Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.     

G13 is an essential mediator of platelet activation in hemostasis and thrombosis

Platelet activation at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. Platelet activators such as adenosine diphosphate, thrombin or thromboxane A(2) (TXA(2)) activate receptors that are coupled to heterotrimeric G proteins. Activation of platelets through these receptors involves signaling through G(q), G(i) and G(z) (refs. 4-6). However, the role and relative importance of G(12) and G(13), which are activated by various platelet stimuli, are unclear. Here we show that lack of Galpha(13), but not Galpha(12), severely reduced the potency of thrombin, TXA(2) and collagen to induce platelet shape changes and aggregation in vitro. These defects were accompanied by reduced activation of RhoA and inability to form stable platelet thrombi under high shear stress ex vivo. Galpha(13) deficiency in platelets resulted in a severe defect in primary hemostasis and complete protection against arterial thrombosis in vivo. We conclude that G(13)-mediated signaling processes are required for normal hemostasis and thrombosis and may serve as a new target for antiplatelet drugs.     

Differential regulation of Rho and Rac through heterotrimeric G-proteins and cyclic nucleotides

Platelets were used to study the activation of Rho and Rac through G-protein-coupled receptors and its regulation by cyclic nucleotides. The thromboxane A(2) (TXA(2)) mimetic rapidly activated both small GTPases independently of integrin alpha(IIb)beta(3) activation., which leads to the activation of G(12)/G(13) and G(q) did not induce Rac activation in G alpha(q)-deficient platelets but was able to activate Rho, to stimulate actin polymerization and phosphatidylinositol 4,5-bisphosphate formation, and to induce shape change. Rac activation by in wild-type platelets could be blocked by chelation of intracellular Ca(2+) and was partially sensitive to apyrase and AR-C69931MX, an antagonist of the G(i)-coupled ADP receptor. Cyclic AMP, which completely blocks platelet function, inhibited the -induced activation of G(q) and G(12)/G(13) as well as of Rac and Rho. In contrast, cGMP, which has no effect on platelet shape change blocked only activation of G(q) and Rac. These data demonstrate that Rho and Rac are differentially regulated through heterotrimeric G-proteins. The G(12)/G(13)-mediated Rho activation is involved in the shape change response, whereas Rac is activated through G(q) and is not required for shape change. Cyclic AMP and cGMP differentially interfere with -induced Rho and Rac activation at least in part by selective effects on the regulation of individual G-proteins through the TXA(2) receptor.     

Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Galpha(q)-deficient platelets that do not respond with activation of integrin alpha(IIb)beta(3) to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Galpha(q)-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin alpha(IIb)beta3-mediated aggregation of Galpha(q)-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin alpha(IIb)beta3 activation. Although G(q)-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin alpha(IIb)beta3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving G(q) and G(i) as well as G12/G13.     

Emerging non-canonical functions for heterotrimeric G proteins in cellular signaling

Abstract

Classically heterotrimeric G proteins have been described as the principal signal transducing machinery for G-protein-coupled receptors. Receptor activation catalyzes nucleotide exchange on the Gα protein, enabling Gα-GTP and Gβγ-subunits to engage intracellular effectors to generate various cellular effects such as second messenger production or regulation of ion channel conductivity. Recent genetic and proteomic screens have identified novel heterotrimeric G-protein-interacting proteins and expanded their functional roles. This review highlights some examples of recently identified interacting proteins and summarizes how they functionally connect heterotrimeric G proteins to previously underappreciated cellular roles.     

Lipid rafts are critical membrane domains in blood platelet activation processes

Among the various hematopoi;etic cells, platelets are critical for maintaining the integrity of the vascular system. They must be rapidly activated by sequential and coordinated mechanisms in order to efficiently prevent haemorrhage upon vascular injury. Several signal transduction pathways lead to platelet activation in vitro and in vivo, among them, several are initiated via receptors or co-receptors containing immuno-receptor tyrosine-based activation motifs (ITAM) which trigger downstream signalling like the immune receptors in lymphocytes. However, in contrast to immune cells for which the role of lipid rafts in signalling has largely been described, the involvement of laterally segregated membrane microdomains in platelet activation has been investigated only recently. The results obtained until now strongly suggest that early steps of platelet activation via the collagen receptor GpVI or via FcgammaRIIa occur preferentially in these microdomains where specific proteins efficiently organize key downstream signalling pathways. In addition, lipid rafts also contribute to platelet activation via heterotrimeric G-protein-coupled receptors. They are sites where the phosphoinositide (PI) metabolism is highly active, leading to a local generation of lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate. Here, evidence is accumulating that cholesterol-enriched membrane microdomains are part of a general process that contributes to the efficiency and the coordination of platelet activation mechanisms. Here we will discuss the biochemical and functional characterizations of human platelet rafts and their potential impact in platelet physiopathology.     

