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)

We have previously shown that the high-affinity binding of substance P (SP) to its receptor is dependent on an interaction with a PTX-insensitive G protein. This G protein couples SP receptor activation to stimulation of its effector, phospholipase C. In this study, we combined photoaffinity labeling, chemical cross-linking techniques, and immunological characterization using sequence-specific antibody probes to identify G proteins that couple to the SP receptor. First we covalently labeled the SP receptor present on rat submaxillary gland membranes with a radioiodinated photoreactive derivative of SP, p-benzoyl-L-phenylalanine(8)-substance P (125I-[Bpa8]SP). Photoincorporation of this SP derivative was susceptible to guanine nucleotide inhibition, indicating that the receptor was coupled to its G protein during labeling. We then used a chemical cross-linking agent to covalently link the photoaffinity labeled SP receptor and its associated G protein. Cross-linking generated a 96 kDa product, formation of which was prevented by the addition of a guanine nucleotide, but not an adenine nucleotide, following photolabeling, but prior to cross-linking. Furthermore, the 96 kDa cross-linked complex was absent in membranes which had been depleted of G proteins by treatment with alkaline buffer prior to addition of the cross-linking agent. Reductive cleavage of the cross-link in the isolated 96 kDa complex yields two products: the 53 kDa SP receptor and a 42 kDa protein identified by immunoblot analysis as either G alpha q or G alpha 11. Antisera against a common sequence within G alpha s, G alpha i, and G alpha o showed no immunoreactivity to the complex or its cleavage products. These results provide the first direct evidence of specific interaction between photoaffinity labeled SP receptor and the alpha subunits of Gq and G11, members of a family of G proteins known to be associated with pertussis toxin-insensitive phospholipase C activation.
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PMID:Chemical cross-linking of the substance P (NK-1) receptor to the alpha subunits of the G proteins Gq and G11. 860 28

Two major intermediaries in signal transduction pathways are pp60v-sre family tyrosine kinases and heterotrimeric guanine nucleotide-binding proteins. In Rat-1 fibroblasts transformed by the v-src oncogene, endothelin-1 (ET-1)-induced inositol 1,4,5-trisphosphate accumulation is increased 6-fold, without any increases in the numbers of ET-1 receptors or in the response to another agonist, thrombin. This ET-1 hyperresponse can be inhibited by an antibody directed against the carboxyl terminus of the Gq/G11 alpha subunit, suggesting that the Gq/G11 protein couples ET-1 receptors to phospholipase C (PLC). While v-src transformation did not increase the expression of the Gq/G11 alpha subunit, immunoblotting with anti-phosphotyrosine antibodies and phosphoamino acid analysis demonstrated that the Gq/G11 alpha subunit becomes phosphorylated on tyrosine residues in v-src-transformed cells. Moreover, when the Gq/G11 protein was extracted from control and transformed cell lines and reconstituted with exogenous PLC, AIF*4-stimulated Gq/G11 activity was markedly increased in extracts from v-src-transformed cells. Our results demonstrate that the process of v-src transformation can increase the tyrosine phosphorylation state of the Gq/G11 alpha-subunit in intact cells and that the process causes an increase in the Gq/G11 alpha-subunit's ability to stimulate PLC following activation with AIF-4.
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PMID:Transformation of Rat-1 fibroblasts with the v-src oncogene increases the tyrosine phosphorylation state and activity of the alpha subunit of Gq/G11. 871 Aug 57

The regulation of the receptor-G protein-phospholipase C (PLC) cascade was investigated in rat myometrium at midgestation (day 12) and at term (day 21) comparatively to the estrogen-treated tissue (day 0). Carbachol-mediated generation of [3H]inositol phosphates was insensitive to pertussis toxin and was enhanced at days 12 and 21 two- and threefold, respectively, with no alteration of muscarinic sites (M3 subtype). A similar increase could be detected in the production of inositol 1,4,5-trisphosphate, indicating the stimulation of a PLC degrading phosphatidylinositol 4,5-bisphosphate. AlF4- also enhanced PLC activation during gestation, suggesting pregnancy-related regulations that bypass receptor activation. Immunoreactive G proteins, Gq alpha and G11 alpha, and PLC-beta 3 were detected in all myometrial preparations. The amount of PLC-beta 3 was similar in day 0 and day 21 myometrium, although decreasing by 75% at midgestation. Of significance was the increased amount of Gq alpha in day 12 and day 21 myometrium (3- and 2-fold, respectively) which coincided with the enhanced phosphoinositide breakdown. The upregulation of Gq alpha may contribute to the enhanced PLC activity during pregnancy and at term.
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PMID:Modulation of phospholipase C pathway and level of Gq alpha/G11 alpha in rat myometrium during gestation. 884 20

