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)

1. We sought to reconstitute and characterize G-protein linked phosphatidyl-D-inositol 4,5-bisphosphate (PIP2)-directed phospholipase C (PLC) isoform activity in pig aortic vascular smooth muscle. 2. Six soluble PLC isoforms, namely gamma 1, delta 1 and beta 1 to beta 4 were partially separated by heparin affinity chromatography and were identified by Western blotting using specific antibodies. 3. In separate experiments, PLC activity was measured in the eluted fractions. Four of the partially resolved PLC isoforms gamma 1, beta 4, beta 2 and beta 1, showed corresponding activity using exogenous [3H]-PIP2 as substrate. 4. The isolated soluble PLC isoforms were reconstituted with receptors and guanyl nucleotide regulatory proteins (G-proteins) by addition of plasma membranes, the phospholipids which had been prelabelled with [3H]-myo-inositol. When so reconstituted PLC beta 2, beta 3 and beta 4 were inhibited (40 +/- 9, 47 +/- 12 and 40 +/- 5% respectively n = 12, +/-s.e.mean and each P < 0.05) by the addition of 1 mM guanosine 5'[beta gamma-imido]triphosphate (p[NH]ppG). 5. By contrast, when plasma membranes were preincubated with pertussis toxin to inhibit the activity of G-protein subunits G alpha i/alpha o the activities of PLC beta 2, beta 3 and beta 4 were stimulated (46 +/- 11, 31 +/- 9 and 37 +/- 8% respectively, n = 12, +/- s.e.mean and each P < 0.05) by the addition of p[NH]ppG. 6. Using well resolved fractions containing only PLC beta 3, time-dependent activity in the presence of p[NH]ppG was measurable only with membranes pretreated with pertussis toxin. 7. PLC beta 3 activity, measured with pertussis pretreated membranes, showed a dose-dependent increase in the presence of p[NH]ppG or guanosine 5'-[gamma-thio]triphosphate (GTP[S]). This increase with 10 microM p[NH]ppG or GTP[S] 10% +/- 4 and 12% +/- 5 respectively (both P < 0.05 vs control without GTP analogue +/- s.e.mean, n = 10) was abolished by 50 microM guanosine 5'-[beta-thio]diphosphate (GDP[S]) which also reduced constitutive PLC beta 3 activity by 9% +/- 4. 8. G-protein antibodies were used to neutralize PLC activity. Antibody to G alpha q/alpha 11, added to membrane fractions pretreated with pertussis toxin and assayed with GTP[S], reduced PLC beta 3 activity by 21% +/- 6 P < 0.02, n = 6, but was without effect on non-pertussis pretreated membranes. Antibodies to G alpha i1/alpha i2 had no effect. Antibodies to G-protein beta subunits had no effect on PLC beta 3 activity with pertussis pretreated preparations but activity without pertussis pretreatment was increased by 30% +/- 10, P < 0.03, n = 6. All results were expressed as % change from controls containing rabbit IgG. 9. In conclusion, pig aortic vascular smooth muscle contains six PLC isoforms. Activation of pertussis sensitive G-protein by GTP analogues results in inhibition of PLC beta 3 activity from liberated G-protein beta gamma subunits. Stimulation of PLC beta 3 activity is associated with a G-protein of the G alpha q family acting through the alpha subunit. The results suggest that the G-protein linked PLC beta isoforms in vascular smooth muscle demonstrate dual regulation by an inhibitory pertussis-sensitive pathway and a stimulatory G-protein of the G alpha q family, which is the case for PLC beta 3. This dual regulation is analogous to that of adenyl cyclase.
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PMID:Phospholipase C isoforms in vascular smooth muscle and their regulation by G-proteins. 879 75

The function of the phosphoinositide signal transduction system and the levels of heterotrimeric G-protein alpha-subunits were examined in postmortem prefrontal cortex regions (8/9) and region (10) from suicide victims with major depression and matched control subjects without psychiatric illness. The hydrolysis of [3H]phosphatidylinositol (PI) stimulated by phospholipase C, GTP-gamma-S, NaF, and neurotransmitter receptor agonists was measured in membrane preparations from both groups. Phospholipase C-beta activity was similar in depressed suicide and control subjects in the two regions of prefrontal cortex. In prefrontal cortex (10), but not in (8/9), the GTP-gamma-S concentration-dependent stimulation of [3H]PI hydrolysis was significantly lower (30%) in the depressed suicide group compared to the control group. Receptor-coupled, G-protein-mediated [3H]PI hydrolysis induced with carbachol, histamine, trans-1-aminocyclopentyl-1, 3-dicarboxylic acid (ACPD, a glutamatergic metabotropic receptor agonist), serotonin, or 2-methylthio-adenosine triphosphate (2mATP, a purinergic receptor agonist) in the presence of GTP-gamma-S stimulated equivalent responses in the two groups of subjects in each brain region. In prefrontal cortex (10) there was a 68% increase in the level of the 45 kDa subtype of G alpha s and in prefrontal cortex (8/9) there was a significant decrease (21%) in the level of G alpha i2 in the depressed suicide group compared to the control group. Levels of other heterotrimeric G-protein alpha-subunits (G alpha q/11, G alpha i1, and G alpha o) were not different in depressed suicide and control subjects in either brain region. Moreover, there were no differences in the levels of phospholipase C-beta or protein kinase C-alpha in the two groups of subjects in either brain region examined. These results demonstrate that in the prefrontal cortex of suicide victims with major depression compared to normal control subjects there is a region-specific alteration of G-protein-induced activation of the phosphoinositide signal transduction system and in the levels of G-protein alpha-subunits involved in cyclic AMP synthesis. These findings provide direct evidence in human brain that these two important signal transduction systems are altered in suicide subjects with major depression.
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PMID:Alterations in phosphoinositide signaling and G-protein levels in depressed suicide brain. 881 80

