Decoding G_q Signaling in Platelets

Most platelet agonists work through G protein coupled receptors (GPCRs), activating pathways that involve members of the G_q, G_i, and G₁₂ families of heterotrimeric G proteins. G_q is critical for most functional responses during platelet activation. A defect in G_q expression in patients leads to impaired platelet secretion and aggregation, which is associated with bleeding diathesis. We have previously shown that the Regulators of G protein Signaling (RGS) proteins function as negative regulators for platelet activation by limiting G protein-dependent signaling, but much remains to be learned about the mechanisms by which G proteins can be regulated during platelet activation.

Here we took advantage of an RGS-insensitive G_q(G188S) mutant mouse line to decipher the regulatory complex of G_q-dependent signaling in platelets. The G188S mutation disrupts RGS18 and G_q interaction in a way similar to what we have observed in an analogous G_i2(G184S) mutation. However, in contrast to increased platelet activation in G_i2(G184S) expressing platelets, G188S expressing platelets have decreased activation in response to thrombin, TxA₂ and ADP, but not to collagen. The hemostatic thrombi formed in G188S mice are significantly reduced with constant embolization as compared with WT controls. Underlying these changes is a decrease in G_q-dependent signaling events, whereas shape change, which is G₁₃-mediated, is unaffected. Our data further reveal that PLCβ3 and GRK2 complex with activated G_q, but not to G_i2. The G188S mutation in G_q not only prevents the binding of RGS proteins to G_q but also impairs the G_q/PLCβ3 interaction, though it does not affect the GRK2 binding to G_q. Instead, there an increase of GRK2 binding to G_q. Finally, the structural analysis shows that PLCβ3 shares a large overlapping binding area with RGS18 to G_q. Computational alanine scanning to predict binding interfaces indicates that the G188S mutation resides in a region important for the binding of both PLC and RGS. The PLC binding interface also overlaps with that of GRK2, which suggests that when PLC is in complex with G_q, it precludes the binding of both RGS proteins and GRK2. In the G188S mutant, however, PLC binding is reduced, which may reduce competition and allow more GRK2 to bind. The binding interfaces of RGS and GRK2 are not predicted to be overlapping, raising the possibility that the two proteins could bind simultaneously to activated WT G_q.

Collectively, these observations 1) indicate that the feedback mechanism of G_q inactivation in platelets is different from that of G_i2, 2) reveal, for the first time, that a G_q/PLCβ/RGS/GRK2 signaling node is present in platelets and plays an important role in G_q-dependent signaling during platelet activation, and 3) show that the G188S mutation affect G_q-mediated signaling by disrupting G_q/PLCβ/RGS interaction.