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)

Release of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) generated by phospholipase C (PLC) upon receptor stimulation plays an important role in the regulation of cell growth and differentiation. A second, completely different, signal transduction system involves retinoic acid (RA) and related derivatives. Binding to intracellular receptor sites can modulate keratinocyte growth and inhibits differentiation. The present study was aimed at characterizing possible interactions between the two signalling pathways in HaCaT keratinocytes. As determined by anion exchange chromatography and HPLC analysis, HaCaT keratinocytes treated with 1 microM RA for up to 72 h showed a marked decrease in Ins(1,4,5)P3 release upon stimulation with 10 microM bradykinin or 10 microM ionomycin. Thin-layer chromatography of phosphatidylinositol phosphates, the substrates of PLC, revealed no differences between RA-treated and untreated cells. Western blot analysis of the PLC isozymes present in HaCaT cells, PLC beta 3 and PLC gamma 1, showed no alterations in the expression of these proteins in RA-treated cells as compared to vehicle-treated controls. In addition, expression of the PLC-activating G protein G alpha q was not affected by RA treatment. Our results show that RA downregulates the PLC-mediated signaling system. The point of interference of this signal transduction crosstalk has yet to be elucidated. Our results suggest, furthermore, that RA-induced attenuation of keratinocyte differentiation might be mediated at least in part by the downregulation of Ins(1,4,5)P3 release.
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PMID:Retinoic acid attenuates phospholipase C-mediated signaling in HaCaT keratinocytes. 934 74

Gastrin-releasing peptide (GRP) causes multiple effects in humans by activating a specific heptaspanning receptor. Within the gastrointestinal tract, GRP receptors (GRP-R) are not normally expressed by mucosal epithelial cells except for those lining the gastric antrum. In contrast, recent studies have shown that up to 40% of resected colon cancers aberrantly express this receptor. This is important because the GRP-R can cause the proliferation of many, but not all, tissues in which it is expressed. Since GRP and other agonists are not known to exist in the colonic lumen, it has not been clear how or even if GRP-R expression in colon cancer contributes to cell proliferation. To evaluate the functional consequence of GRP-R expression on colonic epithelium, we transfected the recently isolated nonmalignant human colon epithelial cell line NCM460 with the cDNA for this receptor. All NCM460 cell lines expressing varying numbers of GRP-R bound selected agonists and antagonists indistinguishably from receptors expressed by other human tissues. Furthermore GRP-R-expressing transfected cell lines, but not wild-type NCM460 cells, proliferated independently of serum or other growth factors. Further evaluation revealed that GRP-R in these cells tonically stimulated G alpha q/11, resulting in increased phospholipase C activation. Since transfected cells do not secrete GRP, nor is their growth influenced by exposure to receptor-specific antagonists, these data indicate that GRP-R ectopically expressed by NCM460 cells are constitutively active. This report provides the first evidence of mutation-independent heptaspanning receptor constitutive activation resulting in cell proliferation, and identifies a potential mechanism whereby the GRP-R may act as an oncogene in human colon cancer.
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PMID:Constitutive activation of the gastrin-releasing peptide receptor expressed by the nonmalignant human colon epithelial cell line NCM460. 936 67

Mutant, guanosine triphosphatase-deficient, alpha-subunits of the G protein, Gs, gsp ocogene have been discovered in 40% of GH-secreting pituitary adenomas. Therefore, we hypothesized that a novel G protein class, G alpha q, involved in pituitary signal transduction, might be involved in pituitary tumorigenesis. Recombinant mutations of G alpha q result in constitutive activation of phospholipase C and have transforming activity. Therefore, we screened tumor samples from 37 pituitary adenomas for the presence of activating mutations of the G alpha q gene. Importantly, our sample contains 8 FSH and LH adenomas. In the pituitary gland, FSH and LH are linked to the GnRH-G alpha q signaling cascade, making these tumors a logical choice for screening for G alpha q mutations. Complementary DNA (cDNA) was synthesized by RT-PCR with G alpha q specific primers to exclude pseudogene transcripts. Fragments of G alpha q cDNA-encompassing residues (Arg183, Gln209) were screened by single-strand conformation polymorphism and then sequenced in both directions. No mutations were detected. We conclude that mutations in these regions of the G alpha q cDNA occur infrequently, if at all, in human pituitary adenomas. Alternative mechanisms underlying pituitary tumorigenesis should be explored.
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PMID:Pituitary adenomas: screening for G alpha q mutations. 939 37

