Anaphylactic shock depends on endothelial Gq/G11.

Anaphylactic shock is a severe allergic reaction involving multiple organs including the bronchial and cardiovascular system. Most anaphylactic mediators, like platelet-activating factor (PAF), histamine, and others, act through G protein-coupled receptors, which are linked to the heterotrimeric G proteins G(q)/G(11), G(12)/G(13), and G(i). The role of downstream signaling pathways activated by anaphylactic mediators in defined organs during anaphylactic reactions is largely unknown. Using genetic mouse models that allow for the conditional abrogation of G(q)/G(11)- and G(12)/G(13)-mediated signaling pathways by inducible Cre/loxP-mediated mutagenesis in endothelial cells (ECs), we show that G(q)/G(11)-mediated signaling in ECs is required for the opening of the endothelial barrier and the stimulation of nitric oxide formation by various inflammatory mediators as well as by local anaphylaxis. The systemic effects of anaphylactic mediators like histamine and PAF, but not of bacterial lipopolysaccharide (LPS), are blunted in mice with endothelial G alpha(q)/G alpha(11) deficiency. Mice with endothelium-specific G alpha(q)/G alpha(11) deficiency, but not with G alpha(12)/G alpha(13) deficiency, are protected against the fatal consequences of passive and active systemic anaphylaxis. This identifies endothelial G(q)/G(11)-mediated signaling as a critical mediator of fatal systemic anaphylaxis and, hence, as a potential new target to prevent or treat anaphylactic reactions.

Galpha12/Galpha13 deficiency causes localized overmigration of neurons in the developing cerebral and cerebellar cortices.

The heterotrimeric G proteins G(12) and G(13) link G-protein-coupled receptors to the regulation of the actin cytoskeleton and the induction of actomyosin-based cellular contractility. Here we show that conditional ablation of the genes encoding the alpha-subunits of G(12) and G(13) in the nervous system results in neuronal ectopia of the cerebral and cerebellar cortices due to overmigration of cortical plate neurons and cerebellar Purkinje cells, respectively. The organization of the radial glia and the basal lamina was not disturbed, and the Cajal-Retzius cell layer had formed normally in mutant mice. Embryonic cortical neurons lacking G(12)/G(13) were unable to retract their neurites in response to lysophosphatidic acid and sphingosine-1-phosphate, indicating that they had lost the ability to respond to repulsive mediators acting via G-protein-coupled receptors. Our data indicate that G(12)/G(13)-coupled receptors mediate stop signals and are required for the proper positioning of migrating cortical plate neurons and Purkinje cells during development.

Lysophospholipids control integrin-dependent adhesion in splenic B cells through G(i) and G(12)/G(13) family G-proteins but not through G(q)/G(11).

Integrin-mediated adhesion is a crucial step in lymphocyte extravasation and homing. We show here that not only the chemokines CXCL12 and CXCL13 but also the lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) enhance adhesion of murine follicular and marginal zone B cells to ICAM-1 in vitro. This process involves clustering of integrin LFA-1 and is blocked by pertussis toxin, suggesting that G(i) family G-proteins are involved. In addition, lysophospholipid-induced adhesion on ICAM-1 depends on Rho and Rhokinase, indicative of an involvement of G(12)/G(13), possibly also G(q)/G(11) family G-proteins. We used G(12)/G(13)- or G(q)/G(11)-deficient B cells to study the role of these G-protein families in lysophospholipid-induced adhesion and found that the pro-adhesive effects of LPA and S1P are completely abrogated in G(12)/G(13)-deficient marginal zone B cells, reduced in G(12)/G(13)-deficient follicular B cells, and normal in G(q)/G(11)-deficient B cells. We also show that loss of lysophospholipid-induced adhesion results in disinhibition of migration in response to the follicular chemokine CXCL13, which might contribute to the abnormal localization of splenic B cell populations observed in B cell-specific G(12)/G(13)-deficient mice in vivo. Taken together, this study shows that lysophospholipids regulate integrin-mediated adhesion of splenic B cells to ICAM-1 through G(i) and G(12)/G(13) family G-proteins but not through G(q)/G(11).

