PAK Membrane Translocation and Phosphorylation Regulate Platelet Aggregation Downstream of Gi and G12/13 Pathways.

Platelet activation plays a pivotal role in physiological hemostasis and pathological thrombosis causing heart attack and stroke. Previous studies conclude that simultaneous activation of Gi and G12/13 signaling pathways is sufficient to cause platelet aggregation. However, using Gq knockout mice and Gq-specific inhibitors, we here demonstrated that platelet aggregation downstream of coactivation of Gi and G12/13 depends on agonist concentrations; coactivation of Gi and G12/13 pathways only induces platelet aggregation under higher agonist concentrations. We confirmed Gi and G12/13 pathway activation by showing cAMP (cyclic adenosine monophosphate) decrease and RhoA activation in platelets stimulated at both low and high agonist concentrations. Interestingly, we found that though Akt and PAK (p21-activated kinase) translocate to the platelet membrane upon both low and high agonist stimulation, membrane-translocated Akt and PAK only phosphorylate at high agonist concentrations, correlating well with platelet aggregation downstream of concomitant Gi and G12/13 pathway activation. PAK inhibitor abolishes Akt phosphorylation, inhibits platelet aggregation in vitro and arterial thrombus formation in vivo. We propose that the PAK-PI3K/Akt pathway mediates platelet aggregation downstream of Gi and G12/13, and PAK may represent a potential antiplatelet and antithrombotic target.

Agonist-induced formation of unproductive receptor-G12 complexes.

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2 receptors (V2R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gs complexes, V2R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V2R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

Podocyte Integrin-ß3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy.

BACKGROUND: Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease-dependent signaling modulates dNP, in part via the G protein-coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. METHODS: A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. RESULTS: The zymogen protein C and aPC bind to podocyte integrin-ß3, a subunit of integrin-αvß3. Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin-αvß3 induces transient binding of integrin-ß3 with G α13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin-ß3via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-ß3, aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC-integrin-ß3 interaction by specifically deleting podocyte integrin-ß3 or by abolishing aPC's integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC's nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPChigh and wild-type mice.Conclusions aPC-integrin-αvß3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin-αvß3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

WNT16 Requires Gα Subunits as Intracellular Partners for Both Its Canonical and Non-Canonical WNT Signalling Activity in Osteoblasts.

In the past years, WNT16 became an interesting target in the field of skeletal research, as it was identified as an essential regulator of the cortical bone compartment, with the ability to increase both cortical and trabecular bone mass and strength in vivo. Even though there are indications that these advantageous effects are coming from canonical and non-canonical WNT-signalling activity, a clear model of WNT signalling by WNT16 is not yet depicted. We, therefore, investigated the modulation of canonical (WNT/ß-catenin) and non-canonical [WNT/calcium, WNT/planar cell polarity (PCP)] signalling in human embryonic kidney (HEK) 293 T and SaOS2 cells. Here, we demonstrated that WNT16 activates all WNT-signalling pathways in osteoblasts, whereas only WNT/calcium signalling was activated in HEK293T cells. In osteoblasts, we therefore, additionally investigated the role of Gα subunits as intracellular partners in WNT16's mechanism of action by performing knockdown of Gα12, Gα13 and Gαq. These studies point out that the above-mentioned Gα subunits might be involved in the WNT/ß-catenin and WNT/calcium-signalling activity by WNT16 in osteoblasts, and for Gα12 in its WNT/PCP-signalling activity, illustrating a novel possible mechanism of interplay between the different WNT-signalling pathways in osteoblasts. Additional studies are needed to demonstrate whether this mechanism is specific for WNT16 signalling or relevant for all other WNT ligands as well. Altogether, we further defined WNT16's mechanism of action in osteoblasts that might underlie the well-known beneficial effects of WNT16 on skeletal homeostasis. These findings on WNT16 and the activity of specific Gα subunits in osteoblasts could definitely contribute to the development of novel therapeutic approaches for fragility fractures in the future.

