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)

Bovine liver cytosol contains a phosphoinositide phospholipase C (PLCcyt) that is activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S)-activated G-proteins from liver plasma membranes. Heparin-Sepharose chromatography indicated that PLCcyt was immunologically distinct from PLC-beta 1, PLC-gamma 1, or PLC-delta 1 from brain. Initial purification of the GTP gamma S-activated G-proteins that stimulated PLCcyt indicated that the beta gamma complex was responsible. G-proteins were subsequently extracted from liver membranes as heterotrimers and purified in the presence of AlCl3, MgCl2, and NaF to allow reversible activation. Immunoblot analysis with an antiserum selective for the beta subunit showed that the stimulatory activity corresponded with the presence of this protein at every chromatographic step. When liver beta gamma complex was purified and separated from all detectable alpha subunits, as shown by immunoblotting and silver staining, it strongly stimulated PLCcyt after removal of the activating ligand [AlF4]- by gel filtration. beta gamma prepared from brain was approximately equipotent with that from liver. beta gamma was half-maximally effective at 33 nM and produced a maximal 50-fold activation of the PLC. Under identical conditions, beta gamma had no effect on brain PLC-gamma 1 or PLC-delta 1 and produced a 2-fold stimulation of PLC-beta 1 activity. Addition of purified GDP-bound alpha o, which had no effect by itself, completely reversed the beta gamma activation of PLCcyt, confirming that beta gamma was the active species. These data provide evidence for a novel mechanism by which beta gamma subunits of pertussis toxin-sensitive or -insensitive G-proteins activate phospholipase C.
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PMID:Activation of cytosolic phosphoinositide phospholipase C by G-protein beta gamma subunits. 133 Oct 76

There is much evidence that G-proteins transduce the signal from receptors for Ca(2+)-mobilizing agonists to the phospholipase C that catalyzes the hydrolysis of phosphoinositides. However, the specific G-proteins involved have not been identified. We have recently purified a 42 kDa protein from liver that activates phosphoinositide phospholipase C and cross-reacts with antisera to a peptide common to G-protein alpha-subunits. It is proposed that this protein is the alpha-subunit of the G-protein that regulates the phospholipase in this tissue. Ca(2+)-mobilizing agonists and certain growth factors also promote the hydrolysis of phosphatidylcholine through the activation of phospholipases C and D in many cell types. This yields a larger amount of diacylglycerol for a longer time than does the hydrolysis of inositol phospholipids. Consequently phosphatidylcholine breakdown is probably a major factor in long-term regulation of protein kinase C. The functions of phosphatidic acid produced by phospholipase D are speculative, but there is evidence that it is a major source of diacylglycerol in many cell types. The regulation of phosphatidylcholine phospholipases is multiple and involves direct activation by G-proteins, and regulation by Ca2+, protein kinase C and perhaps growth factor receptor tyrosine kinases.
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PMID:Cell signalling through phospholipid breakdown. 165 98

The regulatory factors controlling uterine contractile activity during pregnancy remain unclear, although pathways modulating intracellular Ca2+ and prostaglandin production play an important role. Because excitatory hormones raise myometrial Ca2+ levels and prostaglandin output through increasing phosphoinositide hydrolysis, regulation of G protein coupling to phospholipase C activation could be a key site for control. To measure the functional activity of this signaling pathway, we measured formation of [3H]inositol phosphates from prelabeled guinea pig myometrial membranes in response to G protein activation by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and fluoride. Although these agents stimulated a three- to fivefold increase in phosphoinositide phospholipase C activity in nonpregnant myometrium, at 46-47, 53-60, and 66-69 days of pregnancy (full term 67 +/- 2 days) this response fell by 43-83%. Moreover, the half-maximal effective dose (ED50) for GTP gamma S action was increased from 8.11 +/- 0.91 nM (n = 5) in the nonpregnant state to 307.4 +/- 142.3 (n = 9) and 209.7 +/- 155.1 nM (n = 8) at 53-60 and 66-69 days, respectively. Because phospholipase C levels displayed only a limited fall (28%) whether measured by direct Ca2+ activation or by immunoblotting, this study indicates a considerable suppression of G protein functional coupling to myometrial phosphoinositide hydrolysis throughout late gestation. Such a desensitization is likely to contribute to reports of diminished contractile sensitivity during pregnancy and to reflect an essential regulatory event in the processes maintaining uterine quiescence in pregnant guinea pig.
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PMID:Pregnancy suppresses G protein coupling to phosphoinositide hydrolysis in guinea pig myometrium. 216 88

