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Despite fully functional primary hemostasis, platelets of healthy neonates exhibit hypoaggregability and secretion defects, which may be adaptations to specific requirements in this developmental stage. The etiologies for reduced signal transduction vary with the type of agonist. The discovered peculiarities are lower receptor densities, reduced calcium mobilization, and functional impairments of G proteins. Reduced secretion of dense granules has been attributed to lower numbers of granules. Signaling studies with adult platelets have shown a regulating effect of the G

Promiscuous G protein-coupled receptors (GPCRs) engage multiple Gα subtypes with different efficacies to propagate signals in cells. A mechanistic understanding of Gα selectivity by GPCRs is critical for therapeutic design, since signaling can be restrained by ligand-receptor complexes to preferentially engage specific G proteins. However, details of GPCR selectivity are unresolved. Here, we investigated cognate G protein selectivity using the prototypical promiscuous Gαq/11 and Gα12/13 coupling receptors, angiotensin II type I receptor (AT1R) and prostaglandin F2α receptor (FP), bioluminescence resonance energy transfer-based G protein and pathway-selective sensors, and G protein knockout cells. We determined that competition between G proteins for receptor binding occurred in a receptor- and G protein-specific manner for AT1R and FP but not for other receptors tested. In addition, we show that while Gα12/13 competes with Gαq/11 for AT1R coupling, the opposite occurs for FP, and Gαq-mediated signaling regulated G protein coupling only at AT1R. In cells, the functional modulation of biased ligands at FP and AT1R was contingent upon cognate Gα availability. The efficacy of AT1R-biased ligands, which poorly signal through Gαq/11, increased in the absence of Gα12/13. Finally, we show that a positive allosteric modulator of Gαq/11 signaling that also allosterically decreases FP-Gα12/13 coupling, lost its negative modulation in the absence of Gαq/11 coupling to FP. Together, our findings suggest that despite preferential binding of similar subsets of G proteins, GPCRs follow distinct selectivity rules, which may contribute to the regulation of ligand-mediated G protein bias of AT1R and FP.

We could previously show that thromboxane A

The purpose of this study aimed to figure out the role of GNA13 in lung squamous cell carcinoma (LUSC) and the underlying mechanism. Male BALB/c mice were used to construct LUSC mouse model. Cell growth was examined using MTT and colony formation assay. Cell migration and invasion was determined using wound healing and transwell assay. The expression and phosphorylation of protein was detected by Western blotting assay. Immunohistochemistry staining was used to observe the tumor growth and metastasis. GNA13 was overexpressed in both LUSC tissues and LUSC cell lines. Knockdown of GNA13 in LUSC cells reduced cell viability and inhibited the formation of colonies in the SK-MES-1 and NCI-H520 cells. Cell migration and invasion was also prevented by inhibition of GNA13 in the LUSC cells. Phosphorylation of PI3K and AKT was downregulated by silencing GNA13 and upregulated by overexpression of GNA13 in the LUSC cells. In LUSC mouse model, tumor size and tumor weight were significantly decreased in si-GNA13 mice compared to control group. The expression of GNA13, Ki67, MMP2 and phosphorylation of AKT were significantly inhibited in si-GNA13 mice compared to control group. This study has demonstrated that knockdown of GNA13 could inhibit cell survival, migration and metastasis in LUSC.

Dendritic cells (DCs) are crucial for initiating adaptive immune responses. However, the factors that control DC positioning and homeostasis are incompletely understood. We found that type-2 conventional DCs (cDC2s) in the spleen depend on Gα 13 and adhesion G protein–coupled receptor family member-E5 (Adgre5, or CD97) for positioning in blood-exposed locations. CD97 function required its autoproteolytic cleavage. CD55 is a CD97 ligand, and cDC2 interaction with CD55-expressing red blood cells (RBCs) under shear stress conditions caused extraction of the regulatory CD97 N-terminal fragment. Deficiency in CD55-CD97 signaling led to loss of splenic cDC2s into the circulation and defective lymphocyte responses to blood-borne antigens. Thus, CD97 mechanosensing of RBCs establishes a migration and gene expression program that optimizes the antigen capture and presentation functions of splenic cDC2s.