Heterotrimeric G-alpha subunits Gpa11 and Gpa12 define a transduction pathway that control spore size and virulence in Mucor circinelloides.

Mucor circinelloides is one of the causal agents of mucormycosis, an emerging and high mortality rate fungal infection produced by asexual spores (sporangiospores) of fungi that belong to the order Mucorales. M. circinelloides has served as a model genetic system to understand the virulence mechanism of this infection. Although the G-protein signaling cascade plays crucial roles in virulence in many pathogenic fungi, its roles in Mucorales are yet to be elucidated. Previous study found that sporangiospore size and calcineurin are related to the virulence in Mucor, in which larger spores are more virulent in an animal mucormycosis model and loss of a calcineurin A catalytic subunit CnaA results in larger spore production and virulent phenotype. The M. circinelloides genome is known to harbor twelve gpa (gpa1 to gpa12) encoding G-protein alpha subunits and the transcripts of the gpa11 and gpa12 comprise nearly 72% of all twelve gpa genes transcript in spores. In this study we demonstrated that loss of function of Gpa11 and Gpa12 led to larger spore size associated with reduced activation of the calcineurin pathway. Interestingly, we found lower levels of the cnaA mRNAs in sporangiospores from the Δgpa12 and double Δgpa11/Δgpa12 mutant strains compared to wild-type and the ΔcnaA mutant had significantly lower gpa11 and gpa12 mRNA levels compared to wild-type. However, in contrast to the high virulence showed by the large spores of ΔcnaA, the spores from Δgpa11/Δgpa12 were avirulent and produced lower tissue invasion and cellular damage, suggesting that the gpa11 and gpa12 define a signal pathway with two branches. One of the branches controls spore size through regulation of calcineurin pathway, whereas virulences is controlled by an independent pathway. This virulence-related regulatory pathway could control the expression of genes involved in cellular responses important for virulence, since sporangiospores of Δgpa11/Δgpa12 were less resistant to oxidative stress and phagocytosis by macrophages than the ΔcnaA and wild-type strains. The characterization of this pathway could contribute to decipher the signals and mechanism used by Mucorales to produce mucormycosis.

Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release, and coagulation.

It is currently unclear why agonist-stimulated platelets require shear force to efficiently externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles (MVs). We reveal that integrin outside-in signaling is an important mechanism for this requirement. PS exposure and MV release were inhibited in ß3-/- platelets or by integrin antagonists. The impaired MV release and PS exposure in ß3-/- platelets were rescued by expression of wild-type ß3 but not a Gα13 binding-deficient ß3 mutant (E733EE to AAA), which blocks outside-in signaling but not ligand binding. Inhibition of Gα13 or Src also diminished agonist/shear-dependent PS exposure and MV release, further indicating a role for integrin outside-in signaling. PS exposure in activated platelets was induced by application of pulling force via an integrin ligand, which was abolished by inhibiting Gα13-integrin interaction, suggesting that Gα13-dependent transmission of mechanical signals by integrins induces PS exposure. Inhibition of Gα13 delayed coagulation in vitro. Furthermore, inhibition or platelet-specific knockout of Gα13 diminished laser-induced intravascular fibrin formation in arterioles in vivo. Thus, ß3 integrins serve as a shear sensor activating the Gα13-dependent outside-in signaling pathway to facilitate platelet procoagulant function. Pharmacological targeting of Gα13-integrin interaction prevents occlusive thrombosis in vivo by inhibiting both coagulation and platelet thrombus formation.

Loss of Gα12/13 exacerbates apical area dependence of actomyosin contractility.

During development, coordinated cell shape changes alter tissue shape. In the Drosophila ventral furrow and other epithelia, apical constriction of hundreds of epithelial cells folds the tissue. Genes in the Gα12/13 pathway coordinate collective apical constriction, but the mechanism of coordination is poorly understood. Coupling live-cell imaging with a computational approach to identify contractile events, we discovered that differences in constriction behavior are biased by initial cell shape. Disrupting Gα12/13 exacerbates this relationship. Larger apical area is associated with delayed initiation of contractile pulses, lower apical E-cadherin and F-actin levels, and aberrantly mobile Rho-kinase structures. Our results suggest that loss of Gα12/13 disrupts apical actin cortex organization and pulse initiation in a size-dependent manner. We propose that Gα12/13 robustly organizes the apical cortex despite variation in apical area to ensure the timely initiation of contractile pulses in a tissue with heterogeneity in starting cell shape.

