBS containing 0.1% (w/v) glucose at room temperature and then distributed (80 μg proteins/well) in a 96-well microplate (PerkinElmer). For G protein activation experiments (BRET2), cells were incubated in the absence (basal BRET signal) or in the presence of ligand for 1 minute. BRET signal between RLuc8 and GFP2 was measured after addition of the luciferase substrate coelenterazine 400a (5 μM). For β-arrestin 2 recruitment (BRET1), cells were stimulated with ligands for 5 or 15 min. BRET signal was recorded after incubation with coelenterazine h (5 μM) for 8 min. For kinetics studies, BRET signal between RLuc and Venus was recorded at 20-s intervals at basal state for the 2 first minutes and then for 5 min after ligand injection. For saturation curves (BRET1), the expression level of Venus- or RLuc-tagged protein was determined by direct measurement of total fluorescence and luminescence respectively. Total fluorescence was first measured with an excitation filter at 485 nm and an emission filter at 520 nm. Then, the same sample was incubated for 8 minutes with 5 μM coelenterazine h and the total luminescence was measured. BRET2 and BRET1 readings were collected using a modified Infinite F500 (Tecan Group Ltd). The BRET2 signal was calculated by the ratio of emission of GFP2 (510–540 nm) to RLuc8 (370–450 nm) and the BRET1 signal by the ratio of emission of Venus (520–570 nm) to RLuc (370–480 nm). Sometimes, results are expressed as the Net BRET computed by deducting the BRET background signal (obtained in the presence of the energy donor alone) from the BRET signal (acquired from cells expressing both the energy donor and acceptor).
Quantification of cell surface receptors by ELISA
Cell surface receptor quantification was performed as previously described . Briefly, HEK293T/17 cells were split into 24-well plates pre-coated with Poly-D-lysine, transiently transfected with a control empty vector (pcDNA3.1) or increasing amounts of vector encoding N-terminally Myc-tagged P2Y1-R or HA-tagged AT1-R. Forty-eight hours post-transfection, cells were fixed (1% paraformaldehyde), saturated (PBS–1% BSA) and incubated with the primary anti-Myc antibody (Clone 9E10. 1:500. Santa Cruz Biotechnology. Dallas, Texas, USA) or anti-HA (Clone 16B12. 1:2500. BioLegend. San Diego. California, USA) and then with HRP-labeled secondary antibody (Sigma-Aldrich/Merck. 1:1000. Darmstadt, Germany). After washing, cells were incubated for 15 min with HRP substrate: TMB (3,39,5,59-tetramethylbenzidine) (BD Biosciences). The reaction was stopped with HCl 1N, and the plates were read at 450 nm in a microplate reader (Infinite F500. Tecan Group Ltd. Männedorf, Switzerland).
Two-month-old C57BL/6JRccHsd male mice (Envigo) were used for experiments and housed in the Anexplo (Toulouse) vivarium according to institutional guidelines. Ethical approval for animal experiments was obtained from the French Ministry of Research in agreement with European Union guidelines. Mice were housed in conventional cages under specific pathogen-free conditions in a constant temperature (20–22 °C) and humidity (50–60%) animal room with a 12/12 h light/dark cycle and free access to food and water.
Washed murine platelets
Whole blood was drawn from the inferior vena cava of anesthetized mice (100 mg/kg ketamine, 10 mg/kg xylazine) into a syringe containing acid citrate dextrose (ACD) (1 volume anticoagulant/9 volumes blood). Platelet-rich plasma (PRP) was obtained by mixing blood with 1 volume of modified Hepes Tyrode’s buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO3, 0.3 mM NaH2PO4, 1 mM MgCl2, 2 mM CaCl2, 5.5 mM glucose, 5 mM HEPES, and 0.35% (w/v) BSA, pH 6.7) followed by a centrifugation at 150 g for 2 min at 37 °C. Then, platelets were pelleted by centrifugation at 1000 g for 4 min, resuspended in modified Hepes Tyrode’s buffer (pH 7.4), adjusted to 3.108 platelets/mL in the presence of 0.02 U/mL of apyrase and 10 μM indomethacin and rested for 45 minutes at 37°C before platelet stimulation. PGI2 (0.5 μM) was added before centrifugation steps to avoid platelet activation.
