Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transmembrane signaling through G protein-coupled receptors (GPCRs) controls a diverse array of cellular processes including metabolism, growth, motility, adhesion, neuronal signaling and blood coagulation. The numerous GPCRs and their key roles in both normal physiology and disease have made them the target for more than 50% of all prescribed drugs. GPCR agonists and antagonists act on the extracellular side of the receptors, whereas the intracellular surface has not yet been exploited for development of new therapeutic agents. Here, we demonstrate the utility of novel cell-penetrating peptides, termed 'pepducins', that act as intracellular inhibitors of signal transference from receptors to G proteins. Attachment of a palmitate lipid to peptides based on the third intracellular loop of protease-activated receptor 1 (PAR1) or PAR4 (refs. 3-5) yielded potent inhibitors of thrombin-mediated aggregation of human platelets. Infusion of the anti-PAR4 pepducin into mice extended bleeding time and protected against systemic platelet activation, consistent with the phenotype of PAR4-deficient mice. We show that pepducins might be used to ascertain the physiological roles of GPCRs and rapidly determine the potential therapeutic value of blockade of a particular signaling pathway.
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PMID:Pepducin-based intervention of thrombin-receptor signaling and systemic platelet activation. 1464 10

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and by the G protein-coupled receptor agonist thrombin. The glycoprotein VI (GPVI)-specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.
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PMID:Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C. 1241 20

Transactivation of the epidermal growth factor receptor (EGFR) represents the paradigm for cross-talk between G protein-coupled receptors (GPCRs) and receptor tyrosine kinase signaling pathways. In a variety of squamous cell carcinoma cell lines of the head and neck (HNSCCs), we found that treatment with the GPCR agonists lysophosphatidic acid (LPA), bradykinin, thrombin, and carbachol results in rapid tyrosine phosphorylation of the EGFR. In these tumor cells, signal transactivation of the EGFR and the oncoprotein HER2/neu is critically dependent on metalloprotease activity. Using the metalloprotease inhibitor batimastat, the EGFR-specific tyrphostin AG1478, and a dominant-negative EGFR mutant, we show that in HNSCC cell lines, EGFR tyrosine phosphorylation, recruitment of the adaptor proteins SHC and Gab1, and activation of the ERK/mitogen-activated protein kinase pathway in response to LPA depend both on metalloprotease function and EGFR tyrosine kinase activity. Most importantly, critical characteristics of HNSCC cell lines such as DNA synthesis, cell cycle progression and tumor cell migration are stimulated by LPA and can be abrogated by interfering with EGFR signal transmission. Together, our results demonstrate the importance of a mechanism that promotes head and neck cancer cell proliferation and motility by GPCR ligands involving EGFR transactivation. Our findings suggest that highly abundant GPCR ligands such as LPA may function as tumor promoters and determinants of HNSCC progression.
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PMID:Lysophosphatidic acid-induced squamous cell carcinoma cell proliferation and motility involves epidermal growth factor receptor signal transactivation. 1241 65

Proteinase-activated receptor 1 (PAR-1) is a G protein-coupled receptor that is activated by thrombin and is implicated in the pathogenesis of inflammation. Although PAR-1 is expressed on immunocompetent cells within the brain such as astrocytes, little is known about its role in the pathogenesis of inflammatory brain diseases. Herein, we investigated PAR-1 regulation of brain inflammation by stimulating human astrocytic cells with thrombin or the selective PAR-1-activating peptide. Activated cells expressed significantly increased levels of IL-1 beta, inducible NO synthase, and PAR-1 mRNA. Moreover, supernatants of these same cells were neurotoxic, which was inhibited by an N-methyl-D-aspartate receptor antagonist. Striatal implantation of the PAR-1-activating peptide significantly induced brain inflammation and neurobehavioral deficits in mice compared with mice implanted with the control peptide or saline. Since HIV-related neurological disease is predicated on brain inflammation and neuronal injury, the expression of PAR-1 in HIV encephalitis (HIVE) was investigated. Immunohistochemical analysis revealed that PAR-1 and (pro)-thrombin protein expression was low in control brains, but intense immunoreactivity was observed on astrocytes in HIVE brains. Similarly, PAR-1 and thrombin mRNA levels were significantly increased in HIVE brains compared with control and multiple sclerosis brains. These data indicated that activation and up-regulation of PAR-1 probably contribute to brain inflammation and neuronal damage during HIV-1 infection, thus providing new therapeutic targets for the treatment of HIV-related neurodegeneration.
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PMID:Up-regulation of proteinase-activated receptor 1 expression in astrocytes during HIV encephalitis. 1259 92