S1pr2/Gα13 signaling regulates the migration of endocardial precursors by controlling endoderm convergence.

Formation of the heart tube requires synchronized migration of endocardial and myocardial precursors. Our previous studies indicated that in S1pr2/Gα13-deficient embryos, impaired endoderm convergence disrupted the medial migration of myocardial precursors, resulting in the formation of two myocardial populations. Here we show that endoderm convergence also regulates endocardial migration. In embryos defective for S1pr2/Gα13 signaling, endocardial precursors failed to migrate towards the midline, and the presumptive endocardium surrounded the bilaterally-located myocardial cells rather than being encompassed by them. In vivo imaging of control embryos revealed that, like their myocardial counterparts, endocardial precursors migrated with the converging endoderm, though from a more anterior point, then moved from the dorsal to the ventral side of the endoderm (subduction), and finally migrated posteriorly towards myocardial precursors, ultimately forming the inner layer of the heart tube. In embryos defective for endoderm convergence due to an S1pr2/Gα13 deficiency, both the medial migration and the subduction of endocardial precursors were impaired, and their posterior migration towards the myocardial precursors was premature. This placed them medial to the myocardial populations, physically blocking the medial migration of the myocardial precursors. Furthermore, contact between the endocardial and myocardial precursor populations disrupted the epithelial architecture of the myocardial precursors, and thus their medial migration; in embryos depleted of endocardial cells, the myocardial migration defect was partially rescued. Our data indicate that endoderm convergence regulates the medial migration of endocardial precursors, and that premature association of the endocardial and myocardial populations contributes to myocardial migration defects observed in S1pr2/Gα13-deficient embryos. The demonstration that endoderm convergence regulates the synchronized migration of endocardial and myocardial precursors reveals a new role of the endoderm in heart development.

GEP oncogene promotes cell proliferation through YAP activation in ovarian cancer.

G-protein-coupled receptors (GPCRs) and their ligands function in the progression of human malignancies. Gα12 and Gα13, encoded by GNA12 and GNA13, respectively, are referred to as the GEP oncogene and are implicated in tumor progression. However, the molecular mechanisms by which Gα12/13 activation promotes cancer progression are not fully elucidated. Here, we demonstrate elevated expression of Gα12/13 in human ovarian cancer tissues. Gα12/13 activation did not promote cellular migration in the ovarian cancer cell lines examined. Rather, Gα12/13 activation promoted cell growth. We used a synthetic biology approach using chimeric G proteins and GPCRs activated solely by artificial ligands to selectively trigger signaling pathways downstream of specific G proteins. We found that Gα12/13 promotes proliferation of ovarian cancer cells by activating the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway. Furthermore, we reveal that inhibition of YAP by short hairpin RNA or a specific inhibitor prevented the growth of ovarian cancer cells. Therefore, YAP may be a suitable therapeutic target in ovarian cancer.

RGS22 inhibits pancreatic adenocarcinoma cell migration through the G12/13 α subunit/F-actin pathway.

Pancreatic cancer is characterized by the potential for local invasion, allowing it to spread during the early developmental stages of the disease. Regulator of G protein signaling 22 (RGS22) localizes to the cytoplasm in pancreatic adenocarcinoma tissue. We overexpressed RGS22 in the human pancreatic cancer cell line BXPC-3. Cells that overexpressed RGS22 had much lower wound-healing rates and greatly reduced migration compared to the control cells. Conversely, cells in which RGS22 expression had been downregulated had higher wound-healing rates and migration than the control cells. These results confirmed that RGS22 expression suppresses pancreatic adenocarcinoma cell migration. Pull-down and coimmunoprecipitation assays revealed that RGS22 had specific interactions with the heterotrimeric G protein G12 α subunit (GNA12) and GNA13 in the cells. We also demonstrated that in the presence of higher RGS22 expression, the cell deformation and F-actin formation caused by lysophosphatidic acid treatment, is delayed. Constitutively active Gα subunits did not accelerate GTP hydrolysis to GDP. We did not investigate the function of RGS22 as a negative regulator of heterotrimeric G12/13 protein signaling. Our data demonstrate that RGS22 acts as a tumor suppressor, repressing human pancreatic adenocarcinoma cell migration by coupling to GNA12/13, which in turn leads to inhibition of stress fiber formation.