Washed human platelets
Human platelets were prepared from adult healthy volunteers free of antiplatelet or anti-inflammatory medication since at least ten days. After informed consent, venous blood was collected, anticoagulated with 0.105 M citrate, and centrifuged (180 g, 10 min, room temperature) to obtain platelet-rich plasma (PRP). After two steps of centrifugation and suspension in Tyrode’s buffer (140 mM NaCl, 5 mM KCl, 5 mM KH2PO4, 1 mM MgSO4, 10 mM HEPES, 5 mM glucose and 0.35% (w/v) BSA, pH 6,7), washed platelets were suspended in the same buffer adjusted to pH 7.4 and containing 1 mM CaCl2. The final platelet suspension was adjusted to 3.108 platelets/mL and rested for 45 min in the presence of 0.02 U/mL apyrase and 10 μM indomethacin at 37 °C prior experiments. PGI2 (0.5 μM) was added before centrifugation steps to avoid platelet activation.
IP1 accumulation assay
Quantification of intracellular IP1 was performed using the HTRF (Homogeneous Time Resolved Fluorescence) IP1 competitive immunoassay (IP-One Tb kit. Cisbio. France) according to the manufacturer’s instructions. Briefly, 20,000 HEK293T/17 cells or 3.106 platelets were distributed in a 384-well white microplate (Greiner) and incubated with the indicated molecules for 30 min or 2 h at 37 °C in the presence of 50 mM of LiCl to prevent IP1 degradation. After addition of d2-labeled IP1 (acceptor) and anti-IP1-Cryptate (donor) for 1 h, the specific FRET signals were calculated by the fluorescence ratio of the acceptor and donor emission signal (665/620 nm) collected using a modified Infinite F500 (Tecan Group Ltd). Conversion of the HTRF ratio of each sample into IP1 concentrations was performed on the basis of a standard curve to determine the linear dynamic range of the assay.
Flow cytometry analysis
For platelet dense granule secretion, washed human platelets were stimulated with 50 μM TRAP (10 min, 37 °C) and stained with conjugated anti-CD63 FITC-conjugated antibody (BD Biosciences reference 557288. 1:5) for 15 min at room temperature. The platelets were then diluted into 1 mL PBS and samples were then kept in the dark until analysis by flow cytometry. The results are expressed as median fluorescence intensity (MFI).
Data and statistical analysis
Statistical analysis was carried out using the GraphPad Prism 9.1 software (GraphPad Software Inc.). Statistical tests used are indicated in the figure legends. A p value < 0.05 was considered as significant.
Bioluminescence resonance energy transfer
Cyclic adenosine monophosphate
G protein-coupled receptor
Homogeneous time resolved fluorescence
Thrombin receptor activating peptide
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We thank the Anexplo animal Facilities (UMS US006/INSERM/Toulouse. France).
MPG, LOM, BP, JMS, CG2, and VP are supported by the Institut National de la santé et de la Recherche Médicale (INSERM). CG2 is supported by grant from the Fondation Bettencourt-Schueller. BP is supported by Fondation pour la Recherche Médicale (FRM grant DEQ20170336737).
Ethics approval and consent to participate
This study was performed in line with the principles of the Declaration of Helsinki. Healthy donors were recruited under a protocol approved by the Toulouse Hospital Bio-Resources biobank, declared to the Ministry of Higher Education and Research (DC2016-2804). Blood was processed following hospital guidelines.
All procedures in animals were performed in accordance with institutional guidelines for animal research and were approved by the French Ministry of Research in agreement with European Union guidelines.
