Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sequence region 55-74 of the alpha-subunit of the acetylcholine receptor (AChR) from Torpedo californica electroplax comprises the amino-terminal end of a sequence segment--residues alpha 67-76--forming the main immunogenic region (MIR), which is most frequently recognized by anti-AChR autoantibodies in myasthenia gravis. The synthetic sequence alpha 55-74 of Torpedo AChR binds alpha-bungarotoxin (alpha BTX), suggesting that amino acid residues within this sequence region may contribute to formation of an alpha BTX binding site. Using single-residue substituted synthetic analogues of the sequence alpha 55-74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine or alanine, we sought identification of the amino acids involved in interaction with alpha-neurotoxins and with three different anti-MIR monoclonal antibodies (mAbs 6, 22, and 198). Substitution of Arg55, Arg57, Trp60, Arg64, Leu65, Arg66, Trp67, or Asn68 strongly inhibited alpha-toxin binding, whereas substitutions of Ile61, Val63, Pro69, Ala70, Asp71, or Tyr72 had marginal effects. Substitutions within the region alpha 68-72 significantly diminished binding of anti-MIR mAbs, although residue preferences differed among mAbs. Further, substituting Trp60 substantially reduced binding of mAb 198, and moderately affected binding of mAb 6, and substitution of Asp62 slightly but consistently affected binding of mAbs 6 and 22.
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PMID:Amino acid residues within the sequence region alpha 55-74 of Torpedo nicotinic acetylcholine receptor interacting with antibodies to the main immunogenic region and with snake alpha-neurotoxins. 851 74

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

The effect of methyl methanesulfonate (MMS) on the phosphorylation of an acidic 80-kDa myristoylated alanine-rich C kinase substrate (MARCKS) protein was investigated in NIH 3T3 fibroblasts. An alkylating agent, MMS inhibited protein kinase C activity and the phosphorylation of MARCKS. MMS treatment also lowered the cellular amounts of second messengers of inositol-1,4,5-trisphosphate and diacylglycerol. Data suggest that MMS decreased the phosphorylation of phospholipase C, a protein whose activity is influenced by its phosphorylation state. We present here the first report that MMS intervenes in a signal cascade by inhibiting the phosphorylation of phospholipase C, which in turn leads to the inactivation of protein kinase C and the subsequent inhibition of MARCKS phosphorylation.
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PMID:Inhibition of the phosphorylation of a myristoylated alanine-rich C kinase substrate by methyl methanesulfonate in cultured NIH 3T3 cells. 862 10

We have identified previously two amino acids, one in each of the fifth and sixth transmembrane segments of both the alpha1a-adrenergic receptor and the alpha1b-adrenergic receptor (AR), that account almost entirely for the selectivity of agonist binding by these receptor subtypes (Hwa, J., Graham, R. M., and Perez, D. M. (1995) J. Biol. Chem. 270, 23189-23195). Thus reversal of these two residues, from those found in the native receptor of one subtype to those in the other subtype, produces complementary changes in subtype selectivity of agonist binding. Here we show that mutating only one of these residues in either the alpha1b-AR or the alpha1a-AR to the corresponding residue in the other subtype (Ala204 --> Val for the alpha1b; Met292 --> Leu for the alpha1a-AR) results in chimeras that are constitutively active for signaling by both the phospholipase C and phospholipase A2 pathways. This is evident by an increased affinity for agonists, increased basal phospholipase C and phospholipase A2 activation, and increased agonist potency. Although mutation of the other residue involved in agonist binding selectivity, to the corresponding residue in the other subtype (Leu314 --> Met for the alpha1b-AR; Val185 --> Ala for the alpha1a-AR) does not alter receptor binding or signaling, per se, when combined with the corresponding constitutively activating mutations, the resulting chimeras, Ala204 --> Val/Leu314 --> Met ( alpha1b-AR) and Val185 --> Ala/Met292 --> Leu ( alpha1a-AR), display wild type ligand binding and signaling. A simple interpretation of these results is that the alpha1a- and alpha1b-ARs possess residues that critically modulate isomerization from the basal state, R, to the active state R*, and that the native receptor structures have evolved to select residues that repress active state isomerization. It is likely that the residues identified here modulate important interhelical interactions between the fifth and sixth transmembrane segments that inhibit or promote receptor signaling.
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PMID:Chimeras of alpha1-adrenergic receptor subtypes identify critical residues that modulate active state isomerization. 862 75

