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Keywords:

  • G proteins;
  • megakaryocytes;
  • platelets;
  • regulator of G proteins

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

Summary.  Regulators of G protein signaling (RGS) are intracellular signaling regulators that bind activated G protein α subunits (Gα) and increase their intrinsic GTPase activity via their common RGS homology domain. In addition to their GTPase accelerating activity (GAP), RGS proteins also contain other domains that regulate their receptor selectivity, their interaction with other proteins such as adenylyl cyclase or their subcellular localization via interaction with scaffold proteins such as tubulin, 14-3-3 or spinophilin. There are at least 37 different RGS family members in humans and numerous physiological functions have been assigned to these proteins, which have rather a tissue-specific expression pattern. The role of some RGS proteins was shown to be important for hematopoiesis. More recent studies also focused on their expression in platelets, and for R4 RGS subfamily members RGS2, RGS16 and RGS18, it could be demonstrated that they regulate megakaryopoiesis and/or platelet function. These functional studies mostly comprised in vitro experiments and in vivo studies using small animal models. Their role in human pathology related to platelet dysfunction remains still largely unknown, except for a case report with a RGS2 gain of function mutation. In addition to an introduction on RGS signaling and different effectors with a special focus on the R4 subfamily members, we here will give an overview of the studies related to the role of RGS proteins in hematopoiesis, megakaryopoiesis and platelet function.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

G protein coupled receptors (GPCRs) transduce signals by a three-component system: upon (i) ligand binding, (ii) GPCRs stimulate the (iii) G protein alpha subunit (Gα) to exchange GDP for GTP, resulting in stimulation of downstream effectors. Signaling is terminated when Gα hydrolyses bound GTP to GDP. RGS proteins have the ability to increase the intrinsic GTPase (GAP) activity of G proteins [1]. This GAP activity enhances G protein deactivation and promotes their desensitization, thereby interfering with different effectors [2]. There are at least 37 RGS family members found in the human genome that all contain a highly conserved 120 amino acids RGS box that serves as a GAP (Fig. 1). The GAP activity of RGS proteins is largely limited to α subunits of the Gq, Gi/o and G12/13 families of G proteins. RGS-PX1 (SNX13) is the only RGS member that was shown to have a GAP activity specifically for the α subunit of the Gs protein [3]. All other RGS proteins seem not to have GAP activity for this G protein subunit, although it has been shown that RGS proteins can indirectly regulate Gsα signaling via their interaction with adenylyl cyclase subtypes, as intensively studied for RGS2 (Fig. 2A) [4].

image

Figure 1.  Classification of RGS proteins into different subfamilies and their protein structure showing the most important domains. β-Cat, β-catenin-binding; D, dimerization domain; D-AKAP, dual-specificity A-kinase anchoring protein; DEP, dishevelled/EGL-10/pleckstrin; DH, double homology; DIX, dishevelled homology domain; GAIP, G alpha interacting protein; GEF, guanine nucleotide exchange factor; GGL, Gγ-like; GoLoco, Gαi/o-Loco; GRK, G protein-coupled receptor kinase; GSK, glycogen synthase kinase 3β-binding; PDZ, PSD95/Dlg/Z0-1/2; PEST, proline, glutamine, serine, threonine-rich; PH, pleckstrin homology; PP2A, protein phosphatase 2A; PTB, phosphotyrosine-binding; PX, phosphatidylinositol-binding; PXA, PX-associated; RBD, Ras-binding domain; RGS, regulator of G protein signaling domain; SNX, sorting nexin.

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image

Figure 2.  Interactions between RGS proteins and different effectors. (A) RGS proteins (such as RGS2) can directly interact with adenylyl cyclase to interfere with Gs signaling. (B) RGS proteins also interact with GIRK channels. (C) RGS interaction with phospholipase C-β leads to Gqalpha activation to increased intracellular calcium levels. (B and C) RGS proteins also have GAP activity for Gialpha and Gqalpha.

