Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The small GTPases of the Rho family play a key role in a number of signaling pathways activated by lysophosphatidic acid (LPA). However, little is known concerning the mechanism of regulation of these proteins. In this study we demonstrate that in Swiss 3T3 fibroblasts, LPA induces a sustained, time-dependent relocalization of RhoA to the Triton X-100-soluble low speed membrane fraction, which can be reversed by removal of LPA from the medium. Translocation was only observed with micromolar concentrations of LPA and was inhibited by pretreating the cells with pertussis toxin but not with tyrosine kinase inhibitors. LPA also induced translocation of CDC42Hs to the membranes but had no effect on the distribution of Rac1, RhoB, or Rho-GDI. Translocation of RhoA was also induced by endothelin-1. Conversely, platelet-derived growth factor did not cause the translocation of RhoA to any membrane fraction but stimulated relocalization of Rac1 to the high speed membrane fraction. Significantly, incubation of cell lysates with guanosine 5'-O-(thiotriphosphate) was sufficient to translocate RhoA, Rac1, and CDC42Hs from the cytosol to the membranes, whereas incubation with GDP had the opposite effect. These data suggest that the translocation of the Rho family proteins to the membrane fraction is controlled by their activation state and that agonists show selectivity in inducing the activation/translocation of these proteins.
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PMID:Differential translocation of rho family GTPases by lysophosphatidic acid, endothelin-1, and platelet-derived growth factor. 895 54

In a cell-free system from neutrophil cytosol GTP(&ggr ;)S can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTP(&ggr ;)S stimulation was susceptible to an excess of GDP, but not Bordetella pertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPgammaS.Gialpha2 or Gbeta1gamma2 subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPgammaS. When recombinant, constitutively activated and GTPgammaS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations >100 times the endogenous, however, they were ineffective. Purified Cdc42/Rac-interactive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPgammaS. The action of GTPgammaS was blocked by mM [Mg2+], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B-sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPgammaS under physiologic conditions. The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPgammaS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P2-binding peptide did not interfere with the response to GTPgammaS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.
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PMID:GTPgammaS-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor. 958 May 66

We studied the function of the platelet integrin alphaIIb beta3 using a B lymphocyte model in which alphaIIb beta3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate alphaIIb beta3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and alphaIIb beta3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Galphai, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of alphaIIb beta3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that alphaIIb beta3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Galphai, protein kinase C, and the actin cytoskeleton.
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PMID:Regulation of alphaIIb beta3 function in human B lymphocytes. 961 43

In cultured vascular smooth muscle cells (VSMCs), activation of phospholipase D (PLD) by angiotensin II (Ang II) represents a major source of sustained generation of second messengers. Understanding the molecular mechanisms controlling activation of this pathway is essential to clarify the complexities of Ang II signaling, but the most proximal mechanisms coupling AT1 receptors to PLD have not been defined. Here we examine the role of heterotrimeric G proteins in AT1 receptor-PLD coupling. In alpha-toxin permeabilized VSMCs, GTPgammaS enhanced Ang II-stimulated PLD activation. In intact cells, Ang II activation of PLD was pertussis toxin-insensitive and was not additive with sodium fluoride, a cell-permeant activator of heterotrimeric G proteins, indicating that AT1 receptor-PLD coupling requires pertussis toxin-insensitive heterotrimeric G proteins. Ang II-stimulated PLD activity was significantly inhibited in VSMCs electroporated with anti-Gbeta antibody (56 +/- 5%) and in cells overexpressing the Gbetagamma-binding region of the carboxyl terminus of beta-adrenergic receptor kinase1 (79 +/- 8%), suggesting a critical role for Gbetagamma in PLD activation by Ang II. This effect may be mediated by pp60(c-src), because in beta-adrenergic receptor kinase1 overexpressing cells, pp60(c-src) activation was inhibited, and in normal cells anti-pp60(c-src) antibody inhibited Ang II-stimulated PLD activity. Galpha12 may also contribute to AT1 receptor-PLD coupling because electroporation of anti-Galpha12 antibody significantly inhibited PLD activity, whereas anti-Galphai and Galphaq/11 antibodies had no effect. Furthermore, electroporation of anti-RhoA antibody also attenuated Ang II-induced PLD activation, suggesting a role for small molecular weight G protein RhoA in this response. Thus, we provide evidence here that Gbetagamma as well as Galpha12 subunits mediate AT1 receptor coupling to tonic PLD activation via pp60(c-src)-dependent mechanisms, and that RhoA is involved in these signaling pathways in rat VSMCs. These results may provide insight into the molecular mechanisms underlying the highly organized, complex, chronic signaling programs associated with vascular smooth muscle growth and remodeling in response to Ang II.
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PMID:Angiotensin II receptor coupling to phospholipase D is mediated by the betagamma subunits of heterotrimeric G proteins in vascular smooth muscle cells. 988 8