Transformation of cercariae of Schistosoma mansoni into schistosomula is accompanied by release of a soluble 28-kDa serine protease (s28) from the acetabular glands. The postulated activities of s28 include cleavage of skin connective tissue proteins (elastin, etc.), release of the cercarial glycocalyx, and cleavage of complement proteins. Our previous results demonstrated the presence of an antigenically cross-reactive protein on the surface of mechanically transformed schistosomula. As shown here, schistosomula express on their surface a 28-kDa serine protease (m28) which can be immunoprecipitated with anti-s28 antibodies. m28 eluted from the schistosomular tegumental membrane with NP-40 was purified to homogeneity in one step by adsorption on a chymotrypsin inhibitor column: 6-aminocaproyl-D-tryptophan methyl ester-Sepharose. Proteolytic activity of m28 was completely inhibited by the chymotrypsin inhibitor N-succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone. Efficient removal of m28 from schistosomula was achieved with NP-40, deoxycholate, cholate, Tween 20, and phospholipases A2 and C, but not with papain, trypsin, pronase, or proteinase K. Furthermore, treatment with phosphatidyl inositol-specific phospholipase C (PI-PLC) followed by hydroxylamine also released m28. Anti-cross-reactive determinant antibodies which recognize a neo epitope exposed in glycosyl phosphatidyl inositol-containing molecules cleaved by PI-PLC bind to purified m28. The latter results suggest that m28 is anchored to the tegumental membrane of schistosomula by a lipid anchor and that perhaps some of the m28 molecules are bound via glycosylphosphatidyl inositol. Based on inhibitor sensitivity and antigenic cross-reactivity, it is conceivable that s28 and m28 are related, if not identical, proteins. Finally, m28 was detected antigenically also on lung-stage and adult worms of S. mansoni.
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PMID:Schistosoma mansoni: evidence for a 28-kDa membrane-anchored protease on schistosomula. 865 54

Post-translational modifications such as phosphorylation and palmitoylation play important roles for the function and regulation of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins. Here we demonstrate that the human endothelin receptor A (ETA) incorporates [3H]palmitate. Mutation of a cluster of five cysteine residues present in the cytoplasmic tail of ETA into serine or alanine residues completely prevented palmitoylation of the receptor. The ligand binding affinity of the non-palmitoylated ETA mutants was essentially unchanged as compared to the palmitoylated wild type ETA suggesting that the replacement of the cysteine residues did not alter the overall structure of the receptor. Furthermore, the ligand-induced stimulation of adenylyl cyclase by the mutant ETA was unaffected by the mutation. In contrast, the mutated non-palmitoylated receptors but not the wild type receptor failed to stimulate phosphatidylinositol hydrolysis by phospholipase C activation upon challenge by endothelin-1. Furthermore, the mutant receptors failed to stimulate the ligand-induced transient increase in the cytoplasmic calcium seen with the wild type ETA. Endothelin-1 induced mitogenic stimuli via the wild type receptors but not through the mutated receptors suggesting an important role for phospholipase C in this signal transduction pathway. The differential regulation of distinct signal transduction pathways by post-translational modification suggests that palmitoylation of the ETA provides a novel mechanism of modulating ETA receptor activity.
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PMID:Palmitoylation of endothelin receptor A. Differential modulation of signal transduction activity by post-translational modification. 870 36

We recently demonstrated that epidermal growth factor receptor (EGFR)-mediated signaling of cell motility and mitogenesis diverge at the immediate post-receptor level. How these two mutually exclusive cell responses cross-communicate is not known. We investigated a possible role for a phospholipase C (PLC)-dependent feedback mechanism that attenuates EGF-induced mitogenesis. Inhibition of PLC gamma activation by U73122 (1 microM) augmented the EGF-induced [3H]thymidine incorporation by 23-55% in two transduced NR6 fibroblast lines expressing motility-responsive EGFR; increased cell division and mitosis was observed in parallel. The time dependence of this increase revealed that it was due to an increase in maximal incorporation and not a foreshortened cell cycle. Motility-responsive cell lines expressing a dominant-negative PLC gamma fragment (PLCz) also demonstrated augmented mitogenic responses by 25-68% when compared with control cells. PLCz- or U73122-augmented mitogenesis was not observed in three non-PLC gamma activating, nonmotility-responsive EGFR-expressing cell lines. Protein kinase C (PKC), which may be activated by PLC-generated second messengers, has been proposed as mediating feedback attenuation due to its capacity to phosphorylate EGFR and inhibit the receptor's tyrosine kinase activity. Inhibition of PKC by Calphostin C (0.05 microM) resulted in a 57% augmentation in the fold of EGF-induced thymidine incorporation. To further establish PKC's role in this feedback attenuation mechanism, an EGFR point mutation, in which the PKC target threonine654 was replaced by alanine, was expressed. Cells expressing these PKC-resistant EGFR constructs demonstrated EGF-induced motility comparable to cells expressing the threonine-containing EGFR. However, when these cells were treated with U73122 or Calphostin C, the mitogenic responses are not enhanced. These findings suggest a model in which PKC activation subsequent to triggering of motility-associated PLC gamma activity attenuates the EGFR mitogenic response.
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PMID:Mitogenic signaling from the egf receptor is attenuated by a phospholipase C-gamma/protein kinase C feedback mechanism. 881 94