G proteins of the Gq/11 subfamily functionally couple cell surface receptors to phospholipase C beta (PLC beta) isoforms. Stimulation of PLC beta induces Ca2+ elevation by inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release and store-dependent 'capacitative' Ca2+ entry through Ca(2+)-permeable channels. The Drosophila trp gene, as well as some human trp homologs, code for such store-operated channels. The related trp-like (trpl) gene product also forms a Ca(2+)-permeable cation channel, but is not activated by store depletion. Co-expression of the constitutively active Gq subfamily member G alpha 11 (G alpha 11) with trpl enhanced trpl currents 33-fold in comparison with co-expression of trpl with other G alpha isoforms or G beta gamma complexes. This activation could not be attributed to signals downstream of PLC beta. In particular, InsP3 infusion, modulation of protein kinase C activity or elevation of intracellular calcium concentration failed to induce trpl currents. In contrast, purified G alpha 11 (but not other G protein subunits) activated trpl channels in inside-out patches. We conclude that trpl is regulated by G11 proteins in a membrane-confined manner not involving cytosolic factors. Thus, G proteins of the Gq subfamily may induce Ca2+ entry not only indirectly via store-operated mechanisms but also by directly stimulating cation channels.
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PMID:Direct activation of trpl cation channels by G alpha11 subunits. 891 61

Thrombin receptor-G protein coupling was investigated in the human epithelial neuroblastoma cell line, SH-EP. In these cells, both alpha-thrombin and thrombin receptor peptides, SFLLRNP (one-letter amino-acid code), which are newly exposed following cleavage by alpha-thrombin, stimulated GTPase activity about 2-fold over basal activity. Pertussis toxin treatment only partially attenuated alpha-thrombin- and SFLLRNP-stimulated GTPase activity by 50%, whereas antibody raised against synthetic heptapeptide SFLLRNP blocked alpha-thrombin-stimulated phosphoinositide hydrolysis more than 80%. Immunoprecipitation studies using this antibody showed that both Gi2, a subtype of guanine nucleotide-binding regulatory proteins (G proteins) mediating inhibition of adenylyl cyclase, and Gq/G11, a G protein mediating stimulation of phospholipase C, were activated by alpha-thrombin. These data suggest that in these cells the thrombin receptor activates pertussis toxin-sensitive and pertussis toxin-insensitive G proteins simultaneously and directly couples to Gi2 and Gq/G11, which mediate different signaling pathways.
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PMID:Direct evidence for two distinct G proteins coupling with thrombin receptors in human neuroblastoma SH-EP cells. 898 57

The alpha subunits of Gq family G proteins, GL1 alpha and GL2 alpha, are bovine homologues of mouse G14 alpha and G11 alpha, respectively, and are closely related to each other. When expressed in Xenopus oocytes together with metabotropic glutamate receptors, GL2 alpha activates endogenous phospholipase C (PLCx) in response to glutamate stimulation, whereas GL1 alpha inhibits the activation of PLCx. By examining the properties of 10 chimeras between GL1 alpha and GL2 alpha and their mutants, we tried to identify the regions on the G alpha proteins that are important for the activation of PLCx. The results indicated that a necessary (but not sufficient) condition for a chimeric G alpha protein to be able to clearly activate PLCx was that its N-terminal quarter portion should be derived from GL2 alpha. No correlation was found between the origin (GL1 alpha or GL2 alpha) of C-terminal regions of the chimeras and the ability of chimeras to activate PLCx. One of the chimeras is different from GL2 alpha at only four amino acid residues in the N-terminal region, and yet it could not activate PLCx. When one of the four residues, Ser-59, in the chimera was mutated back to Ala as in the original GL2 alpha, the resulting mutant became capable of activating PLCx. This residue is localized in the midst of the N-terminal linker connecting the two major domains in the G alpha proteins. These results indicate that Ala-59 is critical for the activation of PLCx, and that the linker may play important roles in determining functions of G alpha proteins.
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PMID:Importance of N-terminal regions of G protein alpha subunits for the activation of phospholipase C in Xenopus oocytes. 898 68

The ability of muscarinic cholinergic receptors to activate phosphoinositide turnover following agonist-induced internalization has been investigated. Incubation of SH-SY5Y neuroblastoma cells with oxotremorine-M resulted in a time-dependent endocytosis of both muscarinic receptors and alpha subunits of G0 and G11, but not of isoforms of phosphoinositide-specific phospholipase C, into a subfraction of smooth endoplasmic reticulum (V1). Agonist-induced increases in diacylglycerol mass and in 32P-phosphatidate labeling, much of which was of the tetraenoic species, were also observed in the V1 fraction, but these increases persisted when the agonist-induced translocation of receptors into the V1 fraction was blocked. All enzymes of the phosphoinositide cycle were detectable in the V1 fraction. However, with the exception of phosphatidylinositol 4-kinase, none was enriched when compared with cell lysates. Both 32P-labeling studies and enzyme assays point to a very limited capacity of this fraction to synthesize phosphatidylinositol 4,5-bisphosphate, whereas the synthesis of phosphatidylinositol 4-phosphate is robust. These results indicate that endocytosed receptors do not appear to retain their ability to activate phosphoinositide turnover. The availability of the substrate for phospholipase C, phosphatidylinositol 4,5-bisphosphate, may be one factor that limits the activity of muscarinic receptors in this subcellular compartment.
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PMID:Agonist-induced endocytosis of muscarinic cholinergic receptors: relationship to stimulated phosphoinositide turnover. 908 17

