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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mitogenic stimulation of Swiss 3T3 fibroblasts with bombesin results in receptor-mediated activation of a complex array of effectors, including phospholipase C beta and mitogen-activated protein (MAP) kinase. Incubation of Swiss 3T3 fibroblasts with the 11-amino acid [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide inhibited bombesin-stimulated cell proliferation and phospholipase C beta activation even at high bombesin concentrations. The peptide did not inhibit the activation of phospholipase C beta by a GTPase-deficient form of the Gq-like protein, G16, indicating that the peptide does not inhibit phospholipase C beta and is acting at a point upstream of the activated form of the G protein alpha subunit. The peptide inhibited MAP kinase activation at low bombesin concentrations, but unlike phospholipase C beta, this inhibition could be overcome with 30 nM bombesin. In control Swiss 3T3 cells, bombesin did not measurably activate Ras or Raf-1 above basal levels. Following incubation of the cells with the [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide, 50 nM bombesin activated Raf-1 4-6-fold over basal levels. Platelet-derived growth factor-stimulated activities of PLC, Ras, Raf-1, and MAP kinase were unaltered after incubation of Swiss 3T3 cells with the [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide, as was platelet-derived growth factor-stimulated growth of the Swiss 3T3 cells. Thus, the peptide behaves as an antagonist that differentially inhibited phospholipase C beta and MAP kinase signal transduction pathways. The growth arrest observed with the peptide indicates that the bombesin-stimulated activation of MAP kinase is not sufficient to support mitogenesis in Swiss 3T3 cells.
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PMID:Differential modulation of bombesin-stimulated phospholipase C beta and mitogen-activated protein kinase activity by [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P. 753 38

The anaphylatoxin C5a receptor activates the Ras/Raf/mitogen-activated protein (MAP) kinase pathway in human neutrophils. The signal pathways involved in Ras/Raf/MAP kinase activation in response to C5a and other chemoattractant receptors is poorly understood. Stimulation of the C5a receptor expressed in HEK293 cells results in modest MAP kinase activation, which is inhibited by pertussis toxin-catalyzed ADP-ribosylation of G(i). Coexpression of the C5a receptor and the G16 alpha subunit (alpha 16) results in the G16-mediated activation of phospholipase C beta and a robust MAP kinase activation. Pertussis toxin treatment of C5a receptor/alpha 16-cotransfected cells inhibits C5a stimulation of MAP kinase activity approximately 60% relative to the control response. Similarly, the protein kinase C inhibitor, GF109203X inhibits activation of MAP kinase activation in C5a receptor/alpha 16-cotransfected cells by 60%; the protein kinase C inhibitor does not affect the modest C5a receptor response in the absence of alpha 16 expression. These results demonstrate that two independent signals are required for the maximal activation of MAP kinase by G protein-coupled receptors.
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PMID:Mitogen-activated protein kinase activation requires two signal inputs from the human anaphylatoxin C5a receptor. 764 93

The C5a chemoattractant factor receptor, when expressed in COS-7 cells, can stimulate phosphoinositide-specific phospholipase C activity through the activation of the G16 isoform of the heterotrimeric G protein, but not through the G11 isoform. To identify the regions of the G alpha 16 subunit protein that are responsible for its activation by the C5a receptor, a series of chimeras between G alpha 16 and G alpha 11 were constructed and tested for their ability to be activated by the C5a receptor. Co-transfection experiments with chimeras in which the carboxyl-terminal regions of G alpha 11 were replaced with the corresponding regions of G alpha 16 demonstrated that changes in the carboxyl terminus, e.g., replacement of 134 amino acids, were not sufficient to confer receptor specificity. An additional segment encompassing residues 220-240 of G alpha 16 was required to confer C5a-induced activation. Testing of a reciprocal series of chimeras composed of G alpha 16 sequences at the amino terminus and G alpha 11 sequences at the carboxyl terminus revealed that certain sequences extending from the amino terminus to amino acid 209 of G alpha 16 were sufficient to endow the chimera with much of the specificity for C5a-induced activation. These results suggest that receptor specificity may involve specific conformations of the G protein stabilized by concerted interactions of multiple amino acid sequences distributed throughout the G alpha protein.
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PMID:Multiple regions of G alpha 16 contribute to the specificity of activation by the C5a receptor. 787 28

