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Clostridium difficile toxin B differentially affects GPCR-stimulated Ca2+ responses in macrophages: independent roles for Rho and PLA2.
Rebres RA, Moon C, Decamp D, Lin KM, Fraser ID, Milne SB, Roach TI, Brown HA, Seaman WE
(2010) J Leukoc Biol 87: 1041-57
MeSH Terms: Animals, Bacterial Proteins, Bacterial Toxins, Blotting, Western, Calcium, Cells, Cultured, Complement C5a, Cytoskeleton, Female, Macrophages, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Phosphatidylinositol 3-Kinases, Phosphatidylinositols, Phospholipase C beta, Phospholipases A2, Protein Isoforms, Signal Transduction, Uridine Diphosphate, rho GTP-Binding Proteins
Show Abstract · Added March 21, 2013
Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca(2+) responses to Galphai-linked receptors, including the C5aR, but reduced responses to Galphaq-linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca(2+) responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLCbeta isoform-deficient BMDM, we found that ToxB inhibited Ca(2+) signaling through PLCbeta4 but enhanced signaling through PLCbeta3. Effects of ToxB on GPCR Ca(2+) responses correlated with GPCR use of PLCbeta3 versus PLCbeta4. ToxB inhibited UDP Ca(2+) signaling without reducing InsP3 production or the sensitivity of cellular Ca(2+) stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca(2+)coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca(2+) signaling by C5a was prevented by inhibition of PLA(2) or 5-LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca(2+) signaling by different GPCR-linked PLCbeta isoforms in macrophages.
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22 MeSH Terms
RACK1 regulates directional cell migration by acting on G betagamma at the interface with its effectors PLC beta and PI3K gamma.
Chen S, Lin F, Shin ME, Wang F, Shen L, Hamm HE
(2008) Mol Biol Cell 19: 3909-22
MeSH Terms: Cell Movement, Chemotaxis, Class Ib Phosphatidylinositol 3-Kinase, Enzyme Inhibitors, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, GTP-Binding Proteins, Gene Expression Regulation, HL-60 Cells, Humans, Isoenzymes, Jurkat Cells, Models, Molecular, Models, Theoretical, Neoplasm Proteins, Neutrophils, Phosphatidylinositol 3-Kinases, Phospholipase C beta, RNA, Small Interfering, Receptors for Activated C Kinase, Receptors, Cell Surface
Show Abstract · Added December 10, 2013
Migration of cells up the chemoattractant gradients is mediated by the binding of chemoattractants to G protein-coupled receptors and activation of a network of coordinated excitatory and inhibitory signals. Although the excitatory process has been well studied, the molecular nature of the inhibitory signals remains largely elusive. Here we report that the receptor for activated C kinase 1 (RACK1), a novel binding protein of heterotrimeric G protein betagamma (G betagamma) subunits, acts as a negative regulator of directed cell migration. After chemoattractant-induced polarization of Jurkat and neutrophil-like differentiated HL60 (dHL60) cells, RACK1 interacts with G betagamma and is recruited to the leading edge. Down-regulation of RACK1 dramatically enhances chemotaxis of cells, whereas overexpression of RACK1 or a fragment of RACK1 that retains G betagamma-binding capacity inhibits cell migration. Further studies reveal that RACK1 does not modulate cell migration through binding to other known interacting proteins such as PKC beta and Src. Rather, RACK1 selectively inhibits G betagamma-stimulated phosphatidylinositol 3-kinase gamma (PI3K gamma) and phospholipase C (PLC) beta activity, due to the competitive binding of RACK1, PI3K gamma, and PLC beta to G betagamma. Taken together, these findings provide a novel mechanism of regulating cell migration, i.e., RACK1-mediated interference with G betagamma-dependent activation of key effectors critical for chemotaxis.
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21 MeSH Terms
Acute tissue-type plasminogen activator release in human microvascular endothelial cells: the roles of Galphaq, PLC-beta, IP3 and 5,6-epoxyeicosatrienoic acid.
