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)

ADP is an important platelet agonist causing shape change and aggregation required for physiological hemostasis. We recently demonstrated the existence of two distinct G protein-coupled ADP receptors on platelets, one coupled to phospholipase C, P2Y1, and the other to inhibition of adenylyl cyclase, P2TAC. In this study, using specific antagonists for these two receptors, we demonstrated that concomitant intracellular signaling from both the P2TAC and P2Y1 receptors is essential for ADP-induced platelet aggregation. Inhibition of signaling through either receptor, by specific antagonists, is sufficient to block ADP-induced platelet aggregation. Furthermore, signaling through the P2TAC receptor could be replaced by activation of alpha2A-adrenergic receptors. On the other hand, activation of serotonin receptors supplements signaling through the P2Y1 receptor. Moreover, this mechanism of ADP-induced platelet aggregation could be mimicked by coactivation of two non-ADP receptors coupled to Gi and Gq, neither of which can cause platelet aggregation by itself. We propose that platelet aggregation results from concomitant signaling from both the Gi and Gq, a mechanism by which G protein-coupled receptors elicit a physiological response.
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PMID:Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. 965 41

The potential role of protein kinase C (PKC)-promoted phosphorylation has been examined in the G-protein-regulated inositol lipid signalling pathway. Incubation of [32P]Pi-labelled turkey erythrocytes with either the P2Y1 receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) or with PMA resulted in a marked increase in incorporation of 32P into the G-protein-activated phospholipase C PLC-betaT. Purified PLC-betaT also was phosphorylated by PKC in vitro to a stoichiometry (mean+/-S. E.M.) of 1.06+/-0.2 mol of phosphate/mol of PLC-betaT. Phosphorylation by PKC was isoenzyme-specific because, under identical conditions, mammalian PLC-beta2 also was phosphorylated to a stoichiometry near unity, whereas mammalian PLC-beta1 was not phosphorylated by PKC. The effects of PKC-promoted phosphorylation on enzyme activity were assessed by reconstituting purified PLC-betaT with turkey erythrocyte membranes devoid of endogenous PLC activity. Phosphorylation resulted in a decrease in basal activity, AlF4(-)-stimulated activity, and activity stimulated by 2MeSATP plus guanosine 5'-[gamma-thio]triphosphate in the reconstituted membranes. The decreases in enzyme activities were proportional to the extent of PKC-promoted phosphorylation. Catalytic activity assessed by using mixed detergent/phospholipid micelles also was decreased by up to 60% by phosphorylation. The effect of phosphorylation on Gqalpha-stimulated PLC-betaT in reconstitution experiments with purified proteins was not greater than that observed on basal activity alone. Taken together, these results illustrate that PKC phosphorylates PLC-betaT in vivo and to a physiologically relevant stoichiometry in vitro. Phosphorylation is accompanied by a concomitant loss of enzyme activity, reflected as a decrease in overall catalytic activity rather than as a specific modification of G-protein-regulated activity.
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PMID:Phosphorylation by protein kinase C decreases catalytic activity of avian phospholipase C-beta. 1002

The P2Y1 receptor is present in the heart, in skeletal and various smooth muscles, and in platelets, where its activation is linked to aggregation. Adenosine 3',5'- and 2',5'-bisphosphates have been identified as selective antagonists at the P2Y1 receptor (Boyer et al. Mol. Pharmacol. 1996, 50, 1323-1329) and have been modified structurally to increase receptor affinity (Camaioni et al. J. Med. Chem. 1998, 41, 183-190). We have extended the structure-activity relationships to a new series of deoxyadenosine bisphosphates with substitutions in the adenine base, ribose moiety, and phosphate groups. The activity of each analogue at P2Y1 receptors was determined by measuring its capacity to stimulate phospholipase C in turkey erythrocyte membranes (agonist effect) and to inhibit phospholipase C stimulation elicited by 10 nM 2-(methylthio)adenosine 5'-diphosphate (antagonist effect). 2'-Deoxyadenosine bisphosphate analogues containing halo, amino, and thioether groups at the 2-position of the adenine ring were more potent P2Y1 receptor antagonists than analogues containing various heteroatom substitutions at the 8-position. An N6-methyl-2-chloro analogue, 6, was a full antagonist and displayed an IC50 of 206 nM. Similarly, N6-methyl-2-alkylthio derivatives 10, 14, and 15 were nearly full antagonists of IC50 < 0.5 microM. On the ribose moiety, 2'-hydroxy, 4'-thio, carbocyclic, and six-membered anhydrohexitol ring modifications have been prepared and resulted in enhanced agonist properties. The 1,5-anhydrohexitol analogue 36 was a pure agonist with an EC50 of 3 microM, i.e., similar in potency to ATP. 5'-Phosphate groups have been modified in the form of triphosphate, methyl phosphate, and cyclic 3',5'-diphosphate derivatives. The carbocyclic analogue had enhanced agonist efficacy, and the 5'-O-phosphonylmethyl modification was tolerated, suggesting that deviations from the nucleotide structure may result in improved utility as pharmacological probes. The N6-methoxy modification eliminated receptor affinity. Pyrimidine nucleoside 3', 5'-bisphosphate derivatives were inactive as agonists or antagonists at P2Y receptor subtypes.
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PMID:Structure-activity relationships of bisphosphate nucleotide derivatives as P2Y1 receptor antagonists and partial agonists. 1022 31