G alpha q is the alpha subunit of one of the heterotrimeric GTP-binding proteins that mediates stimulation of phospholipase C beta. We report the isolation and characterization of cDNA clones from a frontal cortex cDNA library encoding human G alpha q. The encoded protein is 359 amino acids long and is identical in all but one amino acid residue to mouse G alpha q. Analysis of human genomic DNA reveals an intronless sequence with strong homology to human G alpha q cDNA. In comparison to G alpha q cDNA, this genomic DNA sequence includes several small deletions and insertions that alter the reading frame, multiple single base changes, and a premature termination codon in the open reading frame, hallmarks of a processed pseudogene. Probes derived from human G alpha q cDNA sequence map to both chromosomes 2 and 9 in high-stringency genomic blot analyses of DNA from a panel of human-rodent hybrid cell lines. PCR primers that selectively amplify the pseudogene sequence generate a product only when DNA containing human chromosome 2 is used as the template, indicating that the authentic G alpha q gene (GNAQ) is located on chromosome 9. Regional localization by FISH analysis places GNAQ at 9q21 and the pseudogene at 2q14.3-q21.
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PMID:Molecular cloning of human G alpha q cDNA and chromosomal localization of the G alpha q gene (GNAQ) and a processed pseudogene. 882 33

G alpha q and G alpha 11, members of the Gq family of G-proteins, transduce signals from receptors to the beta isoenzymes of phosphatidyl-inositol-specific phospholipase C (PI-PLC). The receptor specificity of these alpha subunits is unknown. G alpha q and G alpha 11 are ubiquitously expressed in tissues; however, there have been conflicting reports of the presence or absence of G alpha 11 protein in haematopoietic cells. Platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors activate PI-PLC via G alpha q, but the role of G alpha 11 is uncertain. To define their roles in platelet activation we studied G alpha q and G alpha 11 gene expression by immunotransfer blotting and by reverse transcription of mRNA followed by PCR (RT-PCR) and direct sequencing. An antiserum specific for mouse G alpha 11 failed to identify G alpha 11 in dog or human platelets or in dog liver, a tissue known to contain G alpha 11. RT-PCR performed with gene-specific primers demonstrated G alpha q mRNA, but not G alpha 11 mRNA, in normal human and mouse platelets and in thromboxane-sensitive and thromboxane-insensitive dog platelets. Studies of mouse and dog liver and human retina confirmed that the cDNA, primers and probes used could amplify and recognize G alpha 11 in other tissues. However, species-specific oligonucleotide primers and probes were essential to demonstrate G alpha 11, but not G alpha q, mRNA. Compared with mouse cDNA, dog and human G alpha 11 cDNA had twice as many nucleotide substitutions (approx. 12% compared with approx. 6%) as G alpha q, G alpha q mRNA was also found in mature erythrocytes but G alpha 11 mRNA was not identified, whereas both G alpha q and G alpha 11 mRNAs were found in bone marrow stem cells. Therefore G alpha 11 gene expression in haematopoietic cells is linked with cellular differentiation. The lack of G alpha 11 indicates that signal transduction from platelet TXA2/PGH2 receptors to PI-PLC occurs via G alpha q, and that G alpha 11 deficiency is not responsible for defective activation of PI-PLC in thromboxane-insensitive dog platelets. Despite the high degree of similarity that exists between G alpha q and G alpha 11, significantly greater species-specific variation in nucleotide sequence is present in G alpha 11 than in G alpha q. Cellular specificity and species specificity are important characteristics of these Gq family G-proteins.
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PMID:Specificity of G alpha q and G alpha 11 gene expression in platelets and erythrocytes. Expressions of cellular differentiation and species differences. 883 52