Informed consent was obtained from all individual participants included in the study.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
P2Y1-R constitutively activates Gq protein-dependent signaling in HEK293T cells in the presence of high apyrase concentration. a. 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 after incubation or not with 0.2U/mL apyrase for 1, 5 or 10 minutes. Data represent the mean ± s.e.m. of four independent experiments and statistical significance between cells expressing P2Y1-R or not was assessed using one-way ANOVA followed by Sidak’s post-tests (*** p < 0.001). b. HEK293T cells expressing P2Y1-R, P2Y12-R or not (pcDNA3.1) were incubated in the presence of 0.2U/mL apyrase for 30 minutes or 2 hours and basal IP1 accumulation was quantified. Data represent the mean ± s.e.m of five independent experiments and are expressed as the percentage of the control mean (pcDNA3.1) at 30 minutes. The statistical comparison was assessed using one-way ANOVA followed by Sidak’s post-tests (*p < 0.05; **p < 0.01; ns, not statistically significant). (PPT 233 kb)
Relative receptor expression at the cell surface. a. HEK293T cells were transfected with increasing amounts of vectors encoding N-terminally Myc-tagged P2Y1-R (left panel) or HA-tagged AT1-R (right panel). Then, cell surface receptor expression was quantified. Data represent the mean ± s.e.m. of six independent experiments and results are expressed as the optical density (OD450nm) value after subtracting the background value obtained in control cells transfected with an empty vector (pcDNA3.1). Statistical significance was assessed by comparing the values obtained with receptor expression to the background value using one-way ANOVA followed by Dunnett’s post-tests (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). b. HEK293T cells were transfected with N-terminally Myc-tagged P2Y1-R or P2Y12-R. Then, cell surface receptor expression was quantified. Data represent the mean ± s.e.m. of six independent experiments and results are expressed as the optical density (OD450nm) value after subtracting the background value obtained in control cells transfected with an empty vector (pcDNA3.1). Statistical significance between receptor expressions was assessed using an unpaired t-test (**p < 0.01). (PPT 225 kb)
P2Y1-R exhibits constitutive signaling in resting human platelets in the presence of high apyrase concentration. a. Washed human platelets were incubated in the presence of high concentration of apyrase (0.2U/mL) in the absence (basal) or in the presence of MRS2179 (10 μM) for 30 minutes or 2 hours and IP1 accumulation was quantified. Data represent the mean ± s.e.m. of 4 healthy donors and are expressed as the percentage of basal mean at 30 minutes. The statistical comparison between untreated (basal) and treated (MRS2179) platelets was assessed using one-way ANOVA followed by Sidak’s post-tests (*p < 0.05; ***p < 0.001). b. Secretion of platelet dense granules was assessed by flow cytometry using selective anti-CD63 antibody. Washed human platelets were analyzed either in resting conditions (basal) or following 10 minutes stimulation by TRAP (50 μM). Results are expressed as median fluorescence intensity (MFI) and data represent the mean ± s.e.m. of 4 healthy donors. Statistical analysis was performed using a paired t-test (**p < 0.01). (PPT 170 kb)
P2Y1-R constitutively associates with β-arrestin 2 in the presence of high apyrase concentration. Basal BRET signal was evaluated in HEK293T cells expressing β-arrestin 2-RLuc alone (pcDNA3.1) or together with P2Y1-R-Venus or P2Y12-R-Venus in the presence or not of 0.2U/mL apyrase. Data represent the mean ± s.e.m. of four independent experiments and statistical significance between cells expressing receptors or not was assessed using one-way ANOVA followed by Sidak’s post-tests (***p < 0.001; ns, not statistically significant). (PPT 133 kb)
Decreasing P2Y1-R cell surface expression unveils agonist-mediated β-arrestin 2 recruitment. a. Relative expression of β-arrestin 2-RLuc probe was assessed by luminescence measurement. Data represent the mean ± s.e.m. of five independent experiments. b. Basal BRET signal was evaluated in HEK293T cells expressing β-arrestin 2-RLuc and decreasing amounts of vectors encoding of P2Y1-R-Venus. Data represent the mean ± s.e.m. of five independent experiments. c. 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 2MeSADP (10 μM) for 15 minutes. 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; ns, not statistically significant).
Source data for Figs 1-4.
Source data for Figs S1-S6.
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Ribes, A., Garcia, C., Gratacap, MP. et al. Platelet P2Y1 receptor exhibits constitutive G protein signaling and β-arrestin 2 recruitment. BMC Biol 21, 14 (2023). https://doi.org/10.1186/s12915-023-01528-y
- Constitutive signaling
- Inverse agonism
- P2Y receptor