Regulator of G-protein signaling 18 integrates activating and inhibitory signaling in platelets

Abstract

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein for the G-␣-q and G-␣-i subunits of heterotrimeric Gproteins that turns off signaling by G-protein coupled receptors. RGS18 is highly expressed in platelets. In the present study, we show that the 14-3-3␥ protein binds to phosphorylated serines 49 and 218 of RGS18. Platelet activation by thrombin, thromboxane A2, or ADP stimulates the association of 14-3-3 and RGS18, probably by increasing the phosphorylation of serine 49. In contrast, treatment of platelets with prostacyclin and nitric oxide, which trigger inhibitory cyclic nucleotide signaling involving cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI), induces the phosphorylation of serine 216 of RGS18 and the detachment of 14-3-3. Serine 216 phosphorylation is able to block 14-3-3 binding to RGS18 even in the presence of thrombin, thromboxane A2, or ADP. 14-3-3-deficient RGS18 is more active compared with 14-3-3-bound RGS18, leading to a more pronounced inhibition of thrombin-induced release of calcium ions from intracellular stores. Therefore, PKA-and PKGI-mediated detachment of 14-3-3 activates RGS18 to block Gq-dependent calcium signaling. These findings indicate cross-talk between platelet activation and inhibition pathways at the level of RGS18 and Gq. IntroductionIn healthy vasculature, endothelial cells lining the blood vessels constantly produce and release prostacyclin (PGI 2 ) and nitric oxide (NO) into the vessel lumen. The interaction of endothelial factors with platelets plays a fundamental role in controlling hemostasis and in maintaining platelets in a resting state. Platelet inhibition by both PGI 2 and NO has been well established. The signaling pathways of both molecules result in an elevation of cyclic nucleotides that activate cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI). These in turn phosphorylate an unknown number of substrate proteins, resulting in reduced release of calcium ions (Ca 2ϩ ) from intracellular stores and reduced activation of G-proteins such as Rap1, ultimately leading to a block of platelet adhesion, granule release, and aggregation. PKA and PKGI have overlapping substrate specificities, which may explain the synergistic role of the 2 pathways. Few substrates have been established in platelets, among them Rap1GAP2, a GTPase-activating protein (GAP) of the small G-protein Rap1, as we have shown previously. 1 Other substrates include vasodilator-stimulated phospho-protein (VASP), heatshock protein 27 (HSP27), and LIM and SH3 domain protein (LASP), all of which regulate actin dynamics. 2,3 The IP 3 -receptor and the IP 3 -receptor-associated G-kinase substrate (IRAG) are the only PKA and/or PKGI substrates that have been shown to mediate cAMP/cGMP effects on intracellular Ca 2ϩ release. [2][3][4] Limited data are available on the specific substrates and signaling events that translate PKA/G substrate phosphorylation into platelet inhibition.Conversely, bindin...

What Can Proteomics Tell Us About Platelets?

“…93 Gegenbauer et al 140 describe RGS18 as an interface molecule between activating and inhibitory pathways. Whereas RGS18 Ser49 is phosphorylated during platelet activation, Ser216 is phosphorylated on platelet inhibition by protein kinase A (PKA) and protein kinase G. 140 These 2 cyclic nucleotide-dependent protein kinases govern inhibitory pathways in platelets which, despite their important role, have been addressed by only few proteomic studies to date. 115,116,141 Up to now, only 15 PKA and 11 protein kinase G substrates have been confirmed.…”

Cyclic nucleotide‐dependent inhibitory signaling interweaves with activating pathways to determine platelet responses

“…The well established antagonism between activators and endothelial inhibitors in the regulation of Ca 2+ levels has recently been confirmed in a systematic study comparing effects of ADP, thrombin, convulxin and U46619 in combination with activators of cAMP and cGMP pathways 61. The Gq GAP RGS18 contributes to differential control of Ca 2+ levels by platelet activators and inhibitors 7, 35. PKA also inhibits IP 3 ‐induced Ca 2+ ‐release through phosphorylation of the IP 3 ‐receptor and the IP 3 ‐receptor‐associated G‐kinase substrate (IRAG, MRVI1) 1.…”

“…In resting platelets, RGS18 interacts with the scaffold protein spinophilin (neurabin‐2, PPP1R9B), the tyrosine phosphatase SHP‐1, and the phospho‐serine/threonine binding adapter protein 14‐3‐3 (Figure 5, RGS18 complex in transition i). Platelet activation by thrombin and TxA 2 induces a dissociation of RGS18 and SHP‐1 from spinophilin35 and increases binding of 14‐3‐3γ to RGS18 7. 14‐3‐3 bound RGS18 is less active leading to enhanced Gq signaling and Ca 2+ ‐release (Figure 5, RGS18 complex inactive).…”

“…Loss of SHP‐1 during platelet activation leads to binding of the serine/threonine phosphatase PP1 to spinophilin which is associated with reduced PP1 activity towards myosin light chain (MLC) potentially contributing to enhanced MLC phosphorylation and platelet activation 36. Cyclic nucleotide inhibitory pathways also disrupt the RGS18/spinophilin/SHP‐1 complex,37 however, they inhibit binding of 14‐3‐3γ to RGS18 (Figure 5, RGS18 complex active) 7. This free RGS18 binds more effectively to Gαi2 and attenuates Gq signaling leading to reduced Ca 2+ ‐release and thus contributing to platelet inhibition 7, 37.…”

“…However, increasing evidence suggests an alternative model whereby simultaneous regulation of both systems determines platelet function. For example, to prevent platelet activation, inhibitory signaling targets key nodes in activating pathways 7. On the other hand, platelet activation requires active blockage of endothelium‐dependent inhibitory pathways,8, 9, 10, 11 including ADP‐mediated inhibition of cAMP production via P2Y12 and thrombin‐mediated cAMP degradation via PDE type 3A.…”