Gastrin-stimulated Gα13 Activation of Rgnef Protein (ArhGEF28) in DLD-1 Colon Carcinoma Cells.

The guanine nucleotide exchange factor Rgnef (also known as ArhGEF28 or p190RhoGEF) promotes colon carcinoma cell motility and tumor progression via interaction with focal adhesion kinase (FAK). Mechanisms of Rgnef activation downstream of integrin or G protein-coupled receptors remain undefined. In the absence of a recognized G protein signaling homology domain in Rgnef, no proximal linkage to G proteins was known. Utilizing multiple methods, we have identified Rgnef as a new effector for Gα13 downstream of gastrin and the type 2 cholecystokinin receptor. In DLD-1 colon carcinoma cells depleted of Gα13, gastrin-induced FAK Tyr(P)-397 and paxillin Tyr(P)-31 phosphorylation were reduced. RhoA GTP binding and promoter activity were increased by Rgnef in combination with active Gα13. Rgnef co-immunoprecipitated with activated Gα13Q226L but not Gα12Q229L. The Rgnef C-terminal (CT, 1279-1582) region was sufficient for co-immunoprecipitation, and Rgnef-CT exogenous expression prevented Gα13-stimulated SRE activity. A domain at the C terminus of the protein close to the FAK binding domain is necessary to bind to Gα13. Point mutations of Rgnef-CT residues disrupt association with active Gα13 but not Gαq. These results show that Rgnef functions as an effector of Gα13 signaling and that this linkage may mediate FAK activation in DLD-1 colon carcinoma cells.

Gα12 gep oncogene deregulation of p53-responsive microRNAs promotes epithelial-mesenchymal transition of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a poor prognosis owing to aggressive phenotype. Gα12 gep oncogene product couples to G-protein-coupled receptors, whose ligand levels are frequently increased in tumor microenvironments. Here, we report Gα12 overexpression in human HCC and the resultant induction of zinc-finger E-box-binding homeobox 1 (ZEB1) as mediated by microRNA deregulation. Gα12 expression was higher in HCC than surrounding non-tumorous tissue. Transfection of Huh7 cell with an activated mutant of Gα12 (Gα12QL) deregulated microRNA (miRNA or miR)-200b/a/429, -194-2/192 and -194-1/215 clusters in the miRNome. cDNA microarray analyses disclosed the targets affected by Gα12 gene knockout. An integrative network of miRNAs and mRNA changes enabled us to predict ZEB1 as a key molecule governed by Gα12. Decreases of miR-200a/b, -192 and -215 by Gα12 caused ZEB1 induction. The ability of Gα12 to decrease p53 levels, as a result of activating protein-1 (AP-1)/c-Jun-mediated mouse double minute 2 homolog induction, contributed to transcriptional deregulation of the miRNAs. Gα12QL induced ZEB1 and other epithelial-mesenchymal transition markers with fibroblastoid phenotype change. Consistently, transfection with miR-200b, -192 or -215 mimic prevented the ability of Gα12QL to increase tumor cell migration/invasion. In xenograft studies, sustained knockdown of Gα12 decreased the overall growth rate and average volume of tumors derived from SK-Hep1 cell (mesenchymal-typed). In HCC patients, miR-192, -215 and/or -200a were deregulated with microvascular invasion or growth advantage. In the HCC samples with higher Gα12 level, a correlation existed in the comparison of relative changes of Gα12 and ZEB1. In conclusion, Gα12 overexpressed in HCC causes ZEB1 induction by deregulating p53-responsive miRNAs, which may facilitate epithelial-mesenchymal transition and growth of liver tumor. These findings highlight the significance of Gα12 upregulation in liver tumor progression, implicating Gα12 as an attractive therapeutic target.