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All RGS proteins also contain non-GAP activities that are mediated by their non-RGS regions. Emerging data have revealed an expanding network of proteins, lipids and ions that interact with RGS proteins and confer additional regulatory functions [2,5,6]. RGS proteins can be subdivided into several subfamilies (e.g. R4, R7, R12, Rz and others; Fig. 1 for overview) based on their primary sequence homology and the presence of these specific additional domains. These RGS subfamilies have been extensively reviewed elsewhere [7]. The presence of non-RGS domains suggested that RGS proteins have other functions than accelerating GTP hydrolysis. As our review on the role of RGS proteins in megakaryocytes and platelets mainly involves R4 RGS subfamily members, we here also focus on the non-GAP functions that were described for these RGS subfamily members. Except for RGS3, the R4 RGS subfamily members (RGS1-5, 8, 13, 16, 18 and 21) are small proteins (20–30 kDa) that contain only minimal C- and N-terminal extensions flanking the RGS domain (Fig. 1) [2]. Their N-terminus contains an amphipathic alpha helix that serves as a membrane targeting system to bring them close to the different molecules that are involved in GPCR signaling. These R4 RGS members can indeed also interact via these N-terminal domains with other components of the GPCR pathway besides the G protein α subunits, such as receptors and diverse effectors such as adenylyl cyclases (AC), G-protein-gated inwardly rectifying potassium (GIRK) channels and phospholipase C-beta (PLC-β) (for overview see Fig. 2) [2,8]. RGS proteins act as effector stimulators or antagonists via binding to the effector protein directly or via its coupling to the G protein α subunit, which then prevents the physical interaction between the two [9]. Different R4 subfamily RGS proteins (RGS2, 3, 4 and 13) are able to inhibit Gsα-stimulated AC activation [8,10–13]. AC inhibition by RGS proteins could be due to a direct interaction between these RGS proteins and either AC or Gsα (Fig. 2A). It was shown that RGS2 physically interacts via its 19 amino acids long N-terminal non-RGS domain with the C1 cytoplasmic domain of AC type V to inhibit its function [12]. On the other hand, another study showed that RGS2 co-immunoprecipitates with Gsα using cell extracts [14]. These studies show that RGS2 attenuates AC activation by binding to AC and Gsα, though the exact pathway is not yet known and a physiological role for it has not been demonstrated but was later found to be important for platelet morphology and Gs function (see further). R4 subfamily RGS proteins are important modulators of GIRK channel function in response to GPCR activation of Gi/o proteins but the exact mechanism for this still remains unknown (Fig. 2B) [15]. PLC-β is activated by Gq proteins to stimulate calcium release and many R4 subfamily RGS proteins were shown to inhibit PLC-β via their GAP activity on Gq but also could directly inhibit the effector without deactivation of the G protein (Fig. 2C) [5,16]. A role for RGS proteins in the regulation of GIRK and PLC-β for megakaryocytes and/or platelets is not known.

RGS proteins also mediate non-GPCR signaling via interaction of their RGS and non-RGS domains with proteins that will influence their subcellular localization, function and stability. Studies have shown that RGS is able to interact with scaffold proteins (such as spinophilin and 14-3-3) and components of the Wnt signaling pathways [17,18]. Interactions between RGS and scaffolding proteins are not limited to the more complex RGS proteins as binding between R4 subfamily RGS proteins (RGS1, 2, 4 and 16) and spinophilin was reported using pull-down assays [19]. In this study, it was also further demonstrated that spinophilin enhances the RGS2-dependent inhibition of calcium signaling using Xenopus oocytes. 14-3-3 proteins are small (27–32 kDa) and have no functional domains but are essential as chaperones, adaptors and scaffolds to regulate diverse cellular signaling pathways. Recent studies showed that R4 subfamily RGS proteins (RGS3 and 8) bind to 14-3-3 via regions within [20] and/or outside [21,22] their RGS domain. Phosphorylation of the serine residues within the RGS domain binding sites increases their affinity for 14-3-3 [21]. The binding of 14-3-3 to RGS proteins decreases the inhibitory effects of RGS on G protein-mediated signaling [21,22]. Finally, GPCR signaling and RGS proteins have been implicated in Wnt signaling, which is the main regulatory pathway that controls cell fate, cell adhesion, migration, polarity and proliferation of diverse cellular systems. A recent study already showed that the R4 subfamily RGS protein RGS3 has an important role in the non-canonical Wnt pathway (more specifically downstream of Wnt5) that activates calcium release as well as activation of Rho, Rac and other cytoskeletal components [23]. It was also shown that another R4 subfamily member, RGS4, modulated the Wnt8 signaling via a G protein-dependent mechanism [24]. Spinophilin, 14-3-3 and the Wnt pathway were also shown to have an important RGS-dependent role during megakaryopoiesis and in platelet activity (see further for details).

The broad spectrum of physiological functions of RGS proteins is extensively reviewed [25,26]. In the next part of this review, we will specifically focus on the recent discoveries that suggest an important role for RGS proteins (largely from the R4 RGS subfamily) in regulation of different aspects of hematopoiesis, megakaryopoiesis and platelet activation.

