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)

We have identified a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C activity in Dictyostelium discoideum. Addition of Ca2+ (20 microM) results in the rapid formation of Ins(1,4,5)P3 within 5 s and leads to sustained inositol phosphate production for up to 40 min in membranes prepared from [3H]inositol-labelled cells. The phospholipase C activity is primarily membrane-bound under the conditions used to lyse the cells. In addition to this activity we also identified a family of Ca(2+)-regulated phospholipase activities active on a range of phospholipid substrates, using [3H]palmitate labelling. Inositol-specific phospholipase C activity is highest in vegetatively growing cells and in starved cells during the first 6 h in development, during which time Ca2+ elicited a 5-fold stimulation of inositol phosphate formation. After this time, total activity decreased progressively until 15 h, after which the activity remained constant up until 24 h. During this period, Ca2+ was able to stimulate a 2-fold increase in inositol phosphates.
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PMID:Characterization of phospholipase activity in Dictyostelium discoideum. Identification of a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C. 131 14

Surface cAMP receptors on Dictyostelium cells are linked to several second messenger systems and mediate multiple physiological responses, including chemotaxis and differentiation. Activation of the receptor also triggers events which desensitize signal transduction. These events include the following: 1) loss of ligand binding without loss of receptor protein; 2) phosphorylation of the receptor protein, which may lead to impaired signal transduction; 3) redistribution and degradation of the receptor protein; and 4) decrease of cyclic AMP (cAMP) receptor mRNA levels. These mechanisms of desensitization were investigated with the use of mutant synag7, with no activation of adenylyl cyclase; fgdC, with no activation of phospholipase C; and fgdA, with defects in both pathways. cAMP-induced receptor phosphorylation and loss of ligand binding activity was normal in all mutants. In contrast, cAMP-induced degradation of the receptor was absent in all mutants. The cAMP-induced decrease of cAMP-receptor mRNA levels was normal in mutant synag7, but absent in mutant fgdC. Finally, the cAMP analogue (Rp)-cAMPS induced loss of ligand binding without inducing second messenger responses or phosphorylation, redistribution, and degradation of the receptor. We conclude that 1) loss of ligand binding can occur in the absence of receptor phosphorylation; 2) loss of ligand binding and receptor phosphorylation do not require the activation of second messenger systems; 3) cAMP-induced degradation of the receptor may require the phosphorylation of the receptor as well as the activation of at least the synag7 and fgdC gene products; and 4) cAMP-induced decrease of receptor mRNA levels requires the activation of the fgdC gene product and not the synag7 gene product. These results imply that desensitization is composed of multiple components that are regulated by different but partly overlapping sensory transduction pathways.
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PMID:cAMP-induced desensitization of surface cAMP receptors in Dictyostelium: different second messengers mediate receptor phosphorylation, loss of ligand binding, degradation of receptor, and reduction of receptor mRNA levels. 132 48

In Dictyostelium discoideum extracellular cAMP stimulates guanylyl cyclase and phospholipase C; the latter enzyme produces Ins(1,4,5)P3 which releases Ca2+ from internal stores. The following data indicate that intracellular Ca2+ ions inhibit guanylyl cyclase activity. 1) In vitro, Ca2+ inhibits guanylyl cyclase with IC50 = 41 nM Ca2+ and Hill-coefficient of 2.1. 2) Extracellular Ca2+ does not affect basal cGMP levels of intact cells. In electro-permeabilized cells, however, cGMP levels are reduced by 85% within 45 s after addition of 10(-6) M Ca2+ to the medium; halfmaximal reduction occurs at 200 nM extracellular Ca2+. 3) Receptor-stimulated activation of guanylyl cyclase in electro-permeabilized cells is also inhibited by extracellular Ca2+ with half-maximal effect at 200 nM Ca2+. 4) In several mutants an inverse correlation exists between receptor-stimulated Ins(1,4,5)P3 production and cGMP formation. We conclude that receptor-stimulated cytosolic Ca2+ elevation is a negative regulator of receptor-stimulated guanylyl cyclase.
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PMID:Inhibition of receptor-stimulated guanylyl cyclase by intracellular calcium ions in Dictyostelium cells. 135 66