G12/G13 family G proteins regulate marginal zone B cell maturation, migration, and polarization.

G protein-coupled receptors play an important role in the regulation of lymphocyte functions such as migration, adhesion, proliferation, and differentiation. Although the role of G(i) family G proteins has been intensively studied, no in vivo data exist with respect to G12/G13 family G proteins. We show in this study that mice that lack the G protein alpha-subunits G alpha12 and G alpha13 selectively in B cells show significantly reduced numbers of splenic marginal zone B (MZB) cells, resulting in a delay of Ab production in response to thymus-independent Ags. Basal and chemokine-induced adhesion to ICAM-1 and VCAM-1, two adhesion molecules critically involved in MZB localization, is normal in mutant B cells, and the same is true for chemokine-induced migration. However, migration in response to serum and sphingosine 1-phosphate is strongly increased in mutant MZB cells, but not in mutant follicular B cells. Live-cell imaging studies revealed that G alpha12/G alpha13-deficient MZB cells assumed more frequently an ameboid form than wild-type cells, and pseudopod formation was enhanced. In addition to their regulatory role in serum- and sphingosine 1-phosphate-induced migration, G12/G13 family G proteins seem to be involved in peripheral MZB cell maturation, because also splenic MZB cell precursors are reduced in mutant mice, although less prominently than mature MZB cells. These data suggest that G12/G13 family G proteins contribute to the formation of the mature MZB cell compartment both by controlling MZB cell migration and by regulating MZB cell precursor maturation.

Forebrain-specific inactivation of Gq/G11 family G proteins results in age-dependent epilepsy and impaired endocannabinoid formation.

Metabotropic receptors coupled to Gq/G11 family G proteins critically contribute to nervous system functions by modulating synaptic transmission, often facilitating excitation. We investigated the role of Gq/G11 family G proteins in the regulation of neuronal excitability in mice that selectively lack the alpha-subunits of Gq and G11, G alpha q and G alpha 11, respectively, in forebrain principal neurons. Surprisingly, mutant mice exhibited increased seizure susceptibility, and the activation of neuroprotective mechanisms was impaired. We found that endocannabinoid levels were reduced under both basal and excitotoxic conditions and that increased susceptibility to kainic acid could be normalized by the enhancement of endocannabinoid levels with an endocannabinoid reuptake inhibitor, while the competitive cannabinoid type 1 receptor antagonist SR141716A did not cause further aggravation. These findings indicate that Gq/G11 family G proteins negatively regulate neuronal excitability in vivo and suggest that impaired endocannabinoid formation in the absence of Gq/G11 contributes to this phenotype.

GPR109A (PUMA-G/HM74A) mediates nicotinic acid-induced flushing.

Nicotinic acid (niacin) has long been used as an antidyslipidemic drug. Its special profile of actions, especially the rise in HDL-cholesterol levels induced by nicotinic acid, is unique among the currently available pharmacological tools to treat lipid disorders. Recently, a G-protein-coupled receptor, termed GPR109A (HM74A in humans, PUMA-G in mice), was described and shown to mediate the nicotinic acid-induced antilipolytic effects in adipocytes. One of the major problems of the pharmacotherapeutical use of nicotinic acid is a strong flushing response. This side effect, although harmless, strongly affects patient compliance. In the present study, we show that mice lacking PUMA-G did not show nicotinic acid-induced flushing. In addition, flushing in response to nicotinic acid was also abrogated in the absence of cyclooxygenase type 1, and mice lacking prostaglandin D(2) (PGD(2)) and prostaglandin E(2) (PGE(2)) receptors had reduced flushing responses. The mouse orthologue of GPR109A, PUMA-G, is highly expressed in macrophages and other immune cells, and transplantation of wild-type bone marrow into irradiated PUMA-G-deficient mice restored the nicotinic acid-induced flushing response. Our data clearly indicate that GPR109A mediates nicotinic acid-induced flushing and that this effect involves release of PGE(2) and PGD(2), most likely from immune cells of the skin.