Together with the calcium-sensing receptor, the metabotropic glutamate receptors (mGluRs) share no sequence homology with the other G protein-coupled receptors (GPCRs) and therefore constitute a new family of receptors. Recently, it was reported that G alpha 15 and G alpha 16 subunits allow many GPCRs to activate phospholipase C (PLC). Furthermore, the exchange of a few carboxyl-terminal residues of G alpha q by those of G alpha 12 or G alpha o allows the resulting chimeric G alpha subunits (G alpha ql and G alpha qol respectively) to couple Gi-coupled receptors to PLC. We report that mGluR2 and mGluR4, two receptors negatively coupled to adenylyl cyclase, activate PLC when coexpressed with G alpha 15, G alpha ql or G alpha qo. This indicates that the carboxyl-terminal end of the G alpha subunit also plays an important role in the specific interaction between mGluRs and the G proteins. In addition, the measurement of PLC activation by Gi-coupled mGluRs coexpressed with these G alpha subunits constitutes an easy functional assay for the pharmacological characterization of these receptors. The rank order of potency of antagonists was found to be (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine approximately (R,S)- alpha-methyl-4-phosphonophenylglycine > (R,S)-alpha-methyl-4-sulfonophenylglycine > (R,S)-alpha-methyl-4-tetrazolylphenylglycine = (S)-2-amino-2-methyl-4-phosphonobutyrate for mGluR2 and to be (R,S)-alpha-methyl-4-phosphonophenylglycine > or = (S)-2-amino-2-methyl-4-phosphonobutyrate > > (R,S)-alpha-methyl-4-sulfonophenylglycine [(R,S)-alpha-methyl-4-tetrazolylphenylglycine and (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine being inactive at 1 mM] for mGluR4. Using this functional assay, (R,S)-alpha-methyl-4-phosphonophenylglycine was found to have a similar KB value for mGluR2 and mGluR4.
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PMID:Coupling of metabotropic glutamate receptors 2 and 4 to G alpha 15, G alpha 16, and chimeric G alpha q/i proteins: characterization of new antagonists. 886 38

In plasma membranes derived from bovine mesenteric lymphatic smooth muscle cells, guanine nucleotide and forskolin stimulated adenylyl cyclase (AC) activity in a concentration-dependent manner, indicative of the presence of the stimulatory G-protein Gs linked to AC. There was no significant enzyme inhibition by low concentrations of guanine nucleotide and no effect on basal or guanine nucleotide-stimulated activity following pertussis toxin treatment of cells, suggesting the absence of Gi linked to inhibition of AC. Furthermore, there was no effect of adrenaline, isoprenaline or clonidine on basal or forskolin-stimulated activities, nor was there any specific binding of the beta-adrenoceptor ligand [125I]cyanopindolol to membranes, suggesting that catecholamine receptors do not modulate AC activity in these membranes. Pertussis toxin-mediated ADP ribosylation of membrane proteins and Western immunoblotting analysis revealed the presence of G-protein subunits G alpha i2, G alpha q, and G beta 1. In experiments designed to identify a possible effector enzyme for these G-proteins, membranes were screened with a range of antibodies raised against phospholipase C (PLC) beta, gamma and delta isozymes. Though no evidence was obtained by Western blotting for any of these proteins, PLC activity was concentration-dependently stimulated by Ca2+, but not by AIF4-, GTP[S], or purified G beta gamma subunits. Finally, no specific binding to membranes of the alpha 1-adrenoceptor ligand [3H]prazosin or the alpha 2-adrenoceptor ligand [3H]yohimbine was obtained. In conclusion, this study provides evidence for a Gs-dependent stimulation of AC, and for the presence of Gi2 and Gq/11, which do not appear to regulate a PLC activity also identified in lymphatic smooth muscle cell membranes. Furthermore, neither AC nor PLC appear to be associated with catecholamine receptors.
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PMID:Evidence for the presence of G-proteins, adenylyl cyclase and phospholipase C activities in lymphatic smooth muscle cell membranes. 895 44

Accumulated evidence suggests that dopamine and dopamine D1 agonists can activate phospholipase C in both brain and peripheral tissue. The receptor that mediates the hydrolysis of phosphoinositides has not been identified. The cloned dopamine D1A receptor that is generally thought to be linked to adenylyl cyclase, has also been proposed to couple to phospholipase C. However, a number of studies have suggested that this signaling pathway is mediated via a distinct D1-like dopamine receptor. We tested whether the D1A site plays a role in stimulating phosphoinositide hydrolysis by using the dopamine D1A-deficient mutant mice as a test model. Results show that although D1 dopamine receptor-mediated product on of cAMP is completely absent in membranes of D1A-deficient mice, D1 receptor-mediated accumulation of inositol phosphate is identical in tissues of mutant and wild-type animals. Furthermore, the coupling of [3H]SCH23390 binding sites in striatal or frontal cortex membranes to G alpha s is markedly reduced, although coupling of [3H]SCH23390 binding sites to G alpha q was unaltered in tissue taken from D1A mutant mice compared with control animals. These results clearly demonstrate that dopaminergic stimulation of inositol phosphate formation is mediated by a D1 dopamine receptor subtype that is distinct from the D1A receptor that activates adenylyl cyclase.
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PMID:D1-like dopaminergic activation of phosphoinositide hydrolysis is independent of D1A dopamine receptors: evidence from D1A knockout mice. 901 40

