Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets

Abstract

Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3K␣/␤ subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2؊/؊ mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.Arrestins are cytoplasmic proteins that were originally characterized by their ability to associate with agonist-activated G protein-coupled receptors (GPCRs), 2 mediating their internalization and desensitization (1). More recent studies suggest that arrestins play additional roles in GPCR signaling, by serving as scaffolds to recruit signaling complexes to the receptor, thereby facilitating activation of G protein-dependent and -independent pathways (2, 3). One such arrestinmediated pathway is the PI3K-dependent activation of the Ser-Thr kinase, Akt (4, 5). In fibroblasts, colorectal, and gastric carcinoma cells, arrestins have been found to play a critical role in localizing PI3K to GPCR complexes through an interaction with Src family kinases (SFKs) (6 -8). Perhaps most relevant for platelet agonists, thrombin-stimulated Akt phosphorylation involved activation of both G i and G q : G i -dependent signaling to Akt required ras activation, while G q -dependent Akt activation required arrestin-2 (9).Previous work from our laboratory and others has demonstrated that Akt-dependent pathways contribute to platelet activation by G protein-coupled receptors (10, 11). Yet, the mechanisms leading to Akt activation in platelets remain incompletely defined. Multiple laboratories have demonstrated that thrombin-dependent Akt phosphorylation in platelets is reduced by about 90% in the presence of inhibitors for the G i -coupled ADP receptor, P2Y12, and is blocked by inhibi...

Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin

“…The sequential activation of PAR1 and PAR4 by thrombin may allow the receptors to rearrange in the platelet membrane and alter the signaling response. A recent study by Li et al (49) described how PAR4 cooperates with the ADP receptor, P2Y12. In these studies platelets from arrestin-2-deficient mice had a reduction in Akt phosphorylation and fibrinogen binding in response to PAR4 activation.…”

Highly electronegative LDL from patients with ST-elevation myocardial infarction triggers platelet activation and aggregation

“…27 To determine whether L5 activates platelets through the PKCa signaling pathway, we examined the activity and activation of PKCa in human platelets exposed to L5 from STEMI patients. In the lysate of L5-treated platelets, total PKC activity was increased (data not shown).…”

Protease-activated receptors in hemostasis

“…PARs are no exception as evidence exists for both constitutive and agonist-induced dimers. 8,23,[75][76][77][78] The major point of contention regarding the organization of GPCRs is defining a reliable readout for oligomerization of receptors in native tissues. PARs have a distinct advantage over other receptor families because they cooperate in specific ways that are consistent with dimerization or oligomerization.…”

“…23,78,79 In platelets, PAR4 also forms agonist dependent heterodimers with the ADP receptor, P2Y12. 76,77 Li et al showed that simultaneous stimulation of PAR4 and P2Y12 resulted in arrestin-2 recruitment to PAR4. 77 Following recruitment, arrestin-2 served as a scaffold to support Akt signaling.…”

“…76,77 Li et al showed that simultaneous stimulation of PAR4 and P2Y12 resulted in arrestin-2 recruitment to PAR4. 77 Following recruitment, arrestin-2 served as a scaffold to support Akt signaling. There is a growing body of literature suggesting that heterodimers of GPCRs may influence signaling by acting as allosteric modulators.…”