Characteristic defects in neural crest cell-specific Galphaq/Galpha11- and Galpha12/Galpha13-deficient mice.

The endothelin/endothelin receptor system plays a critical role in the differentiation and terminal migration of particular neural crest cell subpopulations. Targeted deletion of the G-protein-coupled endothelin receptors ET(A) and ET(B) was shown to result in characteristic developmental defects of derivatives of cephalic and cardiac neural crest and of neural crest-derived melanocytes and enteric neurons, respectively. Since both endothelin receptors are coupled to G-proteins of the G(q)/G(11)- and G(12)/G(13)-families, we generated mouse lines lacking Galpha(q)/Galpha(11) or Galpha(12)/Galpha(13) in neural crest cells to study their roles in neural crest development. Mice lacking Galpha(q)/Galpha(11) in a neural crest cell-specific manner had craniofacial defects similar to those observed in mice lacking the ET(A) receptor or endothelin-1 (ET-1). However, in contrast to ET-1/ET(A) mutant animals, cardiac outflow tract morphology was intact. Surprisingly, neither Galpha(q)/Galpha(11)- nor Galpha(12)/Galpha(13)-deficient mice showed developmental defects seen in animals lacking either the ET(B) receptor or its ligand endothelin-3 (ET-3). Interestingly, Galpha(12)/Galpha(13) deficiency in neural crest cell-derived cardiac cells resulted in characteristic cardiac malformations. Our data show that G(q)/G(11)- but not G(12)/G(13)-mediated signaling processes mediate ET-1/ET(A)-dependent development of the cephalic neural crest. In contrast, ET-3/ET(B)-mediated development of neural crest-derived melanocytes and enteric neurons appears to involve G-proteins different from G(q)/G(11)/G(12)/G(13).

G13-mediated signaling as a potential target for antiplatelet drugs.

The activation of platelets at sites of vascular injury is essential for primary hemostasis, but also underlies arterial thrombosis leading to myocardial infarction or stroke. The inhibition of platelet function is therefore a major strategy to prevent and treat arterial thrombosis. Platelet stimuli like ADP, thrombin or thromboxane A(2) activate receptors that are coupled to heterotrimeric G proteins to regulate intracellular signaling pathways. Activation of platelets through these receptors has been shown to involve signaling through various heterotrimeric G proteins. The G protein G(13), which is able to link receptors to the Rho/Rho-kinase pathway, has recently been shown to be critically involved in platelet activation and to play an important role in platelet-dependent arterial thrombosis. The G(13)-mediated signaling pathway may therefore be an interesting new target for antiplatelet drugs.

Anti-glycoprotein VI treatment severely compromises hemostasis in mice with reduced alpha2beta1 levels or concomitant aspirin therapy.

BACKGROUND: Platelet inhibition is a major strategy to prevent arterial thrombosis, but it is frequently associated with increased bleeding because of impaired primary hemostasis. The activating platelet collagen receptor, glycoprotein VI (GP VI), may serve as a powerful antithrombotic target because its inhibition or absence results in profound protection against arterial thrombosis but no major bleeding in mice. METHODS AND RESULTS: Mice lacking (-/-) or expressing half-levels (+/-) of the other major platelet collagen receptor, integrin alpha2beta1, were injected with the anti-GP VI antibody JAQ1 and analyzed on day 5. Anti-GP VI treatment resulted in a marked hemostatic defect in alpha2-/- or alpha2+/- mice, as shown by dramatically prolonged tail bleeding times. Platelet adhesion to collagen was studied in an ex vivo whole-blood perfusion system under high shear conditions. Weak integrin activation by thromboxane A2 (TxA2) receptor stimulation restored defective adhesion of anti-GP VI-treated wild-type but not alpha2-/- or alpha2+/- platelets to collagen. This process required the simultaneous activation of the G(q) and G13 signaling pathways, as demonstrated by use of the respective knockout strains. Conversely, inhibition of TxA2 production by aspirin severely compromised hemostasis in anti-GP VI-treated or GP VI/Fc receptor gamma-chain-deficient but not control mice. CONCLUSIONS: Anti-GP VI therapy may result in defective hemostasis in patients with reduced alpha2beta1 levels or concomitant aspirin therapy. These observations may have important implications for a potential use of anti-GP VI-based therapeutics in the prevention of cardiovascular disease.