We have examined the direct effects of oxidant metabolites on cardiac sarcolemmal phosphoinositide phospholipase C which transduces signals from various receptors for the modulation of intracellular Ca2+ levels. The enzyme activity in rat cardiac sarcolemmal membranes that had been preincubated (10 min; 37 degrees C) with xanthine-xanthine oxidase, a superoxide anion generating system, was not significantly affected. The addition to this system of superoxide dismutase, which converts superoxide anion to hydrogen peroxide (H2O2), resulted in a significant decrease of the enzyme activity in comparison with control values. Such decrease was fully prevented by catalase. Preincubation of sarcolemma with hypochlorous acid also gave a significant inhibition of phospholipase C, which was counteracted by the synthetic thiol reducer dithiothreitol. H2O2-pretreatment induced a concentration-dependent inhibition of the enzyme which was prevented by catalase but not by the iron chelator deferoxamine. Dithiothreitol was able to protect against, as well as to recover the enzyme activity from the H2O2 effects. These data suggest that superoxide anions and hydroxyl radicals did not interfere with phospholipase C activity, and that the nonradical oxidants, H2O2 and hypochlorous acid, may have acted through oxidation of thiol (SH) groups. The existence of reactive SH groups associated with the enzyme was confirmed by the inhibitory effects of SH modifiers (p-chloromercuriphenylsulfonic acid, 5'5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide and methyl methanethiosulfonate), which were prevented and in some cases also reversed by dithiothreitol. The biological reducer glutathione (GSH) was not able to recover the H2O2-induced inhibition of phospholipase C, whereas its oxidized form (GSSG) decreased the enzyme activity both in control and H2O2-pretreated membranes. The enzyme was active in a wide range of GSH/GSSG redox states, but H2O2 pretreatment narrowed this range. The results showed that oxidative stress changed the redox state of sarcolemmal phospholipase C, and this deactivated the enzyme. The oxidants' concentrations that significantly impaired phospholipase C in this study were compatible with those occurring in vivo during ischemia-reperfusion [Am. J. Med. 91(Suppl. 3C):235, 1991]. This supports the possibility that alteration of the receptor-associated phospholipase C may be a factor in the oxidant-related dysfunction of the ischemic-reperfused heart.
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PMID:Oxidative stress modifies the activity of cardiac sarcolemmal phospholipase C. 828 Jul 55

A series of chimeras between a constitutively active mutant of the alpha-subunit of Gq and the alpha-subunit of Gs was constructed to identify the domains in alphaq specifically involved in interaction with its effector phosphoinositide phospholipase C (PLC). Transient expression of the chimeric proteins and measurement of the production of inositol phosphates and cAMP in HEK-293 cells revealed that the Ile217-Lys276 sequence of alphaq contained the PLC interaction sites, whereas the residues for activation of adenylyl cyclase were in the Ile235-Leu294 sequence of alphas. Alanine scanning mutagenesis of the Ile217-Lys276 region of alphaq further identified two clusters of amino acids (Asp243,Asn244,Glu245 and Arg256,Thr257) that were specifically required for interaction with PLC. Comparison of the sequences of alphaq, alphas, and alphat showed that the PLC-interacting residues identified in alphaq are different from the corresponding residues in alphas and alphat that are involved in effector activation. Alignment of the sequences of alphaq and alphat, based on the crystal structure of alphat (Noel, J. P., Hamm, H. E., and Sigler, P. D. (1993) Nature 366, 654-663), indicated that the PLC-activating residues of alphaq are located in alpha-helix 3 and its linker to beta-sheet 4, which are adjacent to a switch region whose conformation changes with activation. It is proposed that the selectivity of alphaq for PLC involves relatively few amino acids, but that the effector may interact with other nonselective sequences in the alpha-subunit.
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PMID:Identification of determinants in the alpha-subunit of Gq required for phospholipase C activation. 861 84