Lipid rafts are critical membrane domains in blood platelet activation processes

Among the various hematopo?̈etic cells, platelets are critical for maintaining the integrity of the vascular system. They must be rapidly activated by sequential and coordinated mechanisms in order to efficiently prevent haemorrhage upon vascular injury. Several signal transduction pathways lead to platelet activation in vitro and in vivo, among them, several are initiated via receptors or co-receptors containing immuno-receptor tyrosine-based activation motifs (ITAM) which trigger downstream signalling like the immune receptors in lymphocytes. However, in contrast to immune cells for which the role of lipid rafts in signalling has largely been described, the involvement of laterally segregated membrane microdomains in platelet activation has been investigated only recently. The results obtained until now strongly suggest that early steps of platelet activation via the collagen receptor GpVI or via FcγRIIa occur preferentially in these microdomains where specific proteins efficiently organize key downstream signalling pathways. In addition, lipid rafts also contribute to platelet activation via heterotrimeric G-protein-coupled receptors. They are sites where the phosphoinositide (PI) metabolism is highly active, leading to a local generation of lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate. Here, evidence is accumulating that cholesterol-enriched membrane microdomains are part of a general process that contributes to the efficiency and the coordination of platelet activation mechanisms. Here we will discuss the biochemical and functional characterizations of human platelet rafts and their potential impact in platelet physiopathology.     

Regulation and roles of PI3Kβ, a major actor in platelet signaling and functions

Phosphoinositide 3-kinases (PI3Ks) are important signaling enzymes involved in the regulation of a number of critical cell functions. Significant progress has been made during the last few years in defining the implication of individual PI3K isoforms. The role of the class IA PI3Kβ in different cell types has only been recently uncovered by the use of isoform-selective inhibitors and the development of mouse models harboring p110β catalytic subunit knock-out or germline knock-in of a kinase-dead allele of p110β. Although it is classically admitted that class IA PI3Ks are activated by receptor tyrosine kinases through recruitment of the regulatory subunits to specific tyrosine phosphorylated motifs via their SH2 domains, PI3Kβ is activated downstream of G protein-coupled receptors, and by co-operation between heterotrimeric G proteins and tyrosine kinases. PI3Kβ has been extensively studied in platelets where it appears to play an important role downstream of ITAM signaling, G protein-coupled receptors and aIIbβ3 integrin. Accordingly, mouse exhibiting p110β inactivation selectively in megakaryocyte/platelets are resistant to thromboembolism induced by carotid injury. The present review summarizes recent data concerning the mechanisms of PI3Kβ regulation and the roles of this PI3K isoform in blood platelet functions and other cell types.     

Non-canonical functions of RGS proteins

Regulators of G protein signalling (RGS) proteins are united into a family by the presence of the RGS domain which serves as a GTPase-activating protein (GAP) for various Galpha subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate signalling of numerous G protein-coupled receptors. In addition to the RGS domains, RGS proteins contain diverse regions of various lengths that regulate intracellular localization, GAP activity or receptor selectivity of RGS proteins, often through interaction with other partners. However, it is becoming increasingly appreciated that through these non-RGS regions, RGS proteins can serve non-canonical functions distinct from inactivation of Galpha subunits. This review summarizes the data implicating RGS proteins in the (i) regulation of G protein signalling by non-canonical mechanisms, (ii) regulation of non-G protein signalling, (iii) signal transduction from receptors not coupled to G proteins, (iv) activation of mitogen-activated protein kinases, and (v) non-canonical functions in the nucleus.     