We previously reported a series of macrocyclic analogues of [Pyr

Lysophosphatidic acid (LPA) has disruptive effects on lumbar spinal stenosis (LSS). Recently, LPA has been reported to be involved in spinal cord neuronal injury and toxicity, promoting the pathogenesis of LSS. However, the exact effects of LPA on spinal cord neurons remain unknown. The purpose of this study is to investigate the effects of LPA (18 : 1) on spinal cord neuronal cytotoxicity, apoptosis, DNA damage, and oxidative stress. After clinical detection of LPA secretion, spinal cord neurons were treated with LPA (18 : 1); cell viability was analyzed by MTT assay, and LDH leakage was detected by LDH kit; cell apoptosis was detected by flow cytometry; ROS production was measured by DCFDA staining and MitoSOX Red Staining; the activation of the Gα12/Gα13 signaling pathway was detected by serum response factor response element (SRF-RE) luciferase reporter gene; the relationship among LPA, LPA4/6, and ROCK was examined by western blotting. In spinal cord neurons treated with LPA (18 : 1), cellular activity decreased and LDH release increased. The Rho kinase inhibitor (Y-27632) can attenuate LPA-induced apoptosis, DNA damage, and oxidative stress in spinal cord neurons. Moreover mechanistic investigation indicated that LPA (18 : 1) activates Gα12/13–Rho–ROCK2-induced apoptosis, DNA damage, and oxidative stress in spinal cord neurons by upregulating LPA4/LPA6 receptors. Further, the Rho kinase inhibitor Y-27632 attenuates the effects of LPA by downregulating LPA4/LPA6 receptors. Taken together, the possible mechanism by which LPA secretion in LSS patients aggravates patient injury was further elucidated using an LPA-induced spinal cord neuronal injury cell model in vitro.

During tumorigenesis, tumors must evolve to evade the immune system and do so by disrupting the genes involved in antigen processing and presentation or up-regulating inhibitory immune checkpoint genes. We performed in vivo CRISPR screens in syngeneic mouse tumor models to examine requirements for tumorigenesis both with and without adaptive immune selective pressure. In each tumor type tested, we found a marked enrichment for the loss of tumor suppressor genes (TSGs) in the presence of an adaptive immune system relative to immunocompromised mice. Nearly one-third of TSGs showed preferential enrichment, often in a cancer- and tissue-specific manner. These results suggest that clonal selection of recurrent mutations found in cancer is driven largely by the tumor’s requirement to avoid the adaptive immune system.

Hepatocellular carcinoma (HCC) is the second most common cancer worldwide, demonstrating aggressiveness and mortality more frequently in men than in women. Despite reports regarding the inhibitory ability of estrogen receptor alpha (ERα, ESR1) in certain cancer progression, targets and the basis of underlying gender disparity in HCC worsening remain elusive. Here, we report the ability of ERα to transcriptionally inhibit G protein subunit alpha 12 (Gα12) responsible for HCC worsening. First, using human samples and public database, the expression of ERα and Gα12 in HCC was examined. Then, quantitative real-time PCR, chromatin immunoprecipitation-assay, luciferase assay and immunoblottings of liver cancer cell lines confirmed the inhibitory ability of ERα on Gα12 and HCC progression. Gα12 promoted mesenchymal characteristics and amoeboidal movement, which was antagonized by ERα overexpression. Additionally, we found microRNA-141 and microRNA-200a as downstream targets of the Gα12 signaling axis for cancer malignancy regulation under the control of ERα. As for in-depth mechanism, PTP4A1 was found to be directly inhibited by microRNA-141 and microRNA-200a. Moreover, we found the inhibitory effect of ERα on amoeboidal movement by analyzing the morphology and blebbing of liver cancer cells and the active form of MLC levels. The identified targets and ESR1 levels are inversely correlated with human specimens, as well as with sex-biased survival rates of HCC patients. Collectively, ERα-dependent repression of Gα12 and consequent changes in the Gα12 signaling may explain the gender disparity in HCC, providing pharmacological clues for the control of metastatic HCC.

Gα13 transduces signals from G-protein-coupled receptors. While Gα13 functions as a tumor suppressor in lymphomas, it is not known whether Gα13 is pro-tumorigenic or tumor suppressive in genetically engineered mouse (GEM) models of epithelial cancers. Here, we show that loss of Gα13 in the Kras/Tp53 (KPC) GEM model promotes well-differentiated tumors and reduces survival. Mechanistically, tumors developing in KPC mice with Gα13 loss exhibit increased E-cadherin expression and mTOR signaling. Importantly, human pancreatic ductal adenocarcinoma (PDAC) tumors with low Gα13 expression also exhibit increased E-cadherin expression and mTOR signaling. Treatment with the mTOR inhibitor rapamycin decreases the growth of syngeneic KPC tumors with Gα13 loss by promoting cell death. This work establishes a tumor-suppressive role of Gα13 in pancreatic tumorigenesis in the KPC GEM model and suggests targeting mTOR in human PDAC tumors with Gα13 loss.