Human neutrophils or HL60 cells express P2U receptors and respond to micromolar concentrations of ATP, adenosine 5'-O-(thiotriphosphate) (ATPgammaS), or UTP with immediate increases in intracellular Ca2+ through activation of phosphoinositide phospholipase C (Cowen, D. S., Lazarus, H. M., Shurin, S. B., Stoll, S. E., and Dubyak, G. R. (1989) J. Clin. Invest. 83, 1651-1660). P2U agonists reportedly induce limited enzyme secretion and enhance the respiratory burst in response to chemotactic factors. We demonstrate here that P2U agonists are chemotactic for neutrophils or differentiated HL60 cells. Rhodamine phalloidin staining indicates that ATPgammaS treatment induces actin polymerization and shape changes similar to those seen when these cells are treated with chemotactic peptide fMet-Leu-Phe. Although undifferentiated HL60 cells fail to mount a rise in Ca2+ when challenged with fMet-Leu-Phe, they increase Ca2+ in response to P2U agonists. However, functional expression of phospholipase C-coupled receptors is not sufficient for chemotaxis since HL60 cell migration in response to these agonists or to fMet-Leu-Phe occurs only after exposure to differentiating agents such as BT2cAMP. In addition to the well known G protein-linked receptors for lipid or peptide chemotactic factors, neutrophils apparently also can utilize G protein-linked purino/pyrimidino receptors to recognize nucleotides as chemoattractants. High concentrations of ATP and UTP generated at sites of platelet aggregation and tissue injury could thus be important mediators of inflammation.
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PMID:P2U agonists induce chemotaxis and actin polymerization in human neutrophils and differentiated HL60 cells. 866 69

Platelet activating factor (PAF) is a potent phospholipid mediator which elicits a diverse array of biological actions by interacting with G protein-coupled PAF receptors (PAFR). Binding of PAF to PAFRs leads to activation of G protein(s) that stimulate phosphoinositide phospholipase C and subsequent intracellular signaling responses. To identify the potential role of intracellular domains of the rat PAFR (rPAFR) in signaling, we examined effects of transfecting minigenes encompassing rPAFR intracellular domains 1 (1i), 2 (2i), and 3 (3i) on inositol phosphate (IP) production mediated by the co-transfected rPAFR cDNA. Although transfection of the rPAFR1i and rPAFR2i minigenes had no effects on PAF-stimulated signaling, transfection of the rPAFR3i minigene inhibited PAF-stimulated IP production by approximately 50% compared to controls. The rPAFR3i domain did not inhibit IP production mediated by the multifunctional rat pituitary adenylate cyclase-activating polypeptide receptor (rPACAPR), demonstrating the specificity of the competition by the rPAFR3i domain. In further experiments, the rPAFR3i domain was engineered onto the homologous domain of a monofunctional transmembrane variant of the rPACAPR (rPACAPR2) that activates only adenylyl cyclase. The rPACAPR2/rPAFR3i chimera responded to PACAP with increases in IP production which were attenuated nearly completely in cells cotransfected with the rPAFR3i domain. In contrast, PACAP had no effects on IP production in a receptor chimera expressing a mutated form of the rPAFR3i domain (rPACAPR2/rPAFR3imut). These results demonstrate the ability of the rPAFR3i domain to confer a phospholipase C-signaling phenotype to a receptor deficient in this activity and show that this activity is specific for the engineered rPAFR3i domain. These results suggest that the third intracellular loop of the rPAFR is a primary determinant in its coupling to phosphoinositide phospholipase C-activating G proteins, providing the first insight into the molecular basis of interaction of PAFRs with signal-transducing G proteins.
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PMID:The third intracellular domain of the platelet-activating factor receptor is a critical determinant in receptor coupling to phosphoinositide phospholipase C-activating G proteins. Studies using intracellular domain minigenes and receptor chimeras. 879 8

Heterotrimeric G proteins transmit hormonal and sensory signals received by cell surface receptors to effector proteins that regulate cellular processes. Members of the highly conserved family of alpha subunits specifically modulate the activities of a diverse array of effector proteins. To investigate the determinants of alpha subunit-effector specificity, we localized the effector-specifying regions of alphai2, which inhibits adenylyl cyclase, and alphaq, which stimulates phosphoinositide phospholipase C using chimeric alpha subunits. The chimeras were generated using an in vivo recombination method in Escherichia coli. The effector-specifying regions of both alphai2 and alphaq were localized within the GTPase domain. An alphaq/alphai2/alphaq chimera containing only 78 alphai2 residues within the GTPase domain robustly inhibited adenylyl cyclase. This alphai2 segment includes regions corresponding to two of the three regions of alphas that activate adenylyl cyclase, but does not include any of the alpha subunit regions that switch conformation upon binding GTP. Replacement of the alphaq residues that comprise the helical domain with the homologous alphai2 residues resulted in a chimeric alpha subunit that activated phospholipase C. Combined with previous studies of the effector-specifying residues of alphas and alphat, our results suggest that the effector specificity of alpha subunits is generally determined by the GTPase and not the helical domain.
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PMID:Localization of the effector-specifying regions of Gi2alpha and Gqalpha. 879 40

