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:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel mutant of the LLC-PK1 renal epithelial cell line, VPR1, was isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and selection using a photoactivatable vasopressin analogue [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine] vasopressin. The VPR1 mutant cell line possessed less than 5% parental V2 receptor binding for vasopressin but exhibited normal calcitonin receptor binding. In contrast to LLC-PK1 cells (wild type), VPR1 cells exhibited no response to vasopressin in terms of in vitro adenylate cyclase activation, in vivo cAMP production, or urokinase-type plasminogen activator induction. The responses of VPR1 cells to other agents, such as calcitonin, the adenylate cyclase activator forskolin, the GTP analogue guanosine 5'-[beta, gamma-imino] triphosphate, 8-bromo adenosine-3',5'-monophosphate were comparable to those of the parental cell line. Somatic cell hybrids were derived from the cell lines LLC-PK1 and VPR1 and analyzed for the dominance/recessiveness of the VPR1 mutant phenotype. Hybrids were found to possess normal vasopressin binding activity as well as functional responses to the hormone, indicating that the mutation affecting the V2 receptor in VPR1 cells is recessive. The VPR1 cell line may thus have application as a recipient for the expression of the V2 receptor gene using DNA-transfer.
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PMID:Isolation and genetic characterization of a renal epithelial cell mutant defective in vasopressin (V2) receptor binding and function. 164 58

In the porcine renal epithelial cell line, LLC-PK1, activation of the cAMP-dependent signal transduction pathway induces the urokinase-type plasminogen activator (uPA) gene. We show here that the cAMP response is enhanced when the intracellular calcium concentration is increased. When LLC-PK1 cells were treated with the calcium ionophore ionomycin alone, there was no uPA mRNA accumulation. However, in the presence of ionomycin the dose-response of 8-bromo-cAMP (Br-cAMP) with respect to uPA mRNA accumulation was shifted toward the lower concentrations of Br-cAMP. A Northern blot analysis after the inhibition of RNA synthesis and nuclear run-on assays showed that the synergistic effect of Ca2+ could be attributed to increases in uPA gene transcription and mRNA stability. In the presence of cycloheximide, a protein synthesis inhibitor, uPA mRNA was stabilized, but the effect of ionomycin on Br-cAMP-induced mRNA accumulation was still maintained. The result suggests that the Ca2+, at least on transcription, does not require new protein synthesis. Ionomycin treatment did not modify the activity of the cAMP-dependent protein kinase, suggesting that Ca2+ either affects a step in the pathway between the kinase and the uPA gene, or acts independently of the cAMP-dependent protein kinase pathway. The effect of ionomycin was not suppressed by protein kinase C down-regulation nor by inhibitors of calmodulin. Synergism was also observed when Br-cAMP was replaced with calcitonin, a peptide hormone which is coupled to adenylate cyclase, and when ionomycin was replaced with another ionophore A23187, suggesting that the synergism is due to an interaction between cAMP-dependent and Ca2(+)-dependent signal transduction pathways.
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PMID:Ca2+ potentiates cAMP-dependent expression of urokinase-type plasminogen activator gene through a calmodulin- and protein kinase C-independent mechanism. 170 Nov 76

Plasminogen activator inhibitor-1 (PAI-1) inhibits the tissue plasminogen activator (tPA) and urokinase activation of plasminogen to plasmin, a protease of trypsin-like specificity which is involved in a number of processes, including fibrinolysis, matrix degradation and angiogenesis. Both phorbol esters and cAMP elevating compounds have been shown to modulate PAI-1 and tPA expression in endothelial cell culture. HBGF-1 (previously designated endothelial cell growth factor) stimulates endothelial cell growth in vitro and is angiogenic in vivo. We have reported that removal of HBGF-1 from human umbilical vein endothelial cell (HUVEC) media results in an approximately 5-fold increase in PAI-1 mRNA levels and in PAI-1 protein secreted into the media by 20 h. Here we report the effects of HBGF-1 on the phorbol ester and cAMP modulation of HUVEC PAI-1 expression. The phorbol ester PMA induced an approximate 5-fold increase in PAI-1 mRNA levels at 4 h, which returned to base line by 20 h, with or without HBGF-1 present in the media. This increase in PAI-1 mRNA levels was mediated by an increase in PAI-1 gene transcription and was abated in the presence of cycloheximide. Treatment of cells with the adenylate cyclase activator forskolin or the phosphodiesterase inhibitor HL 725, in the presence of HBGF-1 or immediately after its withdrawal, decreased PAI-1 mRNA levels and protein secreted into the conditioned media by 20 h. However, forskolin or HL 725 addition had little or no effect on PAI-1 mRNA when added 20 h after HBGF-1 withdrawal. Both the PMA and HBGF-1 modulation of PAI-1 were abolished by treatment with the protein kinase inhibitor H-7. Treatment of HUVEC with HBGF-1 had no acute effect on intracellular inositol phosphate hydrolysis or cAMP levels. Further studies on intracellular pathways involved in HBGF-1 modulation of PAI-1 will enhance our understanding of the role these factors play in cellular proliferation and angiogenesis.
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PMID:Heparin-binding growth factor-1 modulation of plasminogen activator inhibitor-1 expression. Interaction with cAMP and protein kinase C-mediated pathways. 170 36