Cross talk between the bombesin neuropeptide receptor and Sonic hedgehog pathways in small cell lung carcinoma.

Small cell lung carcinoma (SCLC) often features the upregulation of the Sonic hedgehog (Shh) pathway leading to activation of Gli transcription factors. SCLC cells secrete bombesin (BBS)-like neuropeptides that act as autocrine growth factors. Here, we show that SCLC tumor samples feature co-expression of Shh and BBS-cognate receptor (gastrin-releasing peptide receptor (GRPR)). We also demonstrate that BBS activates Gli in SCLC cells, which is crucial for BBS-mediated SCLC proliferation, because cyclopamine, an inhibitor of the Shh pathway, hampered the BBS-mediated effects. BBS binding to GRPR stimulated Gli through its downstream Gαq and Gα12/13 GTPases, and consistently, other Gαq and Gα13 coupled receptors (such as muscarinic receptor, m1, and thrombin receptor, PAR-1) and constitutively active GαqQL and Gα12/13QL mutants stimulated Gli. By using cells null for Gαq and Gα12/13, we demonstrate that these G proteins are strictly necessary for Gli activation by BBS. Moreover, by using constitutively active Rho small G-protein (Rho QL) as well as its inhibitor, C3 toxin, we show that Rho mediates G-protein-coupled receptor (GPCR)-, Gαq- and Gα12/13-dependent Gli stimulation. At the molecular level, BBS caused a significant increase in Shh gene transcription and protein secretion that was dependent on BBS-induced GPCR/Gαq-12/13/Rho mediated activation of nuclear factor κB (NFκB), which can stimulate a NF-κB response element in the Shh gene promoter. Our data identify a novel molecular network acting in SCLC linking autocrine BBS and Shh circuitries and suggest Shh inhibitors as novel therapeutic strategies against this aggressive cancer type.

Modeling the role of G12V and G13V Ras mutations in the Ras-GAP-catalyzed hydrolysis reaction of guanosine triphosphate.

Cancer-associated point mutations in Ras, in particular, at glycine 12 and glycine 13, affect the normal cycle between inactive GDP-bound and active GTP-bound states. In this work, the role of G12V and G13V replacements in the GAP-stimulated intrinsic GTP hydrolysis reaction in Ras is studied using molecular dynamics (MD) simulations with quantum mechanics/molecular mechanics (QM/MM) potentials. A model molecular system was constructed by motifs of the relevant crystal structure (Protein Data Bank entry 1WQ1 ). QM/MM optimization of geometry parameters in the Ras-GAP-GTP complex and QM/MM-MD simulations were performed with a quantum subsystem comprising a large fraction of the enzyme active site. For the system with wild-type Ras, the conformations fluctuated near the structure ready to be involved in the efficient chemical reaction leading to the cleavage of the phosphorus-oxygen bond in GTP upon approach of the properly aligned catalytic water molecule. Dynamics of the system with the G13V mutant is characterized by an enhanced flexibility in the area occupied by the γ-phosphate group of GTP, catalytic water, and the side chains of Arg789 and Gln61, which should somewhat hinder fast chemical steps. Conformational dynamics of the system with the G12V mutant shows considerable displacement of the Gln61 side chain and catalytic water from their favorable arrangement in the active site that may lead to a marked reduction in the reaction rate. The obtained computational results correlate well with the recent kinetic measurements of the Ras-GAP-catalyzed hydrolysis of GTP.

Regulation of phospholipase C-ß(1) GTPase-activating protein (GAP) function and relationship to G(q) efficacy.