The key event in receptor-catalyzed activation of heterotrimer G proteins is binding of GTP, which leads to subunit dissociation generating GTP-bound alpha subunits and free beta gamma complexes. We have previously identified a mutation that abolished GTP binding in G alpha o (S47C) and demonstrated that the mutant retained the ability to bind beta gamma and could act in a dominant negative fashion when expressed in Xenopus oocytes (Slepak, V.Z., Quick, M.W., Aragay, A.M., Davidson, N., Lester, H.A., and Simon, M.I. (1993) J. Biol. Chem. 268, 21889-21894). In the current work, we investigated the effects of the homologous mutant of G alpha i2 (S48C) upon signaling pathways reconstituted in transiently transfected COS-7 cells. We found that expression of the G alpha i2 S48C mutant prevented stimulation of phospholipase C (PLC) beta 2 by free beta gamma subunit complexes. This effect of G alpha i S48C was not readily reversible in contrast to the inhibitory effect of wild-type G alpha i2, which could be reversed upon activation of the cotransfected muscarinic M2 receptor, presumably by release of beta gamma from the G protein heterotrimer. Coexpression of G alpha i S48C or the wild-type G alpha i2 also dramatically decreased G16-mediated stimulation of PLC by C5a in the cells transfected with cDNAs encoding C5a receptor and G alpha 16. Activation of PLC via endogenous Gq or G11 in the presence of alpha 1C adrenergic receptors was similarly attenuated by coexpression of G alpha i or G alpha i S48C. Pertussis toxin treatment of the transfected cells enhanced the inhibition of the receptor-stimulated PLC by wild-type G alpha i subunits but did not influence the effects of the dominant negative mutant. The enhancement of the wild-type G alpha i inhibitory effect by pertussis toxin can be explained by stabilization of G alpha i binding to beta gamma as a result of ADP-ribosylation, while G alpha i S48C mutant binds beta gamma irreversibly even without pertussis toxin treatment. Therefore, a feasible mechanism to rationalize the attenuation of the G alpha 16 and Gq/11-mediated activation of PLC by cotransfected G alpha i is the competition between G alpha i and G alpha 16 or Gq/11 for the beta gamma complexes, which are necessary for the G protein coupling with receptors. These experiments provide new evidence for the role of beta gamma in the integration of signals controlling phosphoinositide release through different G alpha families.
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PMID:Functional analysis of a dominant negative mutant of G alpha i2. 787 52

Expression of the GTPase-deficient G alpha 16 polypeptide G alpha 16Q212L, a member of the Gq family of heterotrimeric G proteins, constitutively activated phospholipase C beta activity in Swiss 3T3 cells. Expression of G alpha 16Q212L appears to persistently stimulte a low level of protein kinase C activity which also increases protein kinase A activity in Swiss 3T3 cells. Growth of G alpha 16Q212L expressing cells was significantly inhibited relative to wild-type Swiss 3T3 cells. Bombesin-stimulated DNA synthesis was completely inhibited in G alpha 16Q212L expressing clones, whereas the growth responses to platelet-derived growth factor (PDGF) and serum were inhibited 50-80% relative to wild-type cells. In addition to the inhibition of cell growth, G alpha 16Q212L expression significantly inhibited the stimulation of protein kinase C, Raf-1, MEK, mitogen-activated protein kinase, phospholipase A2 activity, and Ca2+ mobilization in response to PDGF. In contrast, PDGF receptor activation of phospholipase C gamma, phosphatidylinositol 3-kinase, and Ras GTP loading was similar in wild-type and G alpha 16Q212L expressing clones. PDGF regulation of membrane ruffling and actin fiber assembly, responses mediated in part by phosphatidylinositol 3-kinase, were unaffected in G alpha 16Q212L expressing clones. The growth inhibitory action of G alpha 16Q212L expression in Swiss 3T3 cells is downstream of the initial SH2 domain-encoded signal transduction proteins regulated in response to PDGF receptor autophosphorylation. The findings demonstrate that constitutively activated G alpha 16Q212L persistently activates phospholipase C activity and effectively inhibits a subset of cytoplasmic signal transduction pathways involved in growth factor tyrosine kinase receptor stimulation of cell growth. G16/Gq-regulated signal transduction can acutely stimulate specific response pathways involved in mitogenesis; but persistent activation of G16/Gq-regulated effectors, including phospholipase C beta, inhibit tyrosine kinase-initiated mitogenesis. One role for G16/Gq response systems may be to modulate growth factor receptor signaling.
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PMID:Expression of GTPase-deficient G alpha 16 inhibits Swiss 3T3 cell growth. 802 Dec 43