We report herein the DNA sequence analysis of the heat-labile cytotonic enterotoxin gene (alt) from Aeromonas hydrophila and the biological function of the purified hyperproduced toxin (Alt). One large open-reading frame (ORF), comprised of 1104 bp, was detected at positions 804 to 1907 bp on a 4.0-kb Sa/l DNA fragment from Aeromonas. This ORF encodes for a protein having 368 amino acids (aa) with a computed molecular weight of 38 kDa. The aa sequence of the first 15 NH2-terminal residues of the mature native Alt from A. hydrophila matched with the DNA-derived aa sequence of the alt gene expressed in E. coli starting at position 19, which was leucine. The first 18aa residues of the Alt represented a putative signal sequence with alanine at its carboxy terminus. A BLAST search of the entire database showed 45-51% identity of the Alt, starting at position 158 with the carboxy half of the phospholipase C (PLC) and lipase from A. hydrophila; however, the purified Alt had no lipase/PLC activity. The alt gene was hyperexpressed using gene fusion expression vector systems, and the recombinant Alt exhibited a size of 35-40 kDa. The pure recombinant Alt elongated Chinese hamster ovary cells and elicited fluid secretion in rats ligated intestinal loops, indicating its enterotoxicity. Immunization of mice with recombinant Alt resulted in a reduced fluid secretory response when challenged with Aeromonas. The biological activity of the recombinant Alt in E. coli was about 10-fold less, compared to native Alt from Aeromonas, indicating differential processing of the toxin. The antibodies to native Alt neutralized the biological activity of the recombinant toxin, and these antibodies reacted with the same specificity to the native and recombinant Alt in immunoblots. The role of cyclic adenosine monophosphate and prostaglandins in causing a fluid secretory response by Alt also was demonstrated.
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PMID:Molecular and biochemical characterization of a heat-labile cytotonic enterotoxin from Aeromonas hydrophila. 893 43

v-H-Ras harboring the Gly-60 to Ala mutation (G60A) lacks the ability to induce germinal vesicle breakdown in Xenopus oocytes. Moreover, this mutant is capable of inhibiting the activity of v-H-Ras to induce oocyte germinal vesicle breakdown when co-injected. The duration and the extent of inhibition depends on the molar ratio of v-H-Ras(G60A) to v-H-Ras. The inhibition is not due to a general toxicity of v-H-Ras(G60A) to oocytes because oocytes injected with v-H-Ras(G60A) can be readily induced to mature by other mitogenic agents, such as insulin, insulin-like growth factor 1, insulin-like growth factor 2, and phosphatidylcholine-specific phospholipase C. The dominant negative effect of v-H-Ras(G60A) requires proper membrane attachment of v-H-Ras(G60A). By using a competition assay, it was concluded that the dominant negative phenotype of v-H-Ras(G60A) resulted from sequestering H-Ras downstream effector(s). Raf-1 was identified as one of the sequestered targets.
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PMID:The dominant negative effects of H-Ras harboring a Gly to Ala mutation at position 60. 894 23

Structural determinants within the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor that mediate G-protein activation of adenylate cyclase and phospholipase C are unknown. We investigated the role of the N-terminal region of the third intracellular loop of the opossum PTH/PTHrP receptor in coupling to two signal transduction pathways. We mutated residues in this region by tandem-alanine scanning and expressed these mutant receptors in COS-7 cells and/or Xenopus oocytes. All mutant receptors retained high affinity PTH binding in COS-7 cells, indistinguishable from wild-type receptors. Receptors with tandem-alanine substitutions in two N-terminal segments (377RVL379 and 381TKLR384) demonstrated impaired adenylate cyclase and phospholipase C activation. Receptor mutants with single-alanine substitutions scanning these two segments showed three different signaling defects in COS-7 cells. 1) Two mutant receptors (V378A and L379A) had reduced inositol phosphate (IP), but normal cAMP responses to PTH. 2) Mutant receptor T381A showed reduced cAMP, but wild-type IP responses to PTH. 3) Mutant receptor K382A demonstrated both markedly reduced cAMP and IP production due to PTH. In oocytes, mutants T381A and K382A showed decreased PTH-stimulated cAMP accumulation and intracellular Ca2+ mobilization. Thus, the N-terminal region of the third intracellular loop of this receptor plays a critical role in coupling to both Gs- and Gq-mediated second-messenger generation.
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PMID:The N-terminal region of the third intracellular loop of the parathyroid hormone (PTH)/PTH-related peptide receptor is critical for coupling to cAMP and inositol phosphate/Ca2+ signal transduction pathways. 896 99


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