Xenopus laevis oocytes showed no electrophysiological responses to acetylcholine (ACh) and had no significant cholinergic receptor sites when prepared under our conditions. However, they were found to acquire robust electrophysiological responsiveness to ACh when bovine GL2 alpha, which is a member of the Gq alpha family and is highly homologous to mouse G11 alpha, was expressed by mRNA injection. Further analyses indicated that GL2 alpha amplified the activity of endogenous muscarinic ACh receptors that are expressed at an otherwise undetectable level, and thus made their detection possible. Thus, GL2 alpha may prove to be an effective method for detecting the activities of phospholipase C-linked receptors which are only marginally expressed. The usefulness of this method was confirmed in the analyses of a chimeric receptor constructed from metabotropic glutamate receptor subtype 1 alpha and muscarinic ACh receptor subtype M1. The chimeric receptor showed no electrophysiological responses to ACh when expressed alone in oocytes, but became responsive to ACh when co-expressed with GL2 alpha.
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PMID:The G alpha protein GL2 alpha improves the ability to detect the subthreshold expressions of receptors linked to phospholipase C in Xenopus oocytes. 915 44

We investigated the muscarinic activation of Ca(2+)-activated Cl- currents [ICl(Ca)] in voltage-clamped equine tracheal myocytes. The threshold of cytosolic free Ca2+ concentration ([Ca2+]i) required for activation of ICl(Ca) was 202 +/- 22 nM, and full activation of the current occurred at 771 +/- 31 nM. Hexahydro-sila-difenidol (M3 antagonist) inhibited the methacholine-induced phasic [Ca2+]i increase and ICl(Ca) in a concentration-dependent manner, whereas methoctramine (M2 antagonist) only slightly attenuated the [Ca2+]i increase and ICl(Ca) (14.8 and 21.4%, respectively), consistent with incomplete selectivity. Dialysis of heparin (10 mg/ml) blocked methacholine-induced [Ca2+]i and ICl(Ca) but had no effect on the caffeine-induced Ca2+ release or ICl(Ca); inositol 1,4,5-trisphosphate (100 microM) induced ICl(Ca) and blocked the methacholine current. Conversely, ruthenium red (50 microM) prevented the caffeine-induced [Ca2+]i release and ICl(Ca) but had no effect on methacholine-induced [Ca2+]i or current. Intracellular dialysis of the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7, 500 microM) or the Ca2+/calmodulin-dependent protein kinase inhibitor KN93 (5 microM) had no effect on the [Ca2+]i increase or ICl(Ca). Pertussis toxin (0.5 mg/ml) did not affect the increase in [Ca2+]i or ICl(Ca). Dialysis with antibodies directed against the alpha-subunit of Gq/G11 (Gq alpha/ G alpha 11) blocked the methacholine-induced ICl(Ca) in a concentration-dependent manner, whereas anti-G alpha i-1/G alpha 1-2 antibodies (1:35) and anti-G alpha i-3/G(o) alpha antibodies (1:35) were without effect. The results indicate that stimulation of phospholipase C via M3/Gq proteins is the predominant signaling pathway for the activation of ICl(Ca); at high agonist concentrations, Ca(2+)-induced Ca2+ release does not appear to play a prominent role in muscarinic signaling.
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PMID:Muscarinic signaling pathway for calcium release and calcium-activated chloride current in smooth muscle. 927 48

Activating mutations of the TSH receptor and alpha-subunit of Gs (G alpha s) that increase adenylyl cyclase activity have been identified in a subset of hyperfunctioning benign thyroid follicular adenomas and, less commonly, in hypofunctioning adenomas and carcinomas. In addition some thyroid tumors exhibit inappropriate activation of phospholipase C (PLC), a signaling pathway that has been implicated in the growth and dedifferentiation of thyroid cells. We therefore hypothesized that some thyroid tumors might be caused by somatic mutations in the genes encoding the alpha-chain of Gq or G11 that result in constitutive activation of the PLC pathway. We amplified regions of the alpha q and alpha 11 genes that encode amino acids, Q209 and R183, and we screened the DNA for mutations by sequence analysis and denaturing gradient gel electrophoresis. No mutations were identified after analysis of DNA from 38 thyroid tumors and 2 poorly differentiated thyroid carcinoma cell lines, including: 13 follicular adenomas, 10 follicular carcinomas, 5 papillary carcinomas, and 10 hyperplastic nodules from multinodular goiters. We conclude that activating mutations of alpha q and alpha 11 are absent or rare in hypofunctioning thyroid neoplasms and that other mechanisms must explain the elevated PLC activity reported in thyroid carcinoma.
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PMID:Absence of activating mutations of the genes encoding the alpha-subunits of G11 and Gq in thyroid neoplasia. 946 74


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