Muldowney JA, Painter CA, Sanders-Bush E, Brown NJ, Vaughan DE
(2007) Thromb Haemost 97: 263-71
MeSH Terms: 8,11,14-Eicosatrienoic Acid, Aorta, Biological Factors, Cell Proliferation, Cells, Cultured, Dose-Response Relationship, Drug, Endothelial Cells, Epoprostenol, GTP-Binding Protein alpha Subunits, Gq-G11, Humans, Inositol 1,4,5-Trisphosphate, Isoenzymes, Microcirculation, Nitric Oxide, Phospholipase C beta, Potassium, Signal Transduction, Thrombin, Time Factors, Tissue Plasminogen Activator, Type C Phospholipases, Umbilical Veins
Show Abstract · Added December 10, 2013
The acute physiologic release of tissue-type plasminogen activator (t-PA) from the endothelium is critical for vascular homeostasis. This process is prostacyclin- and nitric oxide (NO)-independent in humans. It has been suggested that calcium signaling and endothelial-derived hyperpolarizing factors (EDHF) may play a role in t-PA release. G-protein-coupled receptor-dependent calcium signaling is typically Galphaq-dependent. EDHFs have been functionally defined and in various tissues are believed to be various regioisomers of the epoxyeicosatrienoic acids (EETs). We tested the hypothesis in vitro that thrombin-stimulated t-PA release from human microvascular endothelial cells (HMECs) is both Galphaq- and EDHF-dependent. Conditioned media was harvested following thrombin stimulation, and t-PA antigen was measured by ELISA. Thrombin-induced t-PA release was limited by a membrane-permeable Galphaq inhibitory peptide, the PLC-beta antagonist U73122, and the IP3 receptor antagonist 2-aminoethoxyphenylborane, while the Galphaq agonist Pasteurella toxin modestly induced t-PA release. The cytochrome P450 (CYP450) inhibitor, miconazole, and the arachidonic acid epoxygenase inhibitor MS-PPOH inhibited thrombin-stimulated t-PA release, while 5,6-EET-methyl ester stimulated t-PA release. The 5,6- and 14,15-EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid, inhibited t-PA release at the 100 microM concentration. However, thrombin-stimulated t-PA release was unaffected by the prostacyclin and NO inhibitors ASA and L-NAME, as well as the potassium channel inhibitors TEA, apamin and charybdotoxin. These studies suggest that thrombin-stimulated t-PA release is Galphaq-, PLC-beta-, IP3-, and 5,6-EET-dependent while being prostacyclin-, NO- and K+ channel-independent in HMECs.
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22 MeSH Terms
RACK1 binds to a signal transfer region of G betagamma and inhibits phospholipase C beta2 activation.
Chen S, Lin F, Hamm HE
(2005) J Biol Chem 280: 33445-52
MeSH Terms: Amino Acid Sequence, Animals, Binding Sites, Binding, Competitive, Carrier Proteins, Conserved Sequence, Enzyme Activation, Escherichia coli, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Glutathione Transferase, Isoenzymes, Maltose-Binding Proteins, Mice, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Neuropeptides, Phospholipase C beta, Protein Binding, Protein Isoforms, Receptors for Activated C Kinase, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, Spectrometry, Fluorescence, Spodoptera, Type C Phospholipases
Show Abstract · Added January 20, 2015
Receptor for Activated C Kinase 1 (RACK1), a novel G betagamma-interacting protein, selectively inhibits the activation of a subclass of G betagamma effectors such as phospholipase C beta2 (PLCbeta2) and adenylyl cyclase II by direct binding to G betagamma (Chen, S., Dell, E. J., Lin, F., Sai, J., and Hamm, H. E. (2004) J. Biol. Chem. 279, 17861-17868). Here we have mapped the RACK1 binding sites on G betagamma. We found that RACK1 interacts with several different G betagamma isoforms, including G beta1gamma1, Gbeta1gamma2, and Gbeta5gamma2, with similar affinities, suggesting that the conserved residues between G beta1 and G beta5 may be involved in their binding to RACK1. We have confirmed this hypothesis and shown that several synthetic peptides corresponding to the conserved residues can inhibit the RACK1/G betagamma interaction as monitored by fluorescence spectroscopy. Interestingly, these peptides are located at one side of G beta1 and have little overlap with the G alpha subunit binding interface. Additional experiments indicate that the G betagamma contact residues for RACK1, in particular the positively charged amino acids within residues 44-54 of G beta1, are also involved in the interaction with PLCbeta2 and play a critical role in G betagamma-mediated PLCbeta2 activation. These data thus demonstrate that RACK1 can regulate the activity of a G betagamma effector by competing for its binding to the signal transfer region of G betagamma.