RGS proteins as regulators of hematopoiesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

Hematopoiesis generates a variety of distinct blood cell types from a common hematopoietic stem cell. Understanding the molecular mechanisms that lead to the formation of a specific hematopoietic lineage is of considerable interest as this provides new insights into the mechanism of diseases that affect hematopoietic subpopulations in adults. It is well known that secreted cytokines modulate the survival, proliferation and differentiation of all blood cell lineages, mediated by defined sets of transcription factors. Hematopoiesis is, however, also influenced by extracellular information that by signal transduction will lead to integrated cellular decisions, pointing out a role for GPCR signaling and its regulators during this process. Chemokines, originally designated as chemoattractant cytokines, bind to chemokine GPCR receptors. The two flagship chemokines in the regulation of differentiation and migration of B and T cells are stromal cell-derived factor-1 (SDF-1 or CXCL12) and B cell-attracting chemokine 1 (BCA1 or CXCL13) with their receptors CXCR4 and CXCR5, respectively. Only a few studies reported on the role of RGS proteins in these GPCR signaling pathways during hematopoiesis (Table 1).

Table 1.   RGS functions in hematopoiesis, megakaryopoiesis and platelet function
RGS proteinRole in general hematopoiesis (except megakaryocyte and platelet development)Role in megakaryopoiesisRole in platelet function
  1. ND; not defined.

RGS1B-cell trafficking into and within lymph nodes, B-cell responsiveness to chemokines (Mice) (25–27)NDND
RGS2T-cell proliferation and IL-2 production (Mice) (28)NDPlatelet morphology and Gs function (human) (47) and (mice) (55)
RGS13B-cell responsiveness to chemokines, T-cell migration, and degranulation of mast cells (in vitro, mice) (29–33)NDND
RGS14Adhesion or migration of lympocytes (in vitro) (34)NDND
RGS16NDRegulator of megakaryocyte migration via the SDF-1/CRCR4 dependent pathway (in vitro) (43)ND
RGS18No obvious effect on formation of HSC or lymphoid lineages (zebrafish) (45)Effect on megakaryocyte differentiation and platelet formation (in vitro and zebrafish) (45)Inhibitor of platelet activation via spinophilin and/or 14-3-3 scaffolding complexes (in vitro) (56, 57)

RGS1

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

RGS1 is expressed in B lymphocytes and it has been shown that B cells isolated from RGS1-deficient mice respond hypersensitively to chemokines CXCL12 and CXCL13 [27]. Many of the B-cell follicles of RGS1-deficient mice have germinal centers even in the absence of immune stimulation. This results in an abnormal architecture of the spleen, a loss of the normal delineation of the B and T zone in lymphoid follicles and an abnormal trafficking of these cells [27].

RGS2

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

RGS2 is widely expressed throughout the body and has many functions besides its impact on T-cell proliferation. Targeted mutation of RGS2 in mice leads to reduced T-cell proliferation and IL-2 production, which translates into reduced immune response when these mice are infected with lymphocytic choriomeningitis virus [28].

RGS13

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

RGS13 is expressed in B and T lymphocytes and mast cells. It functions together with RGS1 in the responsiveness of B lymphocytes to chemoattractants as mentioned above [29] but also plays a role in the CXCR4-meditated migration of T cells [30]. Bansal et al. [31] published a study in which they compared degranulation of mast cells from wild-type and RGS13-deficient mice. Degranulation by RGS13-deficient bone marrow-derived mast cells was significantly greater when compared with wild types. These data suggest that RGS13 may control the intensity of mast cell-driven allergic inflammation besides their function in B and T-cell migration and/or differentiation.

RGS proteins as regulators of megakaryopoiesis and platelet formation

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

During megakaryopoiesis, hematopoietic stem cells pass successive lineage commitment steps and a maturation process to finally form platelets that will be released into the vascular sinusoids of the bone marrow. As in hematopoiesis, this process is regulated by a combination of specific transcription factors, cytokines and chemokines (reviewed in [32]). An important and unique chemokine in the homing of megakaryocytes is again SDF-1 (CXCL12) and its GPCR CXCR4 [33]. Interactions of immature megakaryocytes with the endothelial-enriched microenvironment are promoted by SDF-1 [34]. Megakaryocytes and platelets express CXCR4 and the outcome of CXCR4 signaling is reduced during megakaryopoiesis, which indicates an upregulation of negative regulators during this process [35]. Based on studies in B cells where RGS proteins tightly regulate CXCR4 signaling [36], these proteins were hypothesized to be potential candidates for a role such as negative regulators of CXCR4 signaling during megakaryopoiesis. Again we will here review the studies that described a role for RGS proteins in megakaryopoiesis and platelet production (Table 1).