In cardiac myocytes, sarcolemmal Na+/H+ exchanger (NHE) activity is subject to regulation by a variety of G protein-coupled receptor (GPCR) systems. This regulation usually manifests as an increase in NHE activity (e.g. in response to the stimulation of alpha1-adrenergic, angiotensin AT1, endothelin and thrombin receptors), although some GPCR systems have been shown to inhibit sarcolemmal NHE activity (e.g. beta1-adrenergic receptors) or to attenuate its stimulation by other ligands (e.g. adenosine A1 and angiotensin AT2 receptors). The pertinent molecular signalling mechanisms are only now beginning to be unravelled, with the extracellular signal regulated kinase/ribosomal S6 kinase pathway and the protein kinase C pathway both appearing to play critical roles in the stimulation of sarcolemmal NHE activity. GPCR-mediated regulation of sarcolemmal NHE activity is likely to play significant roles in modulating myocardial function in both physiological and pathophysiological conditions. These roles include the regulation of (1) myocardial pH(i) and contractility, (2) myocardial susceptibility to injury and dysfunction during ischaemia and reperfusion, and (3) myocardial hypertrophy in response to neurohormonal and mechanical stimuli. Greater understanding of the pertinent molecular signalling mechanisms distal to GPCR stimulation may reveal novel targets for therapeutic manipulation.
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PMID:Regulatory effects of G protein-coupled receptors on cardiac sarcolemmal Na+/H+ exchanger activity: signalling and significance. 1265 Aug 72

Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap junctions. Pervanadate, an inhibitor of protein tyrosine phosphatases, mimics activated Src in inhibiting Cx43 gap junctional communication, apparently by promoting tyrosine phosphorylation of the Cx43 C-terminal tail. However, the identity of the protein tyrosine phosphatase(s) that may normally prevent Src-induced gap junction closure is unknown. Receptor-like protein tyrosine phosphatases that mediate homotypic cell-cell interaction are attractive candidates. Here we show that receptor protein tyrosine phosphatase mu (RPTPmu) interacts with Cx43 in diverse cell systems. We find that the first catalytic domain of RPTPmu binds to Cx43. Our results support a model in which RPTPmu, or a closely related protein tyrosine phosphatase, interacts with the regulatory C-terminal tail of Cx43 to prevent Src-mediated closure of Cx43 gap junctional channels.
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PMID:Association of connexin43 with a receptor protein tyrosine phosphatase. 1468 Oct 16

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is the prototypic member of a family of protease-activated receptors. PAR1 is irreversibly proteolytically activated; thus, the magnitude and duration of thrombin cellular responses are determined primarily by mechanisms responsible for termination of receptor signaling. Both phosphorylation and beta-arrestins contribute to rapid desensitization of PAR1 signaling. However, the relative contribution of each of these pathways to the termination of PAR1 signaling is not known. Co-expression of PAR1 with beta-arrestin 1 (betaarr1) in COS-7 cells resulted in a marked inhibition of PAR1 signaling, whereas beta-arrestin 2 (betaarr2) was essentially inactive. Strikingly, signaling by a PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation was also attenuated more effectively by betaarr1 compared with betaarr2. In contrast, both beta-arrestin isoforms were equally effective at desensitizing the substance P receptor, a classic reversibly activated GPCR. PAR1 coimmunoprecipitated betaarr1 in an agonist-dependent manner, whereas betaarr2 association was virtually undetectable. Remarkably, betaarr1 also interacted with phosphorylation defective PAR1 mutant, whereas betaarr2 did not. Moreover, constitutively active beta-arrestin mutants, betaarr1 R169E and betaarr2 R170E, that bind to activated receptor independent of phosphorylation failed to enhance either wild type or mutant PAR1 desensitization compared with normal versions of these proteins. In contrast, beta-arrestin mutants displayed enhanced activity at desensitizing the serotonin 5-hydroxytryptamine(2A) receptor. Taken together, these results suggest that, in addition to PAR1 cytoplasmic tail phosphorylation itself, beta-arrestin binding independent of phosphorylation promotes desensitization of PAR1 signaling. These findings reveal a new level of complexity in the regulation of protease-activated GPCR signaling.
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PMID:Termination of protease-activated receptor-1 signaling by beta-arrestins is independent of receptor phosphorylation. 1469 2