How cells regulate Gq efficacy (initiation and termination of Gq signaling) to effect response remains a central question in pharmacology and drug discovery. Phospholipase C-ß1 (PLC-ß1) is an effector and a GTPase activating protein (GAP) specific to Gαq. The physiological function of PLC-ß1 GAP remains unclear and controversial. GAPs are generally thought to function in deactivation of Gq signaling. However, PLC-ß1 GAP has also been shown to increase signaling efficiency through kinetic coupling with the ligand-activated GPCR. GPCRs function as guanine nucleotide exchange factors (GEF) on the G protein activation cycle. This article sets forth a new hypothesis that could unify these conflicting paradigms as it pertains to physiological signaling and native levels of protein. It is proposed that the physiological function of PLC-ß1 GAP is context-dependent and regulated by phosphatidic acid (PA). PA stimulates PLC-ß1 GAP activity. In the absence of ligand, PLC-ß1 GAP does indeed deactivate Gq signaling, limiting leaky activation to set the threshold for stimulation to sharpen signal kinetics. However in the presence of activating ligand, the increase in levels of PA would stimulate PLC-ß1 GAP to kinetically couple with GPCR GEF to increase signaling efficiency. We found that PA-increased Gq efficiency is dependent on signaling via the unique PLC-ß1 PA binding domain.

Apelin-APJ signaling is a critical regulator of endothelial MEF2 activation in cardiovascular development.

RATIONALE: The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. OBJECTIVE: This study aimed to characterize the embryonic lethal phenotype of the Apj-/- mice and to define the involved downstream signaling targets. METHODS AND RESULTS: We report the first characterization of the embryonic lethality of the Apj-/- mice. More than half of the expected Apj-/- embryos died in utero because of cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj-/- embryos surviving to later stages demonstrated incomplete vascular maturation because of a deficiency of vascular smooth muscle cells and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, noncanonical signaling pathway downstream of apelin-APJ involving Gα13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of myocyte enhancer factor 2. Apj-/- mice have greater endocardial Hdac4 and Hdac5 nuclear localization and reduced expression of the myocyte enhancer factor 2 (MEF2) transcriptional target Krüppel-like factor 2. We identify a number of commonly shared transcriptional targets among apelin-APJ, Gα13, and MEF2 in endothelial cells, which are significantly decreased in the Apj-/- embryos and endothelial cells. CONCLUSIONS: Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.

TGFα shedding assay: an accurate and versatile method for detecting GPCR activation.

A single-format method to detect multiple G protein-coupled receptor (GPCR) signaling, especially Gα(12/13) signaling, presently has limited throughput and sensitivity. Here we report a transforming growth factor-α (TGFα) shedding assay, in which GPCR activation is measured as ectodomain shedding of a membrane-bound proform of alkaline phosphatase-tagged TGFα (AP-TGFα) and its release into conditioned medium. AP-TGFα shedding response occurred almost exclusively downstream of Gα(12/13) and Gα(q) signaling. Relying on chimeric Gα proteins and promiscuous Gα(16) protein, which can couple with Gα(s)- and Gα(i)-coupled GPCRs and induce Gα(q) signaling, we used the TGFα shedding assay to detect 104 GPCRs among 116 human GPCRs. We identified three orphan GPCRs (P2Y10, A630033H20 and GPR174) as Gα(12/13)-coupled lysophosphatidylserine receptors. Thus, the TGFα shedding assay is useful for studies of poorly characterized Gα(12/13)-coupled GPCRs and is a versatile platform for detecting GPCR activation including searching for ligands of orphan GPCRs.

5-HT7R/G12 signaling regulates neuronal morphology and function in an age-dependent manner.