RGS16

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

RGS16 and RGS18 are both highly expressed in megakaryocytes though these early studies did not indicate the expression patterns of these proteins during the different differentiation steps of megakaryopoiesis [37–39]. RGS16 was later shown to act as a negative regulator of CXCR4 signaling in megakaryocytes [40]. Overexpression of RGS16 reduced the SDF-1-mediated migration while inhibition of RGS16 via RNA interference resulted in the opposite phenotype with an increase in CXCR4 signaling without inducing any effect on megakaryocyte adhesion to fibronectin or platelet formation. The inhibition of CXCR4 signaling by RGS was considered to be the result of their GAP activity that stimulates the reassociation of Gα proteins with free βγ complexes that otherwise would stimulate MAP kinases as being a downstream signaling event of the SDF-1/CXCR4 pathway [41].

RGS18

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

RGS18 overexpression had no effect on CXCR4 activity, as described for RGS16 [40]. However, RGS18 was found to be highly expressed in megakaryocytes, as well as in hematopoietic progenitors of the myeloerythroid lineage [37–39]. Binding studies using megakaryocytic cell lysates revealed that RGS18 binds both Gq and Gi proteins, suggesting that this RGS protein controls the duration of G protein receptor responses in megakaryocytes [37], but its physiological role in these cells remained completely unknown. We recently described severe thrombocytopenia in RGS18-depleted zebrafish embryos [42]. We also found that RGS18 did not affect the SDF-1-mediated CXCR4 signaling as no early thrombocyte migration defects were found, as also shown previously by Berthebaud et al. [40]. In addition, this study revealed an unexpected role for this protein, as RGS18-depleted embryos have a reduced development of cilia in hair cells of the inner ear and a defect in the migration of neuromast cells. Moreover, a possible role for Wnt5b signaling was suggested in this RGS18-dependent pathway as Wnt5b-depleted embryos phenocopied all RGS18 knockdown effects. This is not completely surprising because it is known that members of the non-canonical Wnt signaling pathway (which includes Wnt5b) mediate intracellular calcium release via activation of heterotrimeric G proteins [42]. A similar interaction between RGS3 and the Wnt pathway was described before using zebrafish [23]. However, it is not yet known whether RGS18 plays the same regulatory role in human megakaryopoiesis and further studies must be undertaken to gain insights into whether RGS18 specifically modulates MK proliferation and/or proplatelet formation.

RGS proteins as regulators of platelet function

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

Platelet activation and subsequent recruitment of additional platelets is strongly dependent on intracellular signaling pathways that are mainly induced via diverse GPCRs combined with Gαs, Gαi, Gαq, G12/13 or Gαz subunits that are all present in platelets. Most platelet agonists (such as thrombin, ADP, thromboxaneA2 and epinephrine) activate GPCRs that mainly signal through activation of their specific G protein to induce calcium release (Gq) and inhibition (Gi) or activation of adenylyl cylases (Gs), which lead to platelet activation (Gq and Gi) or inhibition of platelet activation (Gs). Human platelet pathologies that are related to defects in the G-protein signaling cascade have recently been reviewed [43,44]. The role of RGS proteins in platelet function under normal and pathological conditions, however, is still poorly understood. Nevertheless, RGS molecules could be potential interesting modifiers of the G protein signaling cascade because mRNAs for RGS1, 2, 3, 6, 9, 10, 16, 18 and 19 were shown to be present in human platelets [45] and RGS2, 3, 5, 6, 10, 14, 16 and 18 transcripts could be detected in rat platelets [46]. Intriguingly, most RGS proteins expressed in platelets belong to the R4 subfamily of RGS proteins. A recent proteomic study that focused on the analysis of signaling complexes after platelet activation by the thrombin receptor activating peptide (TRAP) revealed a specific RGS18 phosphorylation site at residue Ser49 that was differentially regulated after TRAP activation and detected also RGS10 as a potential phosphorylated protein [47]. This suggests that these RGS proteins are involved in protease activated receptor 1 (PAR1)/Gi-Gq signaling in platelets. To date, only a few reports have documented the regulation of RGS proteins by phosphorylation but in general this translational modification was shown to modulate their GAP activity [48,49]. The significance of these proteomic data for platelet activation requires some validation studies.