The P2Y1 receptor is a membrane-bound G protein-coupled receptor stimulated by adenine nucleotides. Using alanine scanning mutagenesis, the role in receptor activation of charged amino acids (Asp, Glu, Lys, and Arg) and cysteines in the extracellular loops (EL) of the human P2Y1 receptor has been investigated. The mutant receptors were expressed in COS-7 cells and measured for stimulation of phospholipase C induced by the potent agonist 2-methylthioadenosine-5'-diphosphate (2-MeSADP). In addition to single point mutations, all receptors carried the hemagglutinin epitope at the N- terminus for detection of cell-surface expression. The C124A and C202A mutations, located near the exofacial end of transmembrane helix 3 and in EL2, respectively, ablated phospholipase C stimulation by </=100 microM 2-MeSADP. Surface enzyme-linked immunosorbent assay detection of both mutant receptors showed <10% expression, suggesting that a critical disulfide bridge between EL2 and the upper part of transmembrane 3, as found in many other G protein-coupled receptors, is required for proper trafficking of the P2Y1 receptor to the cell surface. In contrast, the C42A and C296A mutant receptors (located in the N-terminal domain and EL3) were activated by 2-MeSADP, but the EC50 values were >1000-fold greater than for the wild-type receptor. The double mutant receptor C42A/C296A exhibited no additive shift in the concentration-response curve for 2-MeSADP. These data suggest that Cys42 and Cys296 form another disulfide bridge in the extracellular region, which is critical for activation. Replacement of charged amino acids produced only minor changes in receptor activation, with two remarkable exceptions. The E209A mutant receptor (EL2) exhibited a >1000-fold shift in EC50. However, if Glu209 were substituted with amino acids capable of hydrogen bonding (Asp, Gln, or Arg), the mutant receptors responded like the wild-type receptor. Arg287 in EL3 was impaired similarly to Glu209 when substituted by alanine. Substitution of Arg287 by lysine, another positively charged residue, failed to fully restore wild-type activity.
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PMID:The role of amino acids in extracellular loops of the human P2Y1 receptor in surface expression and activation processes. 1032 57

The response of rat submandibular glands to extracellular purines was tested. In crude cellular suspensions, ATP increased the [Ca2+]i mostly by promoting uptake of extracellular calcium. ATP caused the pHi to drop, a response blocked by chloride channel inhibitors. ATP also inhibited the basal and isoproterenol-stimulated activity of the Na+ -K+ -2Cl-cotransporter. These effects were reproduced by benzoyl-ATP, an agonist of ionotropic purinoceptors. In pure ductal suspensions, ATP activated a metabotropic P2Y1 purinergic receptor coupled to phospholipase C and opened a non-specific cation channel coupled to a P2X7 receptor. Activation of these receptors stimulated a Ca2+ -dependent and a Ca2+ -independent phospholipase A2, the latter resulting in kallikrein secretion. We conclude that purinergic agonists can modulate the activity of both acinar and ductal phases of secretion. Activation of metabotropic receptors coupled to phospholipase C could lead to responses resembling those to muscarinic or adrenergic agonists. Activation of ionotropic receptors could stimulate new intracellular responses also involved in secretory function.
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PMID:Purines, a new class of agonists in salivary glands? 1041 54