Expression of the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells can be induced by signals mediated by both cAMP-dependent protein kinase (PKA) and Ca(2+)- and phospholipid-dependent protein kinase (PKC). We have utilized the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) to down-regulate PKC, in order to test for an effect on the PKA-mediated induction of the uPA gene expression. Incubation of cells for 24 h with 100 ng/ml TPA caused a marked decrease of PKC protein, both in cytosolic and particulate fractions, and an 85% reduction of total PKC activity. After down-regulation of PKC, uPA mRNA accumulation induced by 8-Br-cAMP was 5-10-fold higher than in control cells. Both uPA mRNA stability and uPA gene transcription rates induced by 8-Br-cAMP were increased by PKC down-regulation (6- and 1.8-fold, respectively). Although total PKA activity was reduced by 20% in extracts from PKC-depleted cells, activation of PKA by 8-Br-cAMP was 2.5-fold higher than in control cells. This enhanced activation of PKA in PKC-depleted cells also occurred in response to other cAMP derivatives and to cAMP induced endogenously by the activation of adenylate cyclase with forskolin, but was not due to down-regulation-associated changes in the rate of cAMP synthesis. Our results demonstrate that in LLC-PK1 cells, down-regulation of PKC results in an enhanced induction of uPA gene expression by cAMP-mediated signals without alterations in adenylate cyclase activity, suggesting a mechanism distal to adenylate cyclase.
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PMID:Protein kinase C down-regulation enhances cAMP-mediated induction of urokinase-type plasminogen activator mRNA in LLC-PK1 cells. 171 70

The precise mechanistic role of the cAMP-dependent protein kinase (cAMP-PK) in cAMP-mediated gene induction remains unclear. Renal epithelial cell mutants were compared to the LLC-PK1 parental cell line for induction of the cAMP-responsive urokinase-type plasminogen activator (uPA) gene, as quantitated by the technique of mRNA solution hybridization. The FIB4 and FIB6 mutants, which possess less than 10% parental cAMP-PK catalytic (C) subunit activity, showed markedly diminished uPA mRNA induction in response to agents elevating intracellular cAMP such as the cAMP analogue 8-bromo-cAMP and the adenylate cyclase-stimulating hormones vasopressin and calcitonin. In contrast, the mutant cells responded to a similar or greater extent than the parental cells in terms of uPA mRNA induction following treatment with the Ca2+/phospholipid-dependent protein kinase activator phorbol 12-myristate 13-acetate (PMA). Elevation of intracellular cAMP was found to induce a translocation of the cAMP-PK C subunit from the perinuclear Golgi region to the nucleus in both parental and mutant cell lines, as shown by immunocytochemical techniques. Results argue for the role of the cAMP-PK C subunit activity and possibly nuclear translocation of the C subunit in cAMP-mediated uPA induction, which is mechanistically distinct from the PMA-stimulated response.
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PMID:Mechanisms of cAMP-mediated gene induction: examination of renal epithelial cell mutants affected in the catalytic subunit of the cAMP-dependent protein kinase. 189 92

Pro-opiomelanocortin-derived peptides, alpha-MSH and beta-endorphin, are synthesized and secreted by Leydig cells, and are believed to have paracrine effects on Sertoli cells in the testis. Peptides with MSH activity stimulate adenylate cyclase and cAMP accumulation in Sertoli cell-enriched cultures. The purpose of the present study was to determine whether such peptides would affect Sertoli cell parameters, such as aromatase and plasminogen activator activities, that are known to be regulated by cAMP. alpha-MSH stimulated aromatase activity in Sertoli cell-enriched cultures prepared from 10-day-old rats and this effect was potentiated by methyl isobutylxanthine (MIX). The combination of alpha-MSH plus MIX was not as potent as FSH. alpha-MSH, des-acetyl-alpha-MSH, beta-MSH, ACTH(1-13), and ACTH(1-24) stimulated aromatase activity to a similar extent, suggesting that Sertoli cells do not distinguish between the activities of these peptides. alpha-MSH potentiated the action of dbcAMP and forskolin on Sertoli cell aromatase, but unexpectedly had no effect on the action of either half-maximal or maximal doses of FSH. The regulation of plasminogen activator was examined next; urokinase was markedly suppressed by FSH in 10-day-old Sertoli cells. Although neither alpha-MSH nor MIX alone had an effect on urokinase secretion, in combination they were as effective as FSH. In 10-day-old Sertoli cells each of these peptides had little or no effect on tissue plasminogen activator.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Estradiol and plasminogen activator secretion by cultured rat Sertoli cells in response to melanocyte-stimulating hormones. 247 57

A cosmid (cos pUK0322) harboring the complete human urokinase-type plasminogen activator (u-PA) gene and Geneticin resistance as a selectable marker was isolated from a human genomic library and characterized. After transfection of cos pUK0322 into mouse L cells and selection, several plasminogen activator (PA)-expressing clones were obtained and one (LuPA) was chosen for additional study. The PA expressed was identical to human pro-u-PA in enzymatic, electrophoretic, and antigenic properties. The expression of PA was stable over 50 population doublings. The regulation of the transfected gene was studied by treatment of the cells with various hormones and other effectors. Expression of PA activity was inhibited fivefold by dexamethasone and stimulated two- to threefold by agonists of the adenylate cyclase dependent pathway of signal transduction, such as dibutyryl cyclic AMP and cholera and pertussis toxins. The modulation of PA activity was associated with corresponding changes in mRNA steady-state levels. The phenotypic changes associated with pro-u-PA expression were analyzed in vitro by degradation of 3H-labeled extracellular matrix (ECM), invasion of a matrigel basement membrane analogue, and by light and electron microscopy. LuPA cells and reference HT-1080 fibrosarcoma cells, in contrast to control Lneo cells transfected with the neomycin resistance gene, degraded the ECM and invaded the matrigel basement membrane. Matrix degradation correlated with the modulation of pro-u-