The first in vivo data that generated evidence for a role of RGS proteins as inhibitors of robust platelet activation were obtained using mutant mice with a G185S substitution in their Giα subunit, the G protein that couples to the P2Y12 receptor for ADP [50]. This mutation in Giα2 makes it resistant to accelerated turn-off by all RGS proteins but does not change its coupling to the receptor [51] (Fig. 3A). These mice showed enhanced platelet reactivity to different platelet agonists in aggregation studies and an increased thrombus formation at sites of vascular injury [50]. These are the first data that demonstrate an active role for RGS proteins in the regulation of platelet activity to limit the magnitude of the normal hemostatic response. This study did not further focus on the selection of candidate RGS proteins that could fulfill such a role. We will here further discuss some very recent studies on RGS2 and RGS18 for which some in vitro and/or in vivo data are now available that showed their importance in platelet function (Table 1).

image

Figure 3.  Schematic representation illustrating the regulatory function of different RGS molecules related to changes in platelet function. (A) Knock-in mice with the Giα2-G184S mutation are resistant to the signaling termination by all RGS proteins. Therefore, platelets from these mice have low cAMP levels and a prothrombotic phenotype. (B) Patients with the RGS2-G23D mutation show an increased expression of the larger RGS2 isoforms that strongly interact with adenylyl cyclase to induce platelet Gsα hypofunction and low cAMP levels. (C and D) A model for the role of RGS18-scaffolding complexes in platelets. (C) In resting platelets, spinophilin (SPL), SHP-1 and RGS18 exist as a complex in which SPL is phosphorylated on Y398 and Y483, preventing RGS18 binding to Gq. Platelet activation by thrombin leads to phosphorylation of SHP-1 Y536 to activate this phosphatase. This leads to a dephosphorylation of SPL and dissociation of the SPL/RGS18/SHP-1 complex, releasing RGS18 to inhibit Gq signaling. (D) RGS18 is constitutively bound to 14-3-3 via the phosphorylated serine 218. Platelet activation by thrombin leads to the phosphorylation of S49 of RGS18 by an unknown protein kinase. This results in increased RGS18 affinity to 14-3-3. RGS18 bound to 14-3-3 is less efficient as GTPase-activator of Gq, resulting in increased release of calcium ions from intracellular stores, leading to platelet aggregation. Prostacyclin (PGI2) and nitric oxide (NO) are released from intact endothelial cells and stimulate the activation of cAMP- and cGMP-dependent protein kinases (PKA/PKG). PKA and PKG phosphorylate RGS18 at residue S216, leading to the detachment of 14-3-3 from RGS18. Detachment of 14-3-3 causes RGS18 to turn off Gq signaling more efficiently, resulting in decreased intracellular calcium release and platelet inhibition. In this way RGS18 is able to integrate activating and inhibitory signaling in platelets.

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RGS2

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

Evidence for a role of RGS2 in the regulation of Gsα signaling in platelets originated from studies in patients with a heterozygous RGS2 gain-of-function mutation (G23D) that have Gs hypofunction of platelets with reduced cAMP production after stimulation of Gs-coupled receptors [44]. The G23D mutation results in increased expression of larger RGS2 isoforms that interact strongly with adenylyl cyclase to inhibit its function, leading to decreased Gs activity in platelets from the patients (Fig. 3B). This study suggests a negative regulatory function of RGS2 in Gs platelet signaling. Patients also have enlarged rounder platelets with abnormal alpha granules, which suggests that RGS2 also interferes with platelet formation though further studies are needed to illustrate this aspect. In contrast, a new study has been published where time to thrombus formation and the vessel occlusion time in RGS2-deficient mice were studied and found to be equal to normal control mice, suggesting that RGS2 has no impact on primary hemostasis in mice [52]. It is not yet clear whether redundancy of other RGS proteins (that can be species-specific) is responsible for the absence of a platelet phenotype in these mice or whether the difference with the defect found in humans carrying the RGS2 D23G RGS2 mutation is due to the fact that a complete knockout is not the same as having a specific gain-of-function mutation that increases the inhibitory action of RGS2 via its direct interaction with adenylyl cyclase (AC). It is, however, expected that loss of RGS2 expression in platelets from knockout mice would have a loss of function activity as GAP for Gq and/or Gi activity rather than directly modifying the AC activity but this remains to be studied in detail.