Previous studies have demonstrated that the Dictyostelium G alpha subunit G alpha 2 is essential for the cAMP-activation of adenylyl cyclase and guanylyl cyclase and that g alpha 2 null mutants do not aggregate. In this manuscript, we extend the analysis of the function of G alpha 2 in regulating downstream effectors by examining the in vivo developmental and physiological phenotypes of both wild-type and g alpha 2 null cells carrying a series of mutant G alpha 2 subunits expressed from the cloned G alpha 2 promoter. Our results show that wild-type cells expressing G alpha 2 subunits carrying mutations G40V and Q208L in the highly conserved GAGESG (residues 38-43) and GGQRS (residues 206-210) domains, which are expected to reduce the intrinsic GTPase activity, are blocked in multicellular development. Analysis of down-stream effector pathways essential for mediating aggregation indicates that cAMP-mediated activation of guanylyl cyclase and phosphatidylinositol-phospholipase C (PI-PLC) is almost completely inhibited and that there is a substantial reduction of cAMP-mediated activation of adenylyl cyclase. Moreover, neither mutant G alpha 2 subunit can complement g alpha 2 null mutants. Expression of G alpha 2(G43V) and G alpha 2(G207V) have little or no effect on the effector pathways and can partially complement g alpha 2 null cells. Our results suggest a model in which the dominant negative phenotypes resulting from the expression of G alpha 2(G40V) and G alpha 2(Q208L) are due to a constitutive adaptation of the effectors through a G alpha 2-mediated pathway. Analysis of PI-PLC in g alpha 2 null mutants and in cell lines expressing mutant G alpha 2 proteins also strongly suggests that G alpha 2 is the G alpha subunit that directly activates PI-PLC during aggregation. Moreover, overexpression of wild-type G alpha 2 results in the ability to precociously activate guanylyl cyclase by cAMP in vegetative cells, suggesting that G alpha 2 may be rate limiting in the developmental regulation of guanylyl cyclase activation. In agreement with previous results, the activation of adenylyl cyclase, while requiring G alpha 2 function in vivo, does not appear to be directly carried out by the G alpha 2 subunit. Our data are consistent with adenylyl cyclase being directly activated by either another G alpha subunit or by beta gamma subunits released on activation of the G protein containing G alpha 2.
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PMID:Amino acid substitutions in the Dictyostelium G alpha subunit G alpha 2 produce dominant negative phenotypes and inhibit the activation of adenylyl cyclase, guanylyl cyclase, and phospholipase C. 135 76

The presence of G-proteins, interacting with cAMP surface receptors, was investigated in vegetative cells, aggregation-competent cells, and migrating slugs of Dictyostelium discoideum. Our results indicate that G-proteins are present in all stages. In vegetative cells there is a limited number of cAMP receptors but no effect of GTP tau S on cAMP binding could be detected; in addition, no effect of cAMP on GTP tau S binding or GTPase activity was observed. In both aggregation-competent cells and slugs GTP tau S inhibits cAMP binding, while cAMP stimulates GTP tau S binding and high-affinity GTPase. Since the presence of G-proteins coupled to cAMP receptors could be demonstrated in slugs, the involvement of the effector enzymes adenylate cyclase and phospholipase C was investigated. The results show that adenylate cyclase activity is stimulated by GTP tau S in both stages and that in cells from migrating slugs the Ins(1,4,5)P3 production is increased upon stimulation with cAMP. The possible involvement of G-proteins in signal transduction during the slug stage of D. discoideum is discussed.
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PMID:Involvement of cyclic AMP cell surface receptors and G-proteins in signal transduction during slug migration of Dictyostelium discoideum. 185 Mar 66

Dictyostelium discoideum cells that overexpress a ras gene with a Gly12----Thr12 mutation (Dd-ras-Thr12) have an altered phenotype. These cells were labeled with [3H]inositol and the incorporation of radioactivity into inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was analyzed and found to be higher than in control cells. In contrast, the total mass of Ins(1,4,5)P3, as assessed with an assay using a specific Ins(1,4,5)P3-binding protein, was not significantly different between control and Dd-ras-Thr12 cells. Cells were labeled with [3H]inositol and the incorporation of radioactivity in all inositol metabolites was analyzed. Increased levels of radioactivity were observed for phosphatidylinositol phosphate (PtdInsP), phosphatidylinositol bisphosphate (PtdInsP2), Ins(1,4,5)P3, inositol 1,4-bisphosphate, inositol 4,5-bisphosphate, and inositol 4-monophosphate in Dd-ras-Thr12 cells relative to control cells. Decreased levels were found for phosphatidylinositol (PtdIns) and inositol 1-monophosphate. Calculations on the substrate/product relationships [i.e., Ins(1,4,5)P3/PtdInsP2] demonstrate that the observed differences are due only to the increased conversion of PtdIns to PtdInsP; other enzyme reactions, including phospholipase C, are not significantly different between the cell lines. The activity of PtdIns kinase in vitro is not different between Dd-ras-Thr12 and control cells, suggesting that either the regulation of this enzyme is altered or that the translocation of substrate from the endoplasmic reticulum to the kinase in the plasma membrane is modified. The results suggest multiple metabolic compartments of Ins(1,4,5)P3 in Dictyostelium cells. In Dd-ras-Thr12 transformants the increased conversion of PtdIns to PtdInsP leads to increased levels of Ins(1,4,5)P3 in the compartment with a high metabolic turnover. This Ins(1,4,5)P3 compartment is suggested to be involved in the regulation of cytosolic Ca2+ levels.
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PMID:Increased conversion of phosphatidylinositol to phosphatidylinositol phosphate in Dictyostelium cells expressing a mutated ras gene. 217 55

Dictyostelium discoideum amoebae were transformed with an expression vector for the gp80, a protein believed to mediate EDTA-resistant cell adhesion in developmental cells. Vegetative cells, that do not normally contain gp80, expressed the protein and this expression was correlated with the formation of cell-cell adhesions. These contacts exhibited minimal EDTA-resistance. Biochemical analyses of the protein synthesized by vegetative cells suggested that it is identical to that produced by aggregation-competent cells, including the presence of a glycolipid anchor. Additional experiments indicated that the anchor was insensitive to hydrolysis by exogenous (glycosly)phosphatidylinositol-specific phospholipase C [G)PI-PLCs) but was sensitive to the endogenous anchor degrading enzyme. This enzyme, initially described in aggregating cells (da Silva and Klein, Exp. Cell Res., in press) was found to be present also in vegetative amoebae.
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PMID:Cell adhesion in transformed D. discoideum cells: expression of gp80 and its biochemical characterization. 235 14

Dictyostelium discoideum amebae chemotax toward folate during vegetative growth and toward extracellular cAMP during the aggregation phase that follows starvation. Stimulation of starving amebae with extracellular cAMP leads to both actin polymerization and pseudopod extension (Hall et al., 1988, J. Cell. Biochem. 37, 285-299). We have identified an actin nucleation activity (NA) from starving amebae that is regulated by cAMP receptors and controls actin polymerization (Hall et al., 1989, J. Cell Biol., in press). We show here that NA from vegetative cells is also regulated by chemotactic receptors for folate. Our studies indicate that NA is an essential effector in control of the actin cytoskeleton by chemotactic receptors. Guided by a recently proposed model for signal transduction from the cAMP receptor (Snaar-Jagalska et al., 1988, Dev. Genet. 9, 215-225), we investigated which of three signaling pathways activates the NA effector. Treatment of whole cells with a commercial pertussis toxin preparation (PT) inhibited cAMP-stimulated NA. However, endotoxin contamination of the PT appears to account for this effect. The synag7 mutation and caffeine treatment do not inhibit activation of NA by cAMP. Thus, neither activation of adenylate cyclase nor a G protein sensitive to PT treatment of whole cells is necessary for the NA response. Actin nucleation activity stimulated with folate is normal in vegetative fgdA cells. However, cAMP suppresses rather than activates NA in starving fgdA cells. This indicates that the components of the actin nucleation effector are present and that a pathway regulating the inhibitor(s) of nucleation remains functional in starving fgdA cells. The locus of the fgdA defect, a G protein implicated in phospholipase C activation, is directly or indirectly responsible for transduction of the stimulatory chemotactic signal from cAMP receptors to the nucleation effector in Dictyostelium.
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PMID:Transduction of the chemotactic signal to the actin cytoskeleton of Dictyostelium discoideum. 251 Oct 51

Using the technique of HPLC with Partisil SAX columns, we have found that stimulation of amoebae of Dictyostelium discoideum with the chemoattractant cyclic AMP induces the rapid accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 5 s. A smaller HPLC peak (designated P3) that elutes just after the Ins(1,4,5)P3 peak accumulates more slowly to a maximum at 20 s. In control studies, the changes in Ins(1,4,5)P3 were shown not to be due to varying recovery from the cell extracts and a comparison of reverse-phase and Partisil SAX HPLC columns showed similar values for determinations by either method. The involvement of a G-protein in this chemotactic system was confirmed by the finding that accumulation of Ins(1,4,5)P3 was elicited by the addition of GTP gamma S (5'-[gamma-thio]triphosphate) to saponin-permeabilized amoebae. A study of the changes in the lipid-soluble phosphatidyl inositol phosphates demonstrated that cyclic AMP also stimulated a rapid loss of radioactivity from 32P-labelled phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which corresponded in its timing to the rise in Ins(1,4,5)P3, indicating that a phosphoinositidase C (phospholipase C) is present that can be stimulated by occupation of the cell surface cyclic AMP receptors.
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PMID:Inositol tris- and polyphosphate formation during chemotaxis of Dictyostelium. 255 21

The contact site A glycoprotein, a cell adhesion protein of aggregating Dictyostelium cells, was labeled with fatty acid, myo-inositol, phosphate and ethanolamine in vivo, indicating that the protein is anchored in the membrane by a lipid. This lipid was not susceptible to phosphatidyl inositol specific phospholipase C. When cleaved with nitrous acid or when subjected to acetolysis, the anchor released lipids which were different from those released from Trypanosoma variant cell surface glycoprotein, a protein with a known phosphatidyl inositol-glycan anchor. Resistance to weak and sensitivity to strong alkali indicated that the fatty acid in the contact site A glycolipid anchor was in an amide bond. On incubation with sphingomyelinase, a lipid with the chromatographic behavior of ceramide was released. These results suggest that the contact site A glycoprotein is anchored by a ceramide based lipid glycan.
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PMID:The contact site A glycoprotein of Dictyostelium discoideum carries a phospholipid anchor of a novel type. 272 85


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