Lysophosphatidic acid (LPA) stimulates the c-Fos serum response element (SRE) by activating two distinct signal pathways regulated by the small GTPases, Ras and RhoA. Ras activates the ERK cascade leading to phosphorylation of the transcription factors Elk-1 and Sap1a at the Ets/TCF site. RhoA regulates an undefined pathway required for the activation of the SRF/CArG site. Here we have examined the role of the Ras and RhoA pathways in activation of the SRE and c-Fos expression in Rat-1 cells. Pertussis toxin and PD98059 strongly inhibited LPA-stimulated c-Fos expression and activation of a SRE:Luc reporter. C3 toxin completely inhibited RhoA function, partially inhibited SRE:Luc activity, but had no effect on LPA-stimulated c-Fos expression. Thus, in a physiological context the Ras-Raf-MEK-ERK pathway, but not RhoA, is required for LPA-stimulated c-Fos expression in Rat-1 cells. C3 toxin stimulated the stress-activated protein kinases JNK and p38 and potentiated c-Jun expression and phosphorylation; these properties were shared by another cellular stress agonist the protein kinase C inhibitor Ro-31-8220. However, C3 toxin alone or in combination with growth factors did not stimulate AP-1:Luc activity and actually antagonized the synergistic activation of AP-1:Luc observed in response to co-stimulation with growth factors and Ro-31-8220. These data indicate that C3 toxin is a cellular stress which antagonizes activation of AP-1 at a point downstream of stress-activated kinase activation or immediate-early gene induction.
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PMID:C3 toxin activates the stress signaling pathways, JNK and p38, but antagonizes the activation of AP-1 in rat-1 cells. 992 Sep 30

1. We have studied the modulation of volume-regulated anion channels (VRACs) by the small GTPase Rho and by one of its targets, Rho kinase, in calf pulmonary artery endothelial (CPAE) cells. 2. RT-PCR and immunoblot analysis showed that both RhoA and Rho kinase are expressed in CPAE cells. 3. ICl,swell, the chloride current through VRACs, was activated by challenging CPAE cells with a 25 % hypotonic extracellular solution (HTS) or by intracellular perfusion with a pipette solution containing 100 microM GTPgammaS. 4. Pretreatment of CPAE cells with the Clostridium C2IN-C3 fusion toxin, which inactivates Rho by ADP ribosylation, significantly impaired the activation of ICl,swell in response to the HTS. The current density at +100 mV was 49 +/- 13 pA pF-1 (n = 17) in pretreated cells compared with 172 +/- 17 pA pF-1 (n = 21) in control cells. 5. The volume-independent activation of ICl,swell by intracellular perfusion with GTPgammaS was also impaired in C2IN-C3-pretreated cells (31 +/- 7 pA pF-1, n = 11) compared with non-treated cells (132 +/- 21 pA pF-1, n = 15). 6. Activation of ICl,swell was pertussis toxin (PTX) insensitive. 7. Y-27632, a blocker of Rho kinase, inhibited ICl,swell and delayed its activation. 8. Inhibition of Rho and of Rho kinase by the above-described treatments did not affect the extent of cell swelling in response to HTS. 9. These experiments provide strong evidence that the Rho-Rho kinase pathway is involved in the VRAC activation cascade.
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PMID:Role of Rho and Rho kinase in the activation of volume-regulated anion channels in bovine endothelial cells. 1006 23

The signaling mechanisms utilized by bradykinin (BK) to activate the transcription factor nuclear factor kappaB (NF-kappaB) are poorly defined. We previously demonstrated that BK-stimulated NF-kappaB activation requires the small GTPase RhoA. We present evidence that BK-induced NF-kappaB activation both activates and requires phosphatidylinositol 3-kinase (PI 3-kinase) in A549 human epithelial cells. Pre-treatment with the PI 3-kinase-specific inhibitors, wortmannin, and LY294002 effectively blocked BK-induced PI 3-kinase activity. Wortmannin and LY294002 also abolished BK-induced NF-kappaB activation, as did transient transfection with a dominant negative mutant of the p85 subunit. BK-stimulated PI 3-kinase activity and NF-kappaB activation were sensitive to pertussis but not cholera toxin, suggesting that the B2 BK receptors transducing the response were coupled to Galphai or Galphao heterotrimeric G proteins. Tumor necrosis factor alpha (TNFalpha) also stimulated increased PI 3-kinase activity, however TNFalpha-stimulated NF-kappaB activation was not affected by the PI 3-kinase inhibitors or the p85 dominant negative mutant. These findings provide evidence that BK-induced NF-kappaB activation utilizes a signaling pathway that requires activity of both RhoA and PI 3-kinase and is distinct from the signaling pathway utilized by TNFalpha. Furthermore, we show that the p85 regulatory subunit is required for activation of PI 3-kinase activity by this G protein-coupled receptor.
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PMID:Requirement of phosphatidylinositol 3-kinase activity for bradykinin stimulation of NF-kappaB activation in cultured human epithelial cells. 1018 65