Increased expression of protease-activated receptor 1 (PAR1), a G protein-coupled receptor for thrombin, has previously been correlated with breast carcinoma cell invasion. PAR1 is irreversibly proteolytically activated, internalized, and sorted directly to lysosomes, a critical process for the termination of signaling. We determined that activated PAR1 trafficking is severely altered in metastatic breast carcinoma cells but not in nonmetastatic or normal breast epithelial cells. Consequently, the proteolytically activated receptor is not sorted to lysosomes and degraded. Altered trafficking of proteolytically activated PAR1 caused sustained activation of phosphoinositide hydrolysis and extracellular signal-regulated kinase signaling, even after thrombin withdrawal, and enhanced cellular invasion. Thus, our results reveal that a novel alteration in trafficking of activated PAR1 causes persistent signaling and, in addition to other processes and proteins, contributes to breast carcinoma cell invasion.
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PMID:Persistent signaling by dysregulated thrombin receptor trafficking promotes breast carcinoma cell invasion. 1496 79

G protein-coupled receptors can trigger metalloproteinase-dependent shedding of proteins from the cell surface. We now report that G protein-coupled receptors can themselves undergo regulated metalloproteinase-dependent shedding. The N-terminal exodomain of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, displayed regulated shedding in endothelial cells, which normally express this receptor. Cleavage occurred at a site predicted to render the receptor unresponsive to thrombin. A chimeric protein in which the N-terminal exodomain of PAR1 was fused to an unrelated transmembrane segment was shed as efficiently as PAR1, shedding of both proteins was stimulated by phorbol ester and by a PAR1 agonist. TNFalpha protease inhibitor-2 (TAPI-2), phenanthroline, and tissue inhibitor of metalloproteinase-3 (TIMP-3) but not TIMP-1 or -2 inhibited such shedding. These and other data suggest that the information that specifies PAR1 shedding resides within its N-terminal exodomain rather than its heptahelical segment, that activation of protein kinase C or of PAR1 itself can stimulate PAR1 shedding in trans, and that ADAM17/TACE or a metalloproteinase with similar properties mediates PAR1 shedding. Regulated shedding reduced the amount of cell surface PAR1 available for productive cleavage by thrombin by half or more, but thus far we have been unable to demonstrate an effect of PAR1 shedding on cellular responsiveness to thrombin. Nonetheless, regulated shedding of G protein-coupled receptors represents a new mechanism by which signaling by this important class of receptors might be modulated.
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PMID:Regulated shedding of PAR1 N-terminal exodomain from endothelial cells. 1498 36

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly proteolytically activated, internalized, and then sorted to lysosomes and degraded. Internalization and lysosomal sorting of activated PAR1 is critical for termination of receptor signaling. We previously demonstrated that activated PAR1 is rapidly phosphorylated and internalized via a clathrin- and dynamin-dependent pathway that is independent of arrestins. Toward understanding the mechanisms responsible for activated PAR1 internalization through clathrin-coated pits we examined the function of a highly conserved tyrosine-based motif, YXXL, localized in the cytoplasmic carboxyl tail of the receptor. A mutant PAR1 in which tyrosine 383 and leucine 386 were replaced with alanines (Y383A/L386A) was significantly impaired in agonist-triggered internalization and degradation compared with wild-type receptor. In contrast, constitutive internalization, and recycling of unactivated PAR1 Y383A/L386A mutant was not affected, suggesting that tonic cycling of the mutant receptor remained intact. Strikingly, a PAR1 C387Z truncation mutant in which the YXXL motif was exposed at the C terminus constitutively internalized and degraded in an agonist-independent manner, whereas C387Z truncation mutant in which the critical tyrosine and leucine were mutated to alanine (C387Z-Y383A/L386A) failed to internalize. Inhibition of PAR1 C387Z mutant constitutive internalization with dominant-negative K44A dynamin blocked agonist-independent degradation of the mutant receptor. Together these findings strongly suggest that internalization of activated PAR1 is controlled by multiple regulatory mechanisms involving phosphorylation and a highly conserved tyrosine-based motif, YXXL. This study is the first to describe a function for a tyrosine-based motif, YXX, in GPCR internalization and reveal novel complexities in the regulation of GPCR trafficking.
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PMID:A tyrosine-based sorting signal regulates intracellular trafficking of protease-activated receptor-1: multiple regulatory mechanisms for agonist-induced G protein-coupled receptor internalization. 1502 90


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