A cDNA encoding G16 alpha, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16 alpha in membrane extracts of Sf9 cells activated phospholipase C-beta 1 (PLC-beta 1) in the presence of guanosine 5'-[gamma-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16 alpha in the cytosolic fraction of Sf9 cells did not stimulate PLC-beta 1. Concurrent expression of the G-protein beta gamma subunit complex increased the amount of G16 alpha in Sf9 cell membranes. The guanosine 5'-[gamma-thio]triphosphate-activated form of G16 alpha was purified from cholate extracts of membranes from cells expressing G16 alpha, and the G-protein beta 2 and gamma 2 subunits. G16 alpha activated PLC-beta 1, PLC-beta 2, and PLC-beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated activation of PLC-beta 1 and PLC-beta 3 greatly exceeded that of PLC-beta 2. G16 alpha did not activate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to activate the known isoforms of PLC-beta.
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PMID:Purification and characterization of recombinant G16 alpha from Sf9 cells: activation of purified phospholipase C isozymes by G-protein alpha subunits. 841 74

1. HL-60 human leukemia cells are a widely employed model system for the analysis of signal transduction processes mediated via regulatory heterotrimeric guanine nucleotide-binding proteins (G-proteins). HL-60 promyelocytes are pluripotent and can be differentiated into neutrophilic or monocytic cells. 2. HL-60 cells express formyl peptide-, complement C5a-, leukotriene B4 (LTB4)- and platelet-activating factor receptors, receptors for purine and pyrimidine nucleotides, histamine H1- and H2-receptors, beta 2-adrenoceptors and prostaglandin receptors. 3. The major G-proteins in HL-60 cells are pertussis toxin (PTX)-sensitive Gi-proteins (Gi2 > Gi3). Gs-proteins and G-proteins of the Gq-family (e.g., G16) are expressed, too. 4. G-protein-regulated effector systems in HL-60 cells are adenylyl cyclase and phospholipase C-beta 2 (PLC-beta 2) and, possibly, phospholipase D (PLD), nonselective cation (NSC) channels and NADPH oxidase. 5. The expression of signal transduction pathways in HL-60 cells strongly depends on the differentiation state of cells. 6. Formyl peptides, via Gi-proteins, mediate activation of PLC, PLD, NSC channels, NADPH oxidase and azurophilic granule release and are referred to as full secretagogues. In dibutyryl cAMP (Bt2cAMP)-differentiated HL-60 cells, C5a and LTB4 are partial and incomplete secretagogues, respectively. There are substantial differences in the Gi-protein activations induced by formyl peptides, C5a and LTB4. 7. In HL-60 promyelocytes, purine and pyrimidine nucleotides mediate activation of PLC and NSC channels largely via PTX-insensitive G-proteins and induce functional differentiation. In Bt2cAMP-differentiated HL-60 cells, they additionally activate PLD, NADPH oxidase and granule release via PTX-sensitive and -insensitive pathways. ATP and UTP are partial secretagogues. Multiple types of receptors (i.e., P2Y- and P2U-receptors and pyrimidinocyeptors) may mediate the effects of nucleotides in HL-60 cells. 8. Bt2cAMP- and 1 alpha,25-dihydroxycholecalciferol-differentiated HL-60 cells express H1-receptors coupled to Gi-proteins and PTX-insensitive G-proteins. In the former cells, histamine mediates activation of PLC and NSC channels, and in the latter, activation of NSC channels. Histamine is an incomplete secretagogue in these cells. 9. HL-60 promyelocytes express H2-receptors coupled to adenylyl cyclase, PLC, and NSC channels. There are substantial differences in the agonist/antagonist profiles of H2-receptor-mediated cAMP formation and rises in cytosolic Ca2+ concentration, indicative of the involvement of different H2-receptor subtypes. H2-receptors mediate functional differentiation of HL-60 cells. 10. Certain cationic-amphiphilic histamine receptor ligands (i.e., 2-substituted histamines, lipophilic guanidines, and a histamine trifluoromethyl-toluidide derivative) show stimulatory effects in HL-60 cells that are attributable to receptor-independent activation of Gi-proteins.
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PMID:G-protein-coupled receptors in HL-60 human leukemia cells. 874 93