Prostaglandin F2 alpha (PGF2 alpha) has regulatory (mainly luteolytic) effects in the ovary but the mechanism of action is not completely understood. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to demonstrate the presence of mRNA encoding the PGF2 alpha receptor (FP receptor) in human granulosa-lutein cells. Specific primers for the amplification of cDNA were designed and yielded a single product of 696 bp corresponding to the FP receptor. The identity of this product was verified by sequencing. Fluprostenol, a selective FP receptor agonist, activated phospholipase C (PLC) and increased intracellular free calcium concentration, confirming the functional activation of the receptor. We have demonstrated by Western blotting that granulosa cells express PLC-beta and PLC-gamma isoforms. The cells responded to pervanadate with increased PLC activity and increased tyrosine phosphorylation, demonstrating a functional PLC-gamma tyrosine kinase pathway. However, fluprostenol did not provoke any detectable tyrosine phosphorylation. Moreover, the effect of fluprostenol was inhibited through protein kinase C stimulation by phorbol 12, 13-dibutyrate, and was not affected when cells were treated with phenylarsine oxide, which blocks tyrosine phosphorylation. These results suggest that the FP receptor activates PLC-beta rather than PLC-gamma isoforms. Fluprostenol-induced activation was pertussis toxin resistant. Granulosa cells express G proteins of the Gq family (resistant to pertussis toxin) and mRNA for both G alpha q and G alpha 1 l has been identified by RT-PCR. In conclusion, human granulosa cells have a functional FP receptor the effects of which are mediated through PLC-beta activation probably via Gq/1 l.
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PMID:Activation of the prostaglandin FP receptor in human granulosa cells. 946

LLC-PK1, an epithelial cell line derived from the kidney proximal tubule, was used to study the ability of the G protein alpha-subunit, G alpha q, to regulate cell differentiation. A constitutively active mutant protein, alpha qQ209L, was expressed using the LacSwitch-inducible mammalian expression system. Induction of alpha qQ209L expression with isopropyl-beta-D-thiogalactopyranoside (IPTG) enhanced phospholipase C activity maximally by 6- to 7.5-fold. Increasing concentrations of IPTG progressively inhibited the activity of two differentiation markers, Na(+)-dependent hexose transport and alkaline phosphatase activity. Induction of alpha qQ209L expression also caused a change from an epithelial to a spindle-shaped morphology. The effects of alpha qQ209L expression on cell differentiation were similar to those observed with 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment. However, protein kinase C (PKC) levels were downregulated in TPA-treated cells but not in alpha qQ209L-expressing cells, suggesting that the regulation of PKC by G alpha q may be different from regulation by TPA. Interestingly, the PKC inhibitor GF-109203X did not inhibit the effect of IPTG on the development of Na(+)-dependent hexose transport in alpha qQ209L-expressing cells. These data implicate PKC delta and PKC epsilon in the pathway used by G alpha q to block the development of Na(+)-dependent hexose transport in IPTG-treated cells.
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PMID:Inhibition of cell differentiation by G alpha q in the renal epithelial cell line LLC-PK1. 957

