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)

This study was undertaken in order to investigate the newly discovered spontaneously hypertensive rat (SHR)-specific restriction fragment length polymorphism (RFLP) at the genomic locus of (poly)phosphoinositide-specific phospholipase C (PLC)-delta at a DNA sequence level. Our aim was to clone the PLC-delta complimentary DNA (cDNA) from SHR and analyse the genomic DNA obtained from two hypertensive rat strains such as SHR and its stroke-prone substrain (SHR-SP) and three normotensive rat strains such as Sprague-Dawley, Donryu and Wistar-Kyoto (WKY) by preparing an aortic cDNA library of SHR, hybridization cloning of PLC-delta cDNA and an analysis of the genomic DNA by polymerase chain reaction. By digesting with restriction enzyme XhoI, we discovered an RFLP band displaying only in SHR and SHR-SP, not in Sprague-Dawley, Donryu and WKY rats. DNA sequencing of PLC-delta cDNA cloned from an aortic cDNA library of SHR revealed a total of three SHR-specific point mutations, two of which resulted in amino acid substitutions. The first point mutation (A to T) was detected at the XhoI site, changing a threonine(ACG) to a serine(TCG), and the second point mutation (A to G) was discovered in the vicinity of the first one, changing an isoleucine(ATA) to a methionine(ATG). This is the first demonstration of the mutations in the SHR genome changing amino acid sequences. These amino acid substitutions, situated in the putative catalytic X domain of PLC-delta, may be the major cause of the augmented PLC activity observed in the SHR, possibly leading to hypertension-related phenonemoma such as abnormal calcium homeostasis and increased intracellular calcium ion concentrations.
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PMID:Phospholipase C-delta gene of the spontaneously hypertensive rat harbors point mutations causing amino acid substitutions in a catalytic domain. 168 14

Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.
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PMID:Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence. 748 87

We previously reported that phosphatidylinositol 4,5-bisphosphate (PIP2) dramatically increases the gelating activity of smooth muscle alpha-actinin (Fukami, K., Furuhashi, K., Inagaki, M., Endo, T., Hatano, S., and Takenawa, T. (1992) Nature 359, 150-152) and that the hydrolysis of PIP2 on alpha-actinin by tyrosine kinase activation may be important in cytoskeletal reorganization (Fukami, K., Endo, T., Imamura, M., and Takenawa, T. (1994) J. Biol. Chem. 269, 1518-1522). Here we report that a proteolytic fragment with lysylendopeptidase comprising amino acids 168-184 (TAPYRNVNIQNFHLSWK) from striated muscle alpha-actinin contains a PIP2-binding site. A synthetic peptide composed of the 17 amino acids remarkably inhibited the activities of phospholipase C (PLC)-gamma 1 and -delta 1. Furthermore, we detected an interaction between PIP2 and a bacterially expressed alpha-actinin fragment (amino acids 137-259) by PLC inhibition assay. Point mutants in which arginine 172 or lysine 184 of alpha-actinin were replaced by isoleucine reduced the inhibitory effect on PLC activity by nearly half. Direct interactions between PIP2 and the peptide (amino acids 168-184) or the bacterially expressed protein (amino acids 137-259) were confirmed by enzyme-linked immunosorvent assay. We also found this region homologous to the sequence of the PIP2-binding site in spectrin and the pleckstrin homology domains of PLC-delta 1 and Grb7. Synthetic peptides from the homologous regions in spectrin and PLC-delta 1 inhibited PLC activities. These results indicate that residues 168-184 comprise a binding site for PIP2 in alpha-actinin and that similar sequences found in spectrin and PLC-delta 1 may be involved in the interaction with PIP2.
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PMID:Identification of a phosphatidylinositol 4,5-bisphosphate-binding site in chicken skeletal muscle alpha-actinin. 857 35