RGS18

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

RGS18 was originally discovered as a potential new mediator of G protein signaling in human platelets [37] and shows the highest mRNA expression compared with the other RGS proteins [45,46]. Two recent in vitro studies showed that the normal role of RGS18 is to limit platelet activation. A study by Ma et al. [53] discovered how RGS18 modifies platelet signaling as a part of a heterotrimer complex consisting of the scaffolding protein, spinophilin (SPL), and the tyrosine phosphatase, SHP-1. In resting platelets, SPL is constitutively tyrosine phosphorylated (at residues Y398 and Y483) and forms a binding complex with SHP-1 mediated by Y398 and RGS18. This heterotrimer complex prevents RGS18 binding with Gq and executing its function as a negative regulator. However, once platelets become activated by thrombin or thromboxane A2, SHP-1 will be activated via phosphorylation of Y536, which in turn will lead to a decay of the SPL/SHP-1/RGS18 complex. This dissociation leads to a translocation of RGS18 to the Gq protein, where it will downregulate Gq signaling and inhibit platelet activation (Fig. 3C). Interestingly, SPL-deficient mice have no stable SPL/SHP-1/RGS18 complex, which results in continuous RGS18 activity leading to inhibition of Gq signaling. Therefore, these mice have an impaired platelet aggregation to thrombin and thromboxane A2 analogues and a prolonged carotid artery occlusion time using the FeCl3 injury model.

Another study showed that RGS18 crosstalks between platelet activation and inhibition pathways at the level of Gq vs. Gs signaling. It proved that platelet activation by thrombin, thromboxane A2 or ADP stimulates its association with another scaffolding protein, 14-3-3 [54]. In addition, it was shown that RGS18 is phosphorylated at Ser49 and Ser218 and during activation the Ser49 phosphorylation increases to allow the direct binding with 14-3-3. As RGS18 bound to 14-3-3 has less efficient GAP activity for Gq, this stronger association will lead to prolonged Gq signaling and enhanced platelet aggregation. In contrast, endothelial-secreted negative regulators of aggregation such as NO and prostacyclin, which stimulate PKG and PKA activity, phosphorylate RGS18 at Ser216, leading to the detachment of 14-3-3 from RGS18. This detachment causes RGS18 to turn off Gq signaling more efficiently, resulting in decreased intracellular Ca2+-release and platelet inhibition (Fig. 3D). More studies are needed to combine these two independent studies and draw a uniform RGS18 signaling scheme that fulfills a role as inhibitor of platelet activation.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

The role of RGS proteins in hematopoiesis, megakaryopoiesis and platelet function is still a rather unexplored field although the few studies now available already indicate an interaction with GPCR in these processes. As GPCR signaling is one of the most important signaling pathways present in many conditions, these modifying molecules could be an interesting target in fine-tuning and/or modification of a (disturbed) signaling present in many (patho)physiological conditions. Future studies must be undertaken to show the role of RGS proteins in human physiology in relation to a potential role in primary hemostasis in relation to thrombosis or bleeding. In addition, most published studies are limited to the role of the R4-RGS subfamily members in the formation and function of megakaryocytes and platelets and it will be a major challenge to further address the role of the other subfamily members (Fig. 1), which have very specific domains besides the classical RGS domain. A great deal of attention over the last years was focused on the development of novel drug therapies based on anti-RGS mimetics [6,55]. Therefore, such molecules also carry the potential of being a novel antiplatelet drug or even could be developed as a stimulator of megakaryocyte and/or platelet formation for thrombocytopenia treatment.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References

This work was supported by the ‘Excellentie financiering KULeuven’ (EF/05/013), by research grants G.0490.10N and G.0743.09 from the Fund for Scientific Research – Flanders (FWO-Vlaanderen, Belgium) and GOA/2009/13 from the Research Council of the University of Leuven (Onderzoeksraad KULeuven‘ Belgium). CVG is holder of a clinical-fundamental research mandate of the Fund for Scientific Research-Flanders (F.W.O.-Vlaanderen, Belgium) and of the Bayer and Norbert Heimburger (CSL Behring) Chairs.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. RGS proteins as regulators of hematopoiesis
  5. RGS1
  6. RGS2
  7. RGS13
  8. RGS proteins as regulators of megakaryopoiesis and platelet formation
  9. RGS16
  10. RGS18
  11. RGS proteins as regulators of platelet function
  12. RGS2
  13. RGS18
  14. Conclusions
  15. Addendum
  16. Acknowledgements
  17. Disclosure of Conflict of Interests
  18. References
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