Parathyroid hormone (PTH) elicits many of its physiological effects by activating distinct, G-protein-coupled signaling cascades that lead to synthesis of cyclic AMP and hydrolysis of phosphatidylinositol 4,5-bisphosphate. Using the nonhydrolyzable photo-reactive GTP analog [alpha-32P]GTP-gamma-azidoanilide (GTP-AA) and peptide antisera raised against G-protein alpha-subunits, we studied coupling of the PTH receptor to G-proteins in rat osteoblast-like cells (ROS 17/2.8), and in human embryonal kidney cells expressing the cloned human PTH/parathyroid hormone-related peptide (PTHrP) receptor at 40,000 receptors/cell (C20) or 400,000 receptors/cell (C21). Incubation of C21 membranes (but not C20 membranes) with [Nle8,18, Tyr34]-bovine PTH(1-34) amide (bPTH[1-34]) led to concentration-dependent incorporation of GTP-AA into the two isoforms of G alpha s, into G alpha q/11, and to a much lesser extent into G alpha i(1). In ROS 17/2.8 cells, bPTH(1-34) increased the incorporation of GTP-AA into G alpha s, but not into G alpha q/11 or G alpha i. The ability of bPTH(1-34) to increase labeling of G alpha s and G alpha q/11 was correlated with the receptor-dependent sensitivity of the adenylyl cyclase and phospholipase C signaling pathways to the hormone.
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PMID:Coupling of the PTH/PTHrP receptor to multiple G-proteins. Direct demonstration of receptor activation of Gs, Gq/11, and Gi(1) by [alpha-32P]GTP-gamma-azidoanilide photoaffinity labeling. 970 78

The ligand binding signals to a wide variety of seven transmembrane cell surface receptors are transduced into intracellular signals through heterotrimeric G-proteins. Recently, there have been reports which show diverse coupling patterns of ligand-activated receptors to the members of Gq family alpha subunits. In order to shed some light on these complex signal processing networks, interactions between G alpha q family of G protein and neurokinin-2 receptor as well as muscarinic M1 receptor, which are considered to be new therapeutic targets in asthma, were studied. Using washed membranes from Cos-7 cells co-transfected with different G alpha q and receptor cDNAs, the receptors were stimulated with various concentrations of carbachol and neurokinin A and the agonist-dependent release of [3H]inositol phosphates through phospholipase C beta-1 activation was measured. Differential coupling of G alpha q family of G-protein to muscarinic M1 receptor and neurokinin-2 receptor was observed. The neurokinin-2 receptor shows a ligand-mediated response in membranes co-transfected with G alpha q, G alpha 11 and G alpha 14 but not G alpha 16 and the ability of the muscarinic M1 receptor to activate phospholipase C through G alpha q/11 but not G alpha 14 and G alpha 16 was demonstrated. Clearly G alpha q/11 can couple M1 and neurokinin-2 receptor to activate phospholipase C. But, there are differences in the relative coupling of the G alpha 14 and G alpha 16 subunits to these receptors.
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PMID:Differential coupling of G alpha q family of G-protein to muscarinic M1 receptor and neurokinin-2 receptor. 987 70

Oxytocin stimulates an increase in intracellular calcium in uterine myometrium by several mechanisms. Several lines of evidence indicate that the oxytocin receptor is functionally coupled to GTP-binding proteins of the G alpha q/11 class which stimulate phospholipase C activity. The IP3 generated as a result of phospholipase C activation can trigger release of calcium from intracellular stores. The finding that the oxytocin-stimulated increase in intracellular calcium in myometrial cells is greater in the presence of extracellular calcium than that in its absence indicates that oxytocin also has effects on calcium entry. This action is nifedipine-insensitive but may involve indirect stimulation of calcium entry through release-operated channels. An anti-G alpha q/11 antibody inhibits both oxytocin-stimulated GTPase activity and phospholipase C activity in myometrial membranes. The stimulation by oxytocin of phosphoinositide turnover in COS cells transfected with a plasmid expressing the oxytocin receptor is enhanced by cotransfection of G alpha q. Co-transfection of intracellular domains of the oxytocin receptor causes varying degrees of interference with oxytocin-stimulated phosphoinositide turnover. The data suggest that more than one intracellular domain is involved in oxytocin receptor/G-protein coupling. Oxytocin receptor stimulation of phospholipase C is inhibited by cAMP. This occurs in myometrial cells and in COS cells transfected with a plasmid expressing the receptor. The inhibitory mechanism involves the action of protein kinase A and is probably targeted indirectly at the G alpha q/11 /phospholipase C coupling step.
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PMID:Molecular mechanisms regulating the effects of oxytocin on myometrial intracellular calcium. 1002 15