Adaptor proteins of blood platelets

Adaptor proteins play a pivotal role in the regulation of signal transduction events elicited after the engagement of cell surface receptors. Platelets exhibit a number of integral membrane receptors capable of initiating a cellular response. These include collagen receptors, von Willebrand factor receptors, the fibrinogen receptor, and a number of G-protein coupled receptors, such as those for thrombin and ADP. The primary function of platelet receptors is the translation of externally applied signals into appropriate responses leading to platelet activation being a prerequisite for normal hemostasis. Multitude of signalling pathways described in platelets is based on the interaction of compounds of many different categories, such as transmembrane receptors, protein kinases, protein phoshatases, G-proteins, transmembrane and cytosolic adaptor proteins, phosphoinositides, cyclic AMP or GMP. Adaptor proteins lack intrinsic effector function, but contain distinct molecular domains, which mediate protein-protein and protein-lipid interactions. These molecules thus serve as a scaffolding, around which effectors and their substrates are assembled into three-dimensional signaling complexes. Adaptor proteins integrate receptor-mediated signals at intracellular levels and couple signaling receptors to cytosolic signaling pathways. While the function of adaptor proteins is well established in immune cells, the knowledge about their role in platelet activation is still at the onset Over the last decade numerous adaptor proteins have been identified in platelets and shown to be involved in accurate assembly of intracellular signaling complexes. Collagen-induced platelet intracellular signaling through GPVI resembles the functional response of B- and T-cell antigen receptors and is the best described in the literature. This review focuses on the structure and functional role of the most extensively studied adaptor proteins during platelet activation induced by physiological agonists.     

Enhanced epinephrine-induced platelet aggregation in individuals carrying the G protein beta3 subunit 825T allele

The 825T allele of a common C825T polymorphism in the gene encoding the beta3 subunit of heterotrimeric G proteins is associated with enhanced activation of pertussis toxin (PTX)-sensitive G proteins. We investigated responses of human platelets upon stimulation with epinephrine, which activates PTX-sensitive G proteins, and with agonists which activate additionally, or exclusively PTX-insensitive pathways. Slopes and maximum of the secondary aggregation were significantly enhanced in platelets from 825T allele carriers after epinephrine, and after combined epinephrine/ADP. This effect was more pronounced after inhibition of the cyclooxygenase-2 pathway by acetylsalicylic acid. This phenomenon appeared independent of platelet secretion, or inhibition of the adenylyl cyclase.     

Phosphoproteome of resting human platelets

Beside their main physiological function in hemostasis, platelets are also highly involved in pathological processes, such as atherothrombosis and inflammation. During hemostasis, binding of adhesive substrates to tyrosine-kinase-linked adhesion receptors and/or soluble agonists to G-protein coupled receptors leads to a cascade of intracellular signaling processes based on substrate (de)phosphorylation. The same mechanisms are involved in platelet activation at sites of atherosclerotic plaque rupture, contributing to vessel occlusion and consequently to pathologic states, such as myocardial infarction, stroke, or peripheral artery disease. To gain a deeper insight into platelet function, we analyzed the phosphoproteome of resting platelets and identified 564 phosphorylation sites from more than 270 proteins, of which many have not been described in platelets before. Among those were several unknown potential protein kinase A (PKA) and protein kinase G (PKG) substrates. Because platelet inhibition is tightly regulated especially by PKA and PKG activity, these proteins may represent important new targets for cardiovascular research. Thus, our finding that GPIbalpha is phosphorylated at Ser603 in resting platelets may represent a novel mechanism for the regulation of one of the most important platelet receptor (GPIb-IX-V) mediated signaling pathways by PKA/PKG.     

The Gbetagamma dimer drives the interaction of heterotrimeric Gi proteins with nonlamellar membrane structures

Heterotrimeric G proteins are peripheral membrane proteins that propagate signals from membrane receptors to regulatory proteins localized in distinct cellular compartments. To facilitate signal amplification, G proteins are in molar excess with respect to G protein-coupled receptors. Because G proteins are capable of translocating from membrane to cytosol, protein-lipid interactions play a crucial role in signal transduction. Here, we studied the binding of heterotrimeric G proteins (Galphabetagamma) to model membranes (liposomes) and that of the entities formed upon receptor-mediated activation (Galpha and Gbetagamma). The model membranes used were composed of defined membrane lipids capable of organizing into either lamellar or nonlamellar (hexagonal H(II)) membrane structures. We demonstrated that although heterotrimeric G(i) proteins and Gbetagamma dimers can bind to lipid bilayers of phosphatidylcholine, their binding to membranes was markedly and significantly enhanced by the presence of nonlamellar phases of phosphatidylethanolamine. Conversely, activated G protein alpha subunits showed an opposite membrane binding behavior with a marked preference for lamellar membranes. These results have important consequences in cell signaling. First, the binding characteristics of the Gbetagamma dimer account for the lipid binding behavior and the cellular localization of heterotrimeric G proteins. Second, the distinct protein-lipid interactions of heterotrimeric G proteins, Gbetagamma dimers, and Galpha subunits with membrane lipids explain, in part, their different cellular mobilizations during signaling upon receptor activation. Finally, their differential interactions with lipids suggest an active role of the membrane lipid secondary structure in the propagation of signals through G protein-coupled receptors.     