The involvement of basic residues of interleukin(IL)-8 receptors in coupling to the Gi and G16 proteins was investigated by using a series of IL-8 receptor mutants. Substitution of the basic amino acids in the third inner loop of the receptor does not alter the abilities of the receptor mutants to activate recombinant Galpha16 or phosphoinositide-specific phospholipase C (PLC) beta2 expressed in COS-7 cells. However, an IL-8 receptor mutant with double mutations at residues Lys158 and Arg159 of the second inner loop loses its abilities to activate Galpha16 but retains its ability to activate PLC beta2. The activation of PLC beta2 by an IL-8 receptor that is sensitive to pertussis toxin has been previously demonstrated to be mediated through Gbetagamma. Surprisingly, the IL-8 receptor mutants with substitution of Ala for either residue Lys158 or Arg159 can still activate Galpha16, which suggests that either of the two basic residues in the second inner loop of the IL-8 receptor is sufficient for Galpha16 coupling.
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PMID:Two basic amino acids in the second inner loop of the interleukin-8 receptor are essential for Galpha16 coupling. 931 98

The biogenesis of trimeric G proteins was investigated by measurement of the expression of alpha-subunits in the megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11, representing stages of increasing maturation, and compared with platelets. Megakaryoblasts and platelets contained approximately equal amounts of Gi alpha-1/2, Gi alpha-3, Gq alpha, and G12 alpha protein. Maturation was accompanied by (1) downregulation of mRNA for Gs alpha and disappearance of iloprost-induced Ca2+ mobilization, (2) upregulation of the long form of Gs alpha protein (Gs alpha-L) and an increase in iloprost-induced cAMP formation, and (3) upregulation of G16 alpha mRNA and G16 alpha protein and appearance of thromboxane A2-induced signaling (Ca2+ mobilization and stimulation of prostaglandin I2-induced cAMP formation). Gz alpha protein was absent in the megakaryoblasts despite weak expression of Gz alpha mRNA in DAMI and relatively high levels of Gz alpha mRNA and Gz alpha protein in platelets. These findings reveal major changes in G protein-mediated signal transduction during megakaryocytopoiesis and indicate that G16 alpha couples the thromboxane receptor to phospholipase C beta.
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PMID:Signal transduction through trimeric G proteins in megakaryoblastic cell lines. 932 84

The mu-opioid receptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G16 protein. Given the promiscuous nature of G16 and the high degree of resemblance of signaling properties of the three opioid receptors, both delta- and kappa-opioid receptors are likely to activate G16. Interactions of delta- and kappa-opioid receptors with G16 were examined by coexpressing the opioid receptors and G alpha16 in COS-7 cells. The delta-selective agonist [D-Pen2,D-Pen5] enkephalin potently stimulated the formation of inositol phosphates in cells coexpressing the delta-opioid receptor and G alpha16. The delta-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of G alpha16 and exhibited appropriate ligand selectivity and dose dependency. Similar transfection studies revealed only weak stimulation by the mu-opioid receptor, whereas the kappa-opioid receptor produced moderate phospholipase C activity. G alpha16 thus appeared to interact differentially with the three opioid receptors. Radioligand binding assays indicate that the mu-opioid receptor was expressed at a lower level than those of the delta- and kappa-opioid receptors. To examine if differential coupling to G alpha16 is prevalent, a panel of Gs- or Gi-coupled receptors was coexpressed with G alpha16 in COS-7 cells and assayed for agonist-induced stimulation of phospholipase C. Activation of alpha2- and beta2-adrenergic, dopamine D1 and D2, adenosine A1, somatostatin-1 and -2, C5a, formyl peptide, and luteinizing hormone receptors all resulted in stimulation of phospholipase C, with maximal stimulations ranging from 1.5- to almost 17-fold. These findings suggest that the promiscuous G alpha16 can in fact discriminate among different receptors and that such preferential interaction might in part be due to the abundance of receptors.
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PMID:Differential coupling of mu-, delta-, and kappa-opioid receptors to G alpha16-mediated stimulation of phospholipase C. 957 9


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