alpha-latrotoxin (LTX) stimulates massive release of neurotransmitters by binding to a heptahelical transmembrane protein, latrophilin. Our experiments demonstrate that latrophilin is a G-protein-coupled receptor that specifically associates with heterotrimeric G proteins. The latrophilin-G protein complex is very stable in the presence of GDP but dissociates when incubated with GTP, suggesting a functional interaction. As revealed by immunostaining, latrophilin interacts with G alpha q/11 and G alpha o but not with G alpha s, G alpha i or G alpha z, indicating that this receptor may couple to several G proteins but it is not promiscuous. The mechanisms underlying LTX-evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca2+, LTX triggers vesicular exocytosis because botulinum neurotoxins E, Cl or tetanus toxin inhibit the Ca(2+)-dependent component of the toxin-evoked release. Based on (i) the known involvement of G alpha q in the regulation of inositol-1,4,5-triphosphate generation and (ii) the requirement for Ca2+ in LTX action, we tested the effect of inhibitors of Ca2+ mobilization on the toxin-evoked norepinephrine release. It was found that aminosteroid U73122, which inhibits the coupling of G proteins to phospholipase C, blocks the Ca(2+)-dependent toxin's action. Thapsigargin, which depletes intracellular Ca2+ stores, also potently decreases the effect of LTX in the presence of extracellular Ca2+. On the other hand, clostridial neurotoxins or drugs interfering with Ca2+ metabolism do not inhibit the Ca2(+)-independent component of LTX-stimulated release. In the absence of Ca2+, the toxin induces in the presynaptic membrane non-selective pores permeable to small fluorescent dyes; these pores may allow efflux of neurotransmitters from the cytoplasm. Our results suggest that LTX stimulates norepinephrine exocytosis only in the presence of external Ca2+ provided intracellular Ca2+ stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca2+, which then triggers secretion.
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PMID:Norepinephrine exocytosis stimulated by alpha-latrotoxin requires both external and stored Ca2+ and is mediated by latrophilin, G proteins and phospholipase C. 1021 87

Previous in vitro studies of cord blood platelets from full-term and preterm neonates have demonstrated decreased responses to most physiologic agonists. This hyporesponsiveness is, in part, related to both deficient synthesis of, and response to, an important mediator of platelet function, thromboxane A2(TxA2). The poor response of neonatal platelets to TxA2 is not due to differences in TxA2 receptor binding characteristics, when compared with platelets from adult controls. Therefore, the postreceptor signal transduction pathway was investigated. The TxA2 receptor is linked via the trimeric GTP-binding protein, Gq, to phospholipase C-beta (PLC beta), and stimulation of platelets with the stable TxA2 mimetic, U46619, leads to activation of PLC beta and subsequent intracellular signaling events. U46619-induced 32P-phosphatidic acid formation, an index of PLC beta activation, was decreased in platelets of neonates (166 +/- 10%) when compared with adult controls (206 +/- 22%) (p < 0.05). Mobilization of intracellular calcium was impaired in platelets of newborns (175 +/- 49 nM) in comparison to adult controls (506 +/- 130 nM) (p < 0.01), after stimulation with U46619. U46619-stimulated GTPase activity was blunted in platelet membrane fractions from full-term neonates and almost absent in platelet membranes from preterm infants. Immunoblotting studies of the platelet membrane fractions, quantified by densitometric analysis, showed that levels of the G alpha q subunit were not significantly different between adult and neonate, and were not the cause of the marked differences in GTPase activity. These data suggest that signal transduction through the TxA2 receptor is affected by decreased activity of Gq in platelets of neonates, and that this defect in signal transduction through PLC beta contributes to the observed poor response of newborns' platelets to TxA2 and consequently to TxA2-dependent agonists such as collagen.
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PMID:Impaired signal transduction in neonatal platelets. 1023 66


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