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28 MeSH Terms
RACK1 regulates specific functions of Gbetagamma.
Chen S, Dell EJ, Lin F, Sai J, Hamm HE
(2004) J Biol Chem 279: 17861-8
MeSH Terms: Adenylyl Cyclases, Animals, COS Cells, Cell Line, Cell Membrane, Chemotaxis, Cyclic AMP, Cytosol, Dimerization, Dose-Response Relationship, Drug, Enzyme Activation, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, GTP-Binding Proteins, Gene Expression Regulation, Glutathione Transferase, Humans, Hydrolysis, Interleukin-8, Isoenzymes, Lipid Metabolism, MAP Kinase Signaling System, Microscopy, Confocal, Microscopy, Fluorescence, Neoplasm Proteins, Phospholipase C beta, Protein Binding, Protein Transport, Receptors for Activated C Kinase, Receptors, Cell Surface, Signal Transduction, Transfection, Type C Phospholipases
Show Abstract · Added January 20, 2015
We showed previously that Gbetagamma interacts with Receptor for Activated C Kinase 1 (RACK1), a protein that not only binds activated protein kinase C (PKC) but also serves as an adaptor/scaffold for many signaling pathways. Here we report that RACK1 does not interact with Galpha subunits or heterotrimeric G proteins but binds free Gbetagamma subunits released from activated heterotrimeric G proteins following the activation of their cognate receptors in vivo. The association with Gbetagamma promotes the translocation of RACK1 from the cytosol to the membrane. Moreover, binding of RACK1 to Gbetagamma results in inhibition of Gbetagamma-mediated activation of phospholipase C beta2 and adenylyl cyclase II. However, RACK1 has no effect on other functions of Gbetagamma, such as activation of the mitogen-activated protein kinase signaling pathway or chemotaxis of HEK293 cells via the chemokine receptor CXCR2. Similarly, RACK1 does not affect signal transduction through the Galpha subunits of G(i), G(s), or G(q). Collectively, these findings suggest a role of RACK1 in regulating specific functions of Gbetagamma.
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33 MeSH Terms
Differential regulation of metabotropic glutamate receptor 5-mediated phosphoinositide hydrolysis and extracellular signal-regulated kinase responses by protein kinase C in cultured astrocytes.
Peavy RD, Sorensen SD, Conn PJ
(2002) J Neurochem 83: 110-8
MeSH Terms: Animals, Astrocytes, Calcium Signaling, Cells, Cultured, Enzyme Activation, Enzyme Activators, Enzyme Inhibitors, Excitatory Amino Acid Agonists, Glycine, Hydrolysis, Isoenzymes, Mitogen-Activated Protein Kinase 1, Phosphatidylinositols, Phospholipase C beta, Phosphorylation, Protein Kinase C, Rats, Rats, Sprague-Dawley, Receptor, Metabotropic Glutamate 5, Receptors, Metabotropic Glutamate, Resorcinols, Signal Transduction, Type C Phospholipases
Show Abstract · Added February 19, 2015
The metabotropic glutamate receptor 5 (mGluR5) exhibits a rapid loss of receptor responsiveness to prolonged or repeated agonist exposure. This receptor desensitization has been seen in a variety of native and recombinant systems, and is thought to result from receptor-mediated, protein kinase C (PKC)-dependent phosphorylation of the receptor, uncoupling it from the G protein in a negative feedback regulation. We have investigated the rapid PKC-mediated desensitization of mGluR5 in cortical cultured astrocytes by measuring downstream signals from activation of mGluR5. These include activation of phosphoinositide (PI) hydrolysis, intracellular calcium transients, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation. We present evidence that PKC plays an important role in rapid desensitization of PI hydrolysis and calcium signaling, but not in ERK2 phosphorylation. This differential regulation of mGluR5-mediated responses suggests divergent signaling and regulatory pathways which may be important mechanisms for dynamic integration of signal cascades.