Human platelets express two distinct G protein-coupled ADP receptors, one coupled to phospholipase C through Gq, P2Y1, and the other to inhibition of adenylyl cyclase through Gi, P2TAC. We have recently shown that concomitant intracellular signaling from both the P2TAC and P2Y1 receptors is essential for ADP-induced platelet aggregation. Previous studies have tested whether ADP causes a decrease in the basal cAMP level and this reduction promotes platelet aggregation, but did not study the effect of decreased cAMP levels when the Gq pathway is selectively activated. Since we are now aware that platelet aggregation requires activation of two receptors, we investigated whether the function of P2TAC receptor activation, leading to inhibition of platelet adenylyl cyclase, could be replaced by direct inhibition of adenylyl cyclase, when Gq pathway is also activated, a possibility that has not been addressed to date. In the present study, we supplemented the P2Y1 mediated Gq signaling pathway with inhibition of the platelet adenylyl cyclase by using SQ22536 or dideoxyadenosine, or by selective activation of the alpha2A adrenoceptors with epinephrine. Although SQ22536, dideoxyadenosine, and epinephrine reduced the cAMP levels, only epinephrine could mimic the P2TAC receptor mediated signaling events, suggesting that reduction in basal cAMP levels does not directly contribute to ADP-induced platelet activation. Adenosine-5'-phosphate-3'-phosphosulfate, a P2Y1 receptor antagonist, completely blocked ADP-induced inositol 1,4,5-trisphosphate and inositol 1,3.4-trisphosphate formation suggesting that P2TAC-mediated activation of Gi (or other G proteins) does not activate phospholipase C. These results suggest that a signaling event downstream from Gi, independent of the inhibition of platelet adenylyl cyclase, contributes to alphaIIb beta3 activation.
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PMID:Role of intracellular signaling events in ADP-induced platelet aggregation. 1054 22

We characterized the collagen-induced increase in cytosolic Ca2+ ([Ca2+]i) of bovine platelets loaded with the Ca2+ indicator Fura-PE3/AM. Collagen (10 micrograms/ml)-induced increase in [Ca2+]i was only partially inhibited by aspirin, a cyclooxygenase inhibitor, or adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS, a P2Y1 receptor antagonist), while in human platelets it was almost completely suppressed by aspirin. Collagen-induced increase in [Ca2+]i of bovine platelets was inhibited by U73122 (0.3-5 microM), a phospholipase C inhibitor. Collagen (10 micrograms/ml) increased production of inositol 1,4,5-trisphosphate, which was prevented by pretreatment with U73122 (5 microM). Collagen (10 micrograms/ml) accelerated Mn2+ entry, since the rate of Fura-PE3 quenching by Mn2+ was enhanced by 13-fold following stimulation with collagen. U73122 inhibited the acceleration of Mn2+ entry induced by collagen. PGE1 (2.5 microM) partially inhibited the collagen (50 micrograms/ml)-induced increase in [Ca2+]i in bovine platelets but not in human platelets. The data suggest that collagen-induced Ca2+ mobilization in bovine platelets is mediated by phospholipase C. The Ca2+ mobilization in bovine platelets is different from that in human ones as to the dependency on arachidonic acid metabolites and sensitivity to PGE1.
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PMID:Characteristics of collagen-induced Ca2+ mobilization in bovine platelets. 1072 11