WD40-repeat proteins control the flow of Gβγ signaling for directional cell migration

The ability of cells to generate a highly polarized intracellular signal through G protein-coupled receptors (GPCRs) is essential for their migration toward chemoattractants. The Gβγ subunits of heterotrimeric G proteins play a critical role in transmitting chemotactic signals from GPCRs via the activation of diverse effectors, including PLCβ and PI3K, primarily at the leading edge of cells. Although Gβγ can directly activate many of these effectors through protein-protein interactions in vitro, it remains unclear how Gβγ spatially and temporally orchestrates the activation of these effectors in vivo. A yeast two-hybrid screen for Gβ interacting proteins identified two WD40-repeat domain containing proteins, RACK1 and WDR26, which are predicted to serve as scaffolding/adaptor proteins. Previous data indicates that RACK1 negatively regulates Gβγ-mediated leukocyte migration by inhibiting Gβγ-stimulated PLCβ and PI3K activities. In contrast, recently published work by Sun et al. indicates that WDR26 promotes leukocyte migration by enhancing Gβγ-mediated signal transduction. These findings reveal a novel mechanism regulating Gβγ signaling during chemotaxis, namely through the positive and negative regulation of WDR26 and RACK1 on Gβγ to promote and fine tune Gβγ-mediated effector activation, ultimately governing the ability of cells to polarize and migrate toward a chemoattractant gradient.     

Regulation and functional consequences of ADP receptor-mediated ERK2 activation in platelets

We have previously shown that ADP-induced thromboxane generation in platelets requires signalling events from the G(q)-coupled P2Y1 receptor (platelet ADP receptor coupled to stimulation of phospholipase C) and the G(i)-coupled P2Y12 receptor (platelet ADP receptor coupled to inhibition of adenylate cyclase) in addition to outside-in signalling. While it is also known that extracellular calcium negatively regulates ADP-induced thromboxane A2 generation, the underlying mechanism remains unclear. In the present study we sought to elucidate the signalling mechanisms and regulation by extracellular calcium of ADP-induced thromboxane A2 generation in platelets. ERK (extracllular-signal-regulated kinase) 2 activation occurred when outside-in signalling was blocked, indicating that it is a downstream event from the P2Y receptors. However, blockade of either P2Y1 or the P2Y12 receptors with corresponding antagonists completely abolished ERK phosphorylation, indicating that both P2Y receptors are required for ADP-induced ERK activation. Inhibitors of Src family kinases or the ERK upstream kinase MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] abrogated ADP-induced ERK phosphorylation and thromboxane A2 generation. Finally ADP- or G(i)+G(z)-induced ERK phosphorylation was blocked in the presence of extracellular calcium. The present studies show that ERK2 is activated downstream of P2Y receptors through a complex mechanism involving Src kinases and this plays an important role in ADP-induced thromboxane A2 generation. We also conclude that extracellular calcium blocks ADP-induced thromboxane A2 generation through the inhibition of ERK activation.     

Heterotrimeric G proteins in heart disease

Guanine nucleotide binding proteins (G proteins) are largely grouped into three classes: heterotrimeric G proteins, ras-like or small molecular weight GTP binding proteins, and others like Gh. In the heart G proteins transduce signals from a variety of membrane receptors to generate diverse effects on contractility, heart rate, and myocyte growth. This central position of G proteins forming a switchboard between extracellular signals and intracellular effectors makes them candidates possibly involved in the pathogenesis of cardiac hypertrophy, heart failure, and arrhythmia. This review focuses primarily on discoveries of heterotrimeric G protein alterations in heart diseases that help us to understand the pathogenesis and pathophysiology. We also discuss the underlying molecular mechanisms of heterotrimeric G protein signalling.     

Ion channel regulation by Ras, Rho, and Rab small GTPases

Regulation of ion channels by heterotrimeric guanosine triphosphatases (GTPases), activated by heptathelical membrane receptors, has been the focus of several recent reviews. In comparison, regulation of ion channels by small monomeric G proteins, activated by cytoplasmic guanine nucleotide exchange factors, has been less well reviewed. Small G proteins, molecular switches that control the activity of cellular and membrane proteins, regulate a wide variety of cell functions. Many upstream regulators and downstream effectors of small G proteins now have been isolated. Their modes of activation and action are understood. Recently, ion channels were recognized as physiologically important effectors of small GTPases. Recent advances in understanding how small G proteins regulate the intracellular trafficking and activity of ion channels are discussed here. We aim to provide critical insight into physiological control of ion channel function and the biological consequences of regulation of these important proteins by small, monomeric G proteins.