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23 MeSH Terms
Defining G protein beta gamma specificity for effector recognition.
Dell EJ, Blackmer T, Skiba NP, Daaka Y, Shekter LR, Rosal R, Reuveny E, Hamm HE
(2002) Methods Enzymol 344: 421-34
MeSH Terms: Adenylyl Cyclases, Animals, Cell Line, Cell Membrane, Heterotrimeric GTP-Binding Proteins, Humans, Isoenzymes, Models, Molecular, Phospholipase C beta, Potassium Channels, Protein Conformation, Protein Isoforms, Protein Subunits, Recombinant Proteins, Spodoptera, Transfection, Type C Phospholipases
Added December 10, 2013
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17 MeSH Terms
Metabotropic glutamate receptor 5-induced phosphorylation of extracellular signal-regulated kinase in astrocytes depends on transactivation of the epidermal growth factor receptor.
Peavy RD, Chang MS, Sanders-Bush E, Conn PJ
(2001) J Neurosci 21: 9619-28
MeSH Terms: Animals, Astrocytes, Cells, Cultured, Enzyme Inhibitors, ErbB Receptors, Excitatory Amino Acid Agonists, Excitatory Amino Acid Antagonists, GTP-Binding Protein alpha Subunits, Gq-G11, GTP-Binding Proteins, Heterotrimeric GTP-Binding Proteins, Isoenzymes, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases, Peptides, Phosphatidylinositols, Phospholipase C beta, Phosphorylation, Protein Binding, Protein Subunits, Protein Tyrosine Phosphatases, Quinazolines, Rats, Rats, Sprague-Dawley, Receptor, Metabotropic Glutamate 5, Receptors, Metabotropic Glutamate, Signal Transduction, Transcriptional Activation, Type C Phospholipases, Tyrphostins, src-Family Kinases
Show Abstract · Added February 19, 2015
G-protein-coupled receptors (GPCRs) induce the phosphorylation of mitogen-activated protein (MAP) kinase by actions on any of a number of signal transduction systems. Previous studies have revealed that activation of the G(q)-coupled metabotropic glutamate receptor 5 (mGluR5) induces phosphorylation of the MAP kinase extracellular signal-regulated kinase 2 (ERK2) in cultured rat cortical astrocytes. We performed a series of studies to determine the mechanisms underlying mGluR5-induced phosphorylation of MAP kinase in these cells. Interestingly, our studies suggest that mGluR5-mediated ERK2 phosphorylation is dependent on the activation of G(alphaq) but is not mediated by the activation of phospholipase Cbeta1, activation of protein kinase C, or increases in intracellular calcium. Studies with peptide inhibitors suggest that this response is not dependent on G(betagamma) subunits. However, the activation of ERK2 was dependent on activation of the epidermal growth factor (EGF) receptor and activation of a Src family tyrosine kinase. Furthermore, activation of mGluR5 induced an association of this receptor and the EGF receptor, suggesting the formation of a signaling complex involved in the activation of ERK2. These data suggest that mGluR5 increases ERK2 phosphorylation in astrocytes by a novel mechanism involving the activation of G(alphaq) and both receptor and nonreceptor tyrosine kinases but that is independent of the activation of phospholipase Cbeta1.
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31 MeSH Terms
Molecular basis for interactions of G protein betagamma subunits with effectors.