The activation of P2Y1 receptors in platelets contributes to platelet aggregation, and selective antagonists are sought as potential antithrombotic agents. We reported (Kim et al. J. Med. Chem. 2000, 43, 746-755) that acyclic analogues of adenine nucleotides, containing two phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of adenine, are moderately potent P2Y1 receptor antagonists. In this study we have varied the chain structure, to include asymmetric substitution, olefinic, and cyclopropyl groups. These antagonists inhibited the stimulation of phospholipase C in turkey erythrocyte membranes induced by 30 nM 2-MeS-ADP in the micromolar range. In the series of symmetrically branched aliphatic groups substituted with two phosphate groups, the optimal antagonist potency occurred with the 2-methylpropyl group. A 2-chloro-N(6)-methyladenine derivative, 2-[2-(2-chloro-6-methylaminopurin-9-yl)methyl]propane-1,3-bisoxy(diammoniumphosphate) (7), was a full antagonist at the P2Y1 receptor with an IC(50) value of 0.48 microM. Esterification of one of the phosphate groups or substitution with O-acetyl greatly reduced the antagonist potency at the P2Y1 receptor. Removal of a methylene group of 7 or inclusion of an olefinic or cyclopropyl group also reduced potency. A pair of enantiomeric glycerol derivatives demonstrated a 5-fold stereoselectivity for the S-isomer. Stereoisomerically defined analogues of 7 containing a cyclopropyl group in place of the branched carbon were less potent than 7 as antagonists, with IC(50) values of 2-3 microM. No agonist activity was observed for these analogues. A new rhodopsin-based molecular model of the P2Y1 receptor indicated that the optimal docked orientation of the two monophosphate moieties relative to the adenine N(6) (compared to a rigid, bicyclic analogue) was consistent with the dependence of antagonist potency on chain length. The 3'-phosphate was predicted to occupy a restricted space, deeper in the binding cleft than the 5'-phosphate location. In summary, modification of the flexible spacer chain linking bisphosphate groups to the adenine moiety provided many moderately potent antagonists.
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PMID:Acyclic and cyclopropyl analogues of adenosine bisphosphate antagonists of the P2Y1 receptor: structure-activity relationships and receptor docking. 1154 78

We have investigated the role of secretion and intracellular signalling events in aggregation induced by the glycoprotein (GP)VI-selective snake venom toxin convulxin and by collagen. We demonstrate that aggregation induced by threshold concentrations of convulxin undergoes synergy with ADP acting via the P2Y12 receptor whereas there is no synergy via the P2Y1 receptor or with thromboxanes. On the other hand, apyrase, the P2Y12 receptor antagonist, AR-C67085, and indomethacin only marginally inhibit aggregation induced by convulxin. In comparison, these inhibitors severely attenuate the response to collagen. In order to investigate whether the weak inhibitory action against convulxin is due to release of agonists other than ADP from dense granules, experiments were performed on murine platelets deficient in this organelle (pearl mice platelets). A slightly greater reduction in aggregation induced by convulxin was observed in pearl platelets than in the presence of inhibitors of ADP, but a maximal response was still attained. Importantly, inhibition of protein kinase C further reduced the response to convulxin in pearl platelets demonstrating a direct role for the kinase in aggregation. Chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N',N'-tetraacetic acid (acetoxymethyl)ester (BAPTA-AM) abolished aggregation induced by convulxin under all conditions. Activation of phospholipase C by convulxin was potentiated by ADP acting through the P2Y12 receptor. In conclusion, we show that Ca2+ and protein kinase C, but not release of the secondary agonists ADP and thromboxane A2, are required for full aggregation induced by convulxin, whereas the response induced by collagen shows a much greater dependence on secretion of secondary agonists.
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PMID:Signalling events underlying platelet aggregation induced by the glycoprotein VI agonist convulxin. 1160 85

Low concentrations of collagen and anticardiolipin antibodies (ACLA), which were raised in rabbits by immunization with cardiolipin (CL), co-operatively activated human gel-filtrated platelets (GFP). GFP activated by adding ACLA 5 min prior to collagen (ACLA + Col) showed strong responses in cytosolic Ca2+ mobilization and cell aggregation; the responses decreased after 1 min, however, when collagen was added prior to ACLA (Col + ACLA). Col + ACLA was 30% less effective than the ACLA + Col in: (1) the phosphorylation of pleckstrin and myosin light chain; and (2) the secretion of alpha- and dense granules. Indomethacin inhibited Ca2+ mobilization, pleckstrin phosphorylation and cell aggregation in platelets stimulated by ACLA + Col. The thromboxane B2 level in platelets induced by ACLA + Col was similar to that stimulated by low concentrations of collagen alone. ACLA + Col increased the activities of phospholipase C (PLC) as determined by formation of phosphatidic acid (PA), whereas indomethacin and adenosine 2',5'-diphosphate, an antagonist of the ADP P2Y1 receptor, inhibited PA formation. These results suggest that ACLA, thromboxane A2 derived from the collagen pathway and secreted ADP co-operatively augment PLC activity and lead to platelet aggregation.
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PMID:Enhancement of human platelet activation by the combination of low concentrations of collagen and rabbit anticardiolipin antibodies. 1219


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