In the accompanying paper [Khare et al., Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G993-G1004, 1999], activation of protein kinase C-alpha (PKC-alpha) was shown to be involved in the stimulation of phospholipase D (PLD) by 1,25-dihydroxyvitamin D3 [1, 25(OH)2D3] and 12-O-tetradecanoylphorbol 13-acetate (TPA) in Caco-2 cells. Monomeric or heterotrimeric G proteins, as well as pp60(c-src) have been implicated in PLD activation. We therefore determined whether these signal transduction elements were involved in PLD stimulation by 1,25(OH)2D3 or TPA. Treatment with C3 transferase, which inhibits members of the Rho family of monomeric G proteins, markedly diminished the ability of 1,25(OH)2D3, but not TPA, to stimulate PLD. Brefeldin A, an inhibitor of ADP-ribosylation factor proteins, did not, however, significantly reduce the stimulation of PLD by either of these agents. Moreover, 1,25(OH)2D3, but not TPA, activated pp60(c-src) and treatment with PP1, a specific inhibitor of the pp60(c-src) family, blocked the ability of 1,25(OH)2D3 to activate PLD. Pretreatment of cells with pertussis toxin (PTx) markedly reduced the stimulation of PLD by either agonist. PTx, moreover, inhibited the stimulation of pp60(c-src) and PKC-alpha by 1,25(OH)2D3. PTx did not, however, block the membrane translocation of RhoA induced by 1,25(OH)2D3 or inhibit the stimulation of PKC-alpha by TPA. These findings, taken together with those of the accompanying paper, indicate that although 1,25(OH)2D3 and TPA each activate PLD in Caco-2 cells in part via PKC-alpha, their stimulation of PLD differs in a number of important aspects, including the requirement for pp60(c-src) and RhoA in the activation of PLD by 1,25(OH)2D3, but not TPA. Moreover, the requirement for different signal transduction elements by 1,25(OH)2D3 and TPA to induce the stimulation of PLD may potentially underlie differences in the physiological effects of these agents in Caco-2 cells.
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PMID:1,25-dihydroxyvitamin D3 but not TPA activates PLD in Caco-2 cells via pp60(c-src) and RhoA. 1019 45

A major function of Rac2 in neutrophils is the regulation of oxidant production important in bacterial killing. Rac and the related GTPase Cdc42 also regulate the dynamics of the actin cytoskeleton, necessary for leukocyte chemotaxis and phagocytosis of microorganisms. Although these GTPases appear to be critical downstream components of chemoattractant receptor signaling in human neutrophils, the pathways involved in direct control of Rac/Cdc42 activation remain to be determined. We describe an assay that measures the formation of Rac-GTP and Cdc42-GTP based on their specific binding to the p21-binding domain of p21-activated kinase 1. A p21-binding domain glutathione S-transferase fusion protein specifically binds Rac and Cdc42 in their GTP-bound forms both in vitro and in cell samples. Binding is selective for Rac and Cdc42 versus RhoA. Using this assay, we investigated Rac and Cdc42 activation in neutrophils and differentiated HL-60 cells. The chemoattractant fMet-Leu-Phe and the phorbol ester phorbol myristate acetate stimulate formation of Rac-GTP and Cdc42-GTP with distinct time courses that parallel cell activation. We also show that the signaling pathways leading to Rac and Cdc42 activation in HL-60 cells involve G proteins sensitive to pertussis toxin, as well as tyrosine kinase and phosphatidylinositol 3-kinase activities.
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PMID:Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. 1022 76

Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) is a synthetic peptide that stimulates phosphoinositide (PI) hydrolysis in human leukocytes. The peptide binds to a unique cell surface receptor(s). Recently we had demonstrated that human neutrophils, monocytes, and B lymphocytes express this peptide-specific receptor and that stimulation of human leukocytes with the peptide leads to activation of the oxidative respiratory system and the bactericidal activity of neutrophils or monocytes. In this study we showed that the peptide induces chemotaxis of phagocytic leukocytes and studied the signaling pathway leading to chemotaxis in human monocytes. The peptide-induced monocyte chemotaxis is pertussis toxin (PTX)-sensitive. This fact correlates with the peptide's stimulation of PI hydrolysis and intracellular Ca2+ ([Ca2+]i) release, which is also PTX-sensitive. We demonstrate that the peptide-specific receptor is different from receptor(s) for monocyte chemoattractant protein-1 (MCP-1). We also show that intracellular signaling of WKYMVm leading to monocyte chemotaxis is different from that of MCP-1. The peptide-mediated monocyte chemotaxis is insensitive to protein kinase C (PKC) inhibitor (GF109203X) and butan-1-ol, ruling out PKC and phospholipase D participation in this process. On the other hand, a tyrosine kinase inhibitor (genistein) and RhoA inhibitor (C3 transferase) curtailed the peptide-induced chemotaxis in a concentration-dependent manner, implying the involvement of tyrosine kinase and RhoA, respectively. Treatment of human monocytes with the peptide stimulates tyrosine phosphorylation of several cellular proteins, including p125FAK and Pyk2 and translocation of RhoA from the cytosol to the membrane. We conclude that WKYMVm induces chemotaxis of human phagocytic leukocytes via unique receptors and signaling.
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PMID:Trp-Lys-Tyr-Met-Val-D-Met is a chemoattractant for human phagocytic cells. 1061 72


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