The common neurotransmitter serotonin controls different aspects of early neuronal differentiation, although the underlying mechanisms are poorly understood. Here we report that activation of the serotonin 5-HT(7) receptor promotes synaptogenesis and enhances synaptic activity in hippocampal neurons at early postnatal stages. An analysis of Gα(12)-deficient mice reveals a critical role of G(12)-protein for 5-HT(7) receptor-mediated effects in neurons. In organotypic preparations from the hippocampus of juvenile mice, stimulation of 5-HT(7)R/G(12) signaling potentiates formation of dendritic spines, increases neuronal excitability, and modulates synaptic plasticity. In contrast, in older neuronal preparations, morphogenetic and synaptogenic effects of 5-HT(7)/G(12) signaling are abolished. Moreover, inhibition of 5-HT(7) receptor had no effect on synaptic plasticity in hippocampus of adult animals. Expression analysis reveals that the production of 5-HT(7) and Gα(12)-proteins in the hippocampus undergoes strong regulation with a pronounced transient increase during early postnatal stages. Thus, regulated expression of 5-HT(7) receptor and Gα(12)-protein may represent a molecular mechanism by which serotonin specifically modulates formation of initial neuronal networks during early postnatal development.

p2y5/LPA(6) attenuates LPA(1)-mediated VE-cadherin translocation and cell-cell dissociation through G(12/13) protein-Src-Rap1.

AIMS: We investigated the mechanisms of action of lysophosphatidic acid (LPA) to regulate vascular endothelial (VE)-cadherin dynamics and cell-cell contact. METHODS AND RESULTS: While a low concentration of LPA stimulated VE-cadherin internalization and subsequent cell-cell dissociation, a high concentration of LPA masked the disruptive actions on VE-cadherin and protected the barrier function in human vascular endothelial cells. Knockdown experiments of major LPA receptor subtypes, i.e. LPA(1) and p2y5 (also termed LPA(6)), with their specific small interfering RNAs, showed that LPA(1) and LPA(6) mediate the LPA-induced disruptive and protective actions on barrier integrity, respectively. LPA(6)-mediated tube formation, reflecting stabilization of barrier integrity, was confirmed by in vitro angiogenesis assay. The LPA(1)-mediated disruptive actions were inhibited by pertussis toxin, dominant-negative Rac1, and inhibitors for c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), but not by dominant-negative RhoA. In contrast, the LPA(6)-mediated protective actions were associated with activation of Src and Rap1 and attenuated by abrogation of their activities. Further characterization showed that Rap1 is located downstream of Src and dependent on C3G, a Rap1 guanine nucleotide exchange factor. Finally, an LPA antagonist significantly inhibited lactic acid-induced limb lesions in vivo, which may be attributed to dysfunction of endothelial cells. CONCLUSION: LPA induced disruption and protection of VE-cadherin integrity through LPA(1)-G(i) protein-Rac1-JNK/p38MAPK and LPA(6)-G(12/13) protein-Src-C3G-Rap1 pathways, respectively.

Neoplastic transformation induced by the gep oncogenes involves the scaffold protein JNK-interacting leucine zipper protein.

The activated mutants of the α-subunits of G proteins G(12) and G(13) have been designated as the gep oncogenes owing to their ability to stimulate diverse oncogenic signaling pathways that lead to neoplastic transformation of fibroblast cell lines and tumorigenesis in nude mice models. Studies from our laboratory as well as others have shown that the growth-promoting activities of Gα(12) and Gα(13) involve potent activation of c-Jun N-terminal kinases (JNKs). Our previous studies have indicated that the JNK-interacting leucine zipper protein (JLP), a scaffold protein involved in the structural and functional organization of the JNK/p38 mitogen-activated protein kinase module, tethers Gα(12) and Gα(13) to the JNK signaling module. In the present study, in addition to demonstrating the physical association between JLP and Gα(12), we show that this interaction is enhanced by the receptor- or mutation-mediated activation of Gα(12). We also establish that JLP interacts with Gα(12) through the C-terminal domain that has been previously identified to be involved in binding to Gα(13). Furthermore, using this C-terminal domain as a competitively inhibitor of JLP that can disrupt Gα(12)-JLP interaction, we demonstrate that JLP is required for the stimulation of JNK by Gα(12). Our results also indicate that such JLP interaction is required for Gα(12) as well as Gα(13)-mediated neoplastic transformation of JLP. These studies demonstrate for the first time a functional role for JLP in the gep oncogene-regulated neoplastic signaling pathway.