The phospholipase C (PLC)-beta isozymes differ from the PLC-gamma and PLC-delta isozymes in that they possess a long COOH-terminal sequence downstream of their catalytic domain, are activated by alpha subunits of the Gq class of G proteins, associate with the particulate subcellular fraction, and are present in the nucleus. Most of the COOH-terminal domain of PLC-beta isozymes is predicted to be helical, and three regions in this domain, PLC-beta1 residues 911-928 (region 1), 1055-1072 (region 2), and 1109-1126 (region 3), contain a high proportion of basic residues that are highly conserved. Projection of the sequences of these three regions in helical wheels reveals clustering of the basic residues. The role of the COOH terminus and the clustered basic residues in PLC-beta1 was investigated by either truncating the entire COOH-terminal domain (mutant DeltaC) or replacing two or three clustered basic residues with isoleucine (or methionine), and expressing the mutant enzymes in CV-1, Rat-2, or Swiss 3T3 cells. The DeltaC mutant no longer showed the ability to be activated by Gqalpha, to translocate to the nucleus, or to associate with the particulate fraction. Substitution of clusters of basic residues in regions 1 and 2 generally reduced the extent of activation by Gqalpha, whereas substitution of a basic cluster in region 3 had no effect. Substitution of the cluster of lysine residues 914, 921, and 925 in region 1 had the most marked effect, reducing Gqalpha-dependent activity to 10% of that of wild type. All substitution mutants, with the exception of that in which lysine residues 1056, 1063, and 1070 in region 2 were substituted with isoleucine, behaved like the wild-type enzyme in showing an approximately equal distribution between cytoplasm and nucleus; only 12% of the region 2 mutant was present in the nucleus. None of the basic clusters appeared critical for particulate association; however, replacement of each cluster reduced the amount of PLC-beta1 in the particulate fraction by some extent, suggesting that all the basic residues contribute to the association, presumably by interacting with acidic residues in the particulate fraction. Membrane localization of PLC-beta isozymes is therefore likely mediated by both the COOH-terminal domain and the pleckstrin homology domain, the latter of which is known to bind phosphatidylinositol 4,5-biphosphate.
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PMID:The role of carboxyl-terminal basic amino acids in Gqalpha-dependent activation, particulate association, and nuclear localization of phospholipase C-beta1. 870 89

The growth rate of rodent embryonic neuroblasts and human neuroblastoma cell lines is regulated in part by autocrine or paracrine actions of neuropeptides of the family that includes vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and pituitary adenylate cyclase-activating peptide (PACAP). These peptides act via seven transmembrane G-protein-linked receptors coupled to cAMP elevation, phospholipase C activation, intracellular Ca2+ release, and/or of mitogen-activated protein (MAP) kinase activation. Here we investigated the action of these peptides on the mouse neuroblastoma cell line Neuro2a. PHI and VIP inhibited proliferation at concentrations as low as 10(-13) M and 10(-10) M, respectively. In contrast, PACAP action was biphasic, with stimulation occurring at subnanomolar doses and inhibition at higher doses. Peptide actions were studied further by measuring cAMP and ERK1/2 MAP kinase activity and by assessing 3H-thymidine incorporation in conjunction with a panel of signal transduction pathways inhibitors. The data obtained indicated that the PHI-inhibitory and PACAP-stimulatory activities were mediated by corresponding changes in activity of the MAP kinase pathway and independent of protein kinase A (PKA) or protein kinase C (PKC). In contrast, the inhibitory actions of VIP and PACAP were specifically blocked by antagonists of PKA. Northern blot analysis revealed gene expression for only the PACAP-preferring (PAC1) receptor. However, binding experiments using 125I-labeled PACAP27, PHI, and VIP, demonstrated the presence of PACAP-preferring sites, bivalent VIP/PACAP sites, and PHI-binding sites that did not interact with VIP. The studies demonstrate potent regulatory actions of PACAP, PHI, and VIP on neuroblastoma cell proliferation which appear to be mediated by multiple subsets of receptors which differentially couple to MAP kinase and PKA signaling pathways.
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PMID:Differential effects of peptide histidine isoleucine (PHI) and related peptides on stimulation and suppression of neuroblastoma cell proliferation. A novel VIP-independent action of PHI via MAP kinase. 967 97