Ford CE, Skiba NP, Bae H, Daaka Y, Reuveny E, Shekter LR, Rosal R, Weng G, Yang CS, Iyengar R, Miller RJ, Jan LY, Lefkowitz RJ, Hamm HE
(1998) Science 280: 1271-4
MeSH Terms: Adenosine Diphosphate Ribose, Adenylyl Cyclases, Binding Sites, Calcium Channels, Cell Line, Cyclic AMP-Dependent Protein Kinases, G Protein-Coupled Inwardly-Rectifying Potassium Channels, GTP-Binding Proteins, Guanosine Diphosphate, Heterotrimeric GTP-Binding Proteins, Humans, Isoenzymes, Models, Molecular, Mutation, Phospholipase C beta, Potassium Channels, Potassium Channels, Inwardly Rectifying, Protein Conformation, Rhodopsin, Signal Transduction, Transducin, Type C Phospholipases, beta-Adrenergic Receptor Kinases
Show Abstract · Added December 10, 2013
Both the alpha and betagamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. Gbetagamma subunits can regulate a diverse array of effectors, including ion channels and enzymes. Galpha subunits bound to guanine diphosphate (Galpha-GDP) inhibit signal transduction through Gbetagamma subunits, suggesting a common interface on Gbetagamma subunits for Galpha binding and effector interaction. The molecular basis for interaction of Gbetagamma with effectors was characterized by mutational analysis of Gbeta residues that make contact with Galpha-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-beta2, and beta-adrenergic receptor kinase revealed the Gbeta residues required for activation of each effector and provides evidence for partially overlapping domains on Gbeta for regulation of these effectors. This organization of interaction regions on Gbeta for different effectors and Galpha explains why subunit dissociation is crucial for signal transmission through Gbetagamma subunits.
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23 MeSH Terms
RGS4 inhibits signaling by group I metabotropic glutamate receptors.
Saugstad JA, Marino MJ, Folk JA, Hepler JR, Conn PJ
(1998) J Neurosci 18: 905-13
MeSH Terms: Age Factors, Animals, Calcium, Calcium Channels, Calcium-Calmodulin-Dependent Protein Kinases, Cell Membrane, Chloride Channels, G Protein-Coupled Inwardly-Rectifying Potassium Channels, GTP-Binding Proteins, Glioma, Hippocampus, Hybrid Cells, Inositol 1,4,5-Trisphosphate, Isoenzymes, Mice, Neurons, Oocytes, Patch-Clamp Techniques, Phospholipase C beta, Potassium Channels, Potassium Channels, Inwardly Rectifying, Proteins, RGS Proteins, RNA, Messenger, Rats, Receptors, Metabotropic Glutamate, Receptors, Muscarinic, Signal Transduction, Synapses, Tritium, Type C Phospholipases, Xenopus
Show Abstract · Added February 19, 2015
Metabotropic glutamate receptors (mGluRs) couple to heterotrimeric G-proteins and regulate cell excitability and synaptic transmission in the CNS. Considerable effort has been focused on understanding the cellular and biochemical mechanisms that underlie regulation of signaling by G-proteins and their linked receptors, including the mGluRs. Recent findings demonstrate that regulators of G-protein signaling (RGS) proteins act as effector antagonists and GTPase-activating proteins for Galpha subunits to inhibit cellular responses by G-protein-coupled receptors. RGS4 blocks Gq activation of phospholipase Cbeta and is expressed broadly in rat brain. The group I mGluRs (mGluRs 1 and 5) couple to Gq pathways to regulate several effectors in the CNS. We examined the capacity of RGS4 to regulate group I mGluR responses. In Xenopus oocytes, purified RGS4 virtually abolishes the mGluR1a- and mGluR5a-mediated but not the inositol trisphospate-mediated activation of a calcium-dependent chloride current. Additionally, RGS4 markedly attenuates the mGluR5-mediated inhibition of potassium currents in hippocampal CA1 neurons. This inhibition is dose-dependent and occurs at concentrations that are virtually identical to those required for inhibition of phospholipase C activity in NG108-15 membranes and reconstituted systems using purified proteins. These findings demonstrate that RGS4 can modulate mGluR responses in neurons, and they highlight a previously unknown mechanism for regulation of G-protein-coupled receptor signaling in the CNS.
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32 MeSH Terms