Heterotrimeric G proteins of the Gq/11 family are crucial for the induction of maternal behavior in mice.

Heterotrimeric G proteins of the G(q/11) family transduce signals from a variety of neurotransmitter receptors and have therefore been implicated in several functions of the central nervous system. To investigate the potential role of G(q/11) signaling in behavior, we generated mice which lack the alpha-subunits of the two main members of the G(q/11) family, Galpha(q) and Galpha(11), selectively in the forebrain. We show here that forebrain Galpha(q/11)-deficient females do not display any maternal behavior such as nest building, pup retrieving, crouching, or nursing. However, olfaction, motor behavior and mammary gland function are normal in forebrain Galpha(q/11)-deficient females. We used c-fos immunohistochemistry to investigate pup-induced neuronal activation in different forebrain regions and found a significant reduction in the medial preoptic area, the bed nucleus of stria terminalis, and the lateral septum both in postpartum females and in virgin females after foster pup exposure. Pituitary function, especially prolactin release, was normal in forebrain Galpha(q/11)-deficient females, and activation of oxytocin receptor-positive neurons in the hypothalamus did not differ between genotypes. Our findings show that G(q/11) signaling is indispensable to the neuronal circuit that connects the perception of pup-related stimuli to the initiation of maternal behavior and that this defect cannot be attributed to either reduced systemic prolactin levels or impaired activation of oxytocin receptor-positive neurons of the hypothalamus.

Unresponsiveness of platelets lacking both Galpha(q) and Galpha(13). Implications for collagen-induced platelet activation.

The diffusible platelet stimuli ADP and thromboxane A(2) activate multiple G protein-mediated signaling pathways and function as important secondary mediators of platelet activation as they are released from activated platelets. Because they can also increase their own formation and release, their effects are amplified; eventually, all major G protein-mediated signaling pathways are activated. The multiple positive feedback mechanisms operating during platelet activation have obscured the exact analysis of the roles individual G protein-mediated signaling pathways play during the platelet activation process. In this report, we show that platelets lacking G(q) and G(13) are completely unresponsive to diffusible stimuli such as ADP, thromboxane A(2), or thrombin, even when applied at very high concentrations in combination, whereas all stimuli are able to induce platelet aggregation, shape change, and RhoA activation in platelets lacking only one Galpha subunit. This shows that G(q) or G(13) is required to induce some platelet activation, whereas the activation of G(i)-mediated signaling alone is not sufficient to induceactivation of mouse platelets. In addition, platelets lacking Galpha(q) and Galpha(13) adhered normally to collagen under high shearbut did not aggregate any more in response to collagen, indicating that collagen-induced platelet activation but not platelet adhesion requires intact G protein-mediated signaling pathways.

Mouse models to study G-protein-mediated signaling.

The G-protein-mediated signaling system has evolved as one of the most widely used transmembrane signaling mechanisms in eukaryotic organisms. Mammalian cells express many G-protein-coupled receptors as well as several types of heterotrimeric G-proteins and effectors. This review focuses on recent data from studies in mutant mice, which have elucidated some of the roles of G-protein-mediated signaling in physiology and pathophysiology.

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.