This paper examines the importance of the calcium-mobilizing inositol phosphate pathway in mediating the effects of FMRFamide and its gene-related neuropeptides on the myogenic heart beat of the pond snail Lymnaea stagnalis. These peptides are encoded on a single exon of the FMRFamide gene and mediate diverse physiological effects in the isolated heart. The rate of production of inositol-1,4, 5-trisphosphate [Ins(1,4,5)P(3)] and inositol-1,3,4, 5-tetrakisphosphate [Ins(1,3,4,5)P(4)], measured using an HPLC method, were both significantly elevated in a concentration-dependent manner by FMRFamide (and were also elevated by FLRFamide). The threshold for increasing inositol phosphate production was low (100 pmol l(-1)) with a peak response occurring at 1 micromol l(-1) FMRFamide. The shape of the dose-response curve for FMRFamide-induced elevation of heart-beat frequency, obtained in pharmacological experiments on the isolated whole heart, was similar to that for stimulation of inositol phosphate levels in homogenized heart tissue. FMRFamide and Ins(1,4,5)P(3) produced similar effects on the rate of heart beat in permeabilized whole hearts. In addition, the phospholipase C inhibitor, neomycin (2.5 mmol l(-)(1)), blocked the stimulatory effects of FMRFamide on Ins(1, 4,5)P(3) production in heart homogenate, and attenuated the excitatory effects of this neuropeptide in the isolated heart. The 'isoleucine' pentapeptides, EFLRIamide and pQFYRIamide, also encoded by the FMRFamide gene, produced no significant effects on inositol phosphate production when applied alone or in combination with FMRFamide. These results suggested that FMRFamide (and FLRFamide), but not EFLRIamide and pQFYRIamide, mediated their main effects on heart beat via the inositol phosphate pathway. The fifth peptide, SEQPDVDDYLRDVVLQSEEPLY ('SEEPLY') had no effect when applied alone but appeared to modulate the effects of FMRFamide by delaying the time-to-peak of the Ins(1,4,5)P(3) response from 5 s to 20 s by an unknown mechanism.
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PMID:Inositol-1,4,5-trisphosphate and inositol-1,3,4,5-tetrakisphosphate are second messenger targets for cardioactive neuropeptides encoded on the FMRFamide gene. 1048 18

Two heterozygous PTH/PTH-related peptide (PTHrP) receptor missense mutations were previously identified in patients with Jansen's metaphyseal chondrodysplasia (JMC), a rare form of short limb dwarfism associated with hypercalcemia and normal or undetectable levels of PTH and PTHrP. Both mutations, H223R and T410P, resulted in constitutive activation of the cAMP signaling pathway and provided a plausible explanation for the abnormalities in skeletal development and mineral ion homeostasis. In the present study we analyzed genomic DNA from four additional sporadic cases with JMC to search for novel activating mutations in the PTH/PTHrP receptor, to determine the frequency of the two previously identified missense mutations, H223R and T410P, and to determine whether different mutations present with different severity of the disease. The H223R mutation was identified in three novel JMC patients and is, therefore, to date the most frequent cause of JMC. In the fourth patient, a novel heterozygous missense mutation was found that changes isoleucine 458 in the receptor's seventh membrane-spanning region to arginine (I458R). In COS-7 cells expressing the human PTH/PTHrP receptor with the I458R mutation, basal cAMP accumulation was approximately 8 times higher than that in cells expressing the wild-type receptor despite impaired surface expression of the mutant receptor. Furthermore, the I458R mutant showed higher responsiveness to PTH than the wild-type receptor in its ability to activate both downstream effectors, adenylyl cyclase and phospholipase C. Like the H223R and the T410P mutants, the I458R mutant had no detectable effect on basal inositol phosphate accumulation. Overall, the patient with the I458R mutation exhibited clinical and biochemical abnormalities similar to those in patients with the previously identified H223R and T410P mutations.
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PMID:A novel parathyroid hormone (PTH)/PTH-related peptide receptor mutation in Jansen's metaphyseal chondrodysplasia. 1048 64

The phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits several types of interfacial activation. In the crystal structure of the closely related Bacillus cereus PI-PLC, the rim of the active site is flanked by a short helix B and a loop that show an unusual clustering of hydrophobic amino acids. Two of the seven tryptophans in PI-PLC are among the exposed residues. To test the importance of these residues in substrate and activator binding, we prepared several mutants of Trp-47 (in helix B) and Trp-242 (in the loop). Two other tryptophans, Trp-178 and Trp-280, which are not near the rim, were mutated as controls. Kinetic (both phosphotransferase and cyclic phosphodiesterase activities), fluorescence, and vesicle binding analyses showed that both Trp-47 and Trp-242 residues are important for the enzyme to bind to interfaces, both activating zwitterionic and substrate anionic surfaces. Partitioning of the enzyme to vesicles is decreased more than 10-fold for either W47A or W242A, and removal of both tryptophans (W47A/W242A) yields enzyme with virtually no affinity for phospholipid surfaces. Replacement of either tryptophan with phenylalanine or isoleucine has moderate effects on enzyme affinity for surfaces but yields a fully active enzyme. These results are used to describe how the enzyme is activated by interfaces.
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PMID:Role of tryptophan residues in interfacial binding of phosphatidylinositol-specific phospholipase C. 1191 6

Mouse prostacyclin (mIP) receptors transiently expressed in Chinese hamster ovary (CHO) cells activated both adenylyl cyclase and phospholipase C, with a 33-fold preference for signaling through Gs. The prostacyclin (IP) receptor agonists cicaprost, iloprost, carbacyclin, and prostaglandin E1 showed a similar order of potency for activation of both signaling pathways in cells transiently transfected with the mIP and the chimeric prostacyclin/prostaglandin D2 (IPN-VII/DPC and IPN-V/DPVI-C) receptors. Substitution of the carboxyl-terminal tail of the prostacyclin receptor with the corresponding region of the mDP receptor (IPN-VII/DPC) produced a receptor with increased coupling to both Gs and Gq. However, this increased G-protein coupling was lost in the IPN-V/DPVI-C receptor. The observation that both these chimeric receptors can activate phospholipase C indicates that the carboxyl-terminal tail of the IP receptor is not entirely responsible for its ability to couple to Gq. Site-directed mutagenesis studies suggest that isoleucine at position 323 in the IPN-VII/DPC receptor plays an important role in mediating the increased potency of this chimeric receptor.
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PMID:Properties of chimeric prostacyclin/prostaglandin D2 receptors: site-directed mutagenesis reveals the significance of the isoleucine residue at position 323. 1268 May 91

We investigated the effects of branched-chain amino acids on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Of the branched-chain amino acids, only leucine (10(-5)-10(-3) M) induced hepatocyte DNA synthesis and proliferation in a time- and dose-dependent manner. The addition of valine or isoleucine on its own had no significant effects on the hepatocyte DNA synthesis and proliferation. When combined, isoleucine competitively antagonized leucine-stimulated hepatocyte mitogenesis. U73122 (10(-6) M), AG1478 (10(-7) M), wortmannin (10(-7) M), PD98059 (10(-6) M) and rapamycin (10 ng/ml) inhibited the ability of leucine to stimulate the hepatocyte DNA synthesis and proliferation, suggesting that phospholipase C, tyrosine kinase, phosphatidylinositol 3-kinase, mitogen-activated protein (MAP) kinase, and p70 S6 kinase are involved in leucine signaling. The mitogenic effects of leucine are completely abolished by the addition of anti-transforming growth factor-alpha (TGF-alpha) antibody to the culture medium. Furthermore, leucine stimulated TGF-alpha secretion into the culture medium and the leucine effect was inhibited by U73122. Isoleucine alone had no significant effect on TGF-alpha secretion but this agent blocked leucine-induced TGF-alpha secretion. The results suggest that leucine triggers TGF-alpha secretion through a putative leucine receptor. The secreted TGF-alpha then stimulates hepatocyte DNA synthesis and proliferation through activation of TGF-alpha receptor to induce tyrosine kinase/MAP kinase activity and other downstream growth-related signal transducers.
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PMID:Effects of branched-chain amino acids on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. 1576 40


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