As disturbances in guanine nucleotide binding (G) protein-coupled phosphoinositide second messenger systems have been implicated in bipolar disorder, we examined whether the abundance of G alpha q/11 and phospholipase C (PLC)-beta 1 two key transducing proteins in this signaling pathway, are altered in this disorder. Compared with the controls, immunoreactive levels of G alpha q/11 were significantly elevated by 62% (p = .047) in occipital cortex of bipolar subjects. A similar increase (52%) in the PLC-beta 1 immunolabeling was also found in the occipital cortex of the bipolar subjects, but only reached marginal statistical significance (p = .07). In contrast, frontal and temporal cortex G alpha q/11 or PLC-beta 1 immunolabeling did not differ between bipolar and control subjects. Cerebral cortical immunoreactive levels of G beta 1 or G beta 2, included as a negative control, were not different between comparison groups. These findings support and extend earlier observations suggesting that disturbances in G protein-coupled second messenger signaling pathways may play an important role in the pathophysiology of bipolar affective disorder.
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PMID:Increased G alpha q/11 immunoreactivity in postmortem occipital cortex from patients with bipolar affective disorder. 906 88

Prolonged treatment with guanosine 5'-[gamma-thio]triphosphate (GTP gamma S; 5-16 h, 50 microM) of smooth muscle permeabilized with Staphylococcus aureus alpha-toxin down-regulated (abolished) the acute Ca2+ sensitization of force by GTP gamma S, AIF-4, phenylephrine, and endothelin, but not the response to phorbol dibutyrate or a phosphatase inhibitor, tautomycin. Down-regulation also abolished the GTP gamma S-induced increase in myosin light chain phosphorylation at constant [Ca2+] and was associated with extensive translocation of p21rhoA to the particulate fraction, prevented its immunoprecipitation, and inhibited its ADP ribosylation without affecting the immunodetectable content of G-proteins (p21rhoA, p21ras, G alpha q/11, G alpha i3, and G beta) or protein kinase C (types alpha, beta 1, beta 2, delta, epsilon, eta, theta, and zeta). We conclude that the loss of GTP gamma S- and agonist-induced Ca2+ sensitization through prolonged treatment with GTP gamma S is not due to a decrease in the total content of either trimeric (G alpha q/11, G alpha i3, and G beta) or monomeric (p21rhoA and p21ras) G-protein or protein kinase C but may be related to a structural change of p21rhoA and/or to down-regulation of its (yet to be identified) effector.
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PMID:Down-regulation of G-protein-mediated Ca2+ sensitization in smooth muscle. 919 Feb 7

Glucocorticoids regulate responsiveness of many cells to hormones that bind to G protein-coupled receptors. We examined the effect of glucocorticoids on parathyroid hormone (PTH) activation of two G protein-activated signal transduction pathways, phospholipase C (PLC) and adenylyl cyclase, in osteosarcoma UMR-106-01 cells. Dexamethasone (100 nM) increased PTH-stimulated and NaF-stimulated PLC activity by > 100% over 4 days (223 +/- 8 and 293 +/- 8.2% of control after 4 days for PTH and NaF-stimulated activity, respectively). The increase in PTH-stimulated adenylyl cyclase response in the same cells was more modest (162 +/- 5.4 and 171 +/- 6.8% of control after 4 days for PTH and NaF-stimulated activity, respectively). PTH activation of PLC was blocked by antiserums to G alpha q-11 and activation of adenylyl cyclase by G alpha s antiserums. Quantification of these G protein subunits in control and dexamethasone-treated cells showed a 78% increase in G alpha q-11 (from 18.1 +/- 1.2 to 32.2 +/- 1.5 pmol/mg), whereas G alpha s was increased only 34% (from 6.2 +/- 0.5 to 8.2 +/- 0.3 pmol/mg) and G beta-subunits were increased 40% (from 54 +/- 2.3 to 75.2 +/- 3.8 pmol/mg). These results suggest that glucocorticoids are more potent regulators of PLC activity than adenylyl cyclase activity in UMR cells, and this is mediated, at least in part, by differential increases in G alpha q-11 proteins.
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PMID:Dexamethasone increases G alpha q-11 expression and hormone-stimulated phospholipase C activity in UMR-106-01 cells. 931 42


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