We have previously suggested the involvement of a Ca(2+)-phosphatidylinositol 4,5-bisphosphate (PIP2) complex in the phospholipid transmembrane redistribution triggered by cytosolic Ca2+ in erythrocytes. Indeed, the lipid scrambling was induced by extracellular Ca2+ in erythrocytes loaded with PIP2 and was abolished in inside-out vesicles prepared from PIP2-depleted erythrocytes (Sulpice, J.C., Zachowski, A., Devaux, P.F., & Giraud, F. (1994) J. Biol. Chem. 269, 6347-6354). Here, we show that Ca2+ triggers a partial redistribution of spin-labeled phospholipids in protein-free large unilamellar vesicles (LUVs), only when they contain PIP2. Spermine, a polyamine known to interact with PIP2 and reported to inhibit lipid scrambling in resealed ghosts, was found to inhibit also the Ca(2+)-induced scrambling in LUVs and in PIP2-loaded erythrocytes, presumably by interacting with PIP2 and preventing the formation of Ca(2+)-PIP2 complexes. A similar mechanism can account for spermine inhibition in natural membranes, confirming the role of PIP2 in the scrambling process without excluding the participation of proteins. In erythrocytes, activation of the phosphoinositide phospholipase C (PLC) or a 20 h ATP depletion, which both led to a reduction in the PIP2 content by 40-60%, did not affect Ca(2+)-induced phospholipid scrambling. In contrast, longer ATP depletion, resulting in a 80% reduction in the PIP2 content, did induce a significant decrease in lipid scrambling, suggesting that only the PIP2 pool resistant to the PLC was involved. Spermine was able to inhibit hydrolysis of this pool by an exogenous PLA2. It is thus likely that spermine antagonized the Ca(2+)-induced scrambling in resealed ghosts by interacting with the PLC-resistant pool of PIP2.
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PMID:Antagonist effects of Ca2+ and spermine on phosphatidylinositol 4,5-bisphosphate-mediated transmembrane redistribution of phospholipids in large unilamellar vesicles and in erythrocytes. 887 1

Muscarinic receptor activation of phosphoinositide phospholipase C (PLC) has been examined in rat cerebellar granule cells under conditions that modify intracellular Ca2+ stores. Exposure of cells to medium devoid of Ca2+ for various times reduced carbachol stimulation of PLC with a substantial loss (88%) seen at 30 min. A progressive recovery of responses was observed following the reexposure of cells to Ca2+-containing medium (1.3 mM). However, these changes did not appear to result exclusively from changes in the cytosolic Ca2+ concentration ([Ca2+]i), which decreased to a lower steady level (approximately 25 nM decrease in 1-3 min after extracellular omission) and rapidly returned (within 1 min) to control values when extracellular Ca2+ was restored. Only after loading of the intracellular Ca2+ stores through a transient 1-min depolarization of cerebellar granule cells with 40 mM KCl, followed by washing in nondepolarizing buffer, was carbachol able to mobilize intracellular Ca2+. However, the same treatment resulted in an 80% enhancement of carbachol activation of PLC. In other experiments, partial depletion of the Ca2+ stores by pretreatment of cells with thapsigargin and caffeine resulted in an inhibition (18 and 52%, respectively) of the PLC response. Furthermore, chelation of cytosolic Ca2+ with BAPTA/AM did not influence muscarinic activation of PLC in either the control or predepolarized cells. These conditions, however, inhibited both the increase in [Ca2+]i and the PLC activation elicited by 40 mM KCl and abolished carbachol-induced intracellular Ca2+ release in predepolarized cells. Overall, these results suggest that muscarinic receptor activation of PLC in cerebellar granule cells can be modulated by changes in the loading state of the Ca2+ stores.
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PMID:Intracellular Ca2+ stores regulate muscarinic receptor stimulation of phospholipase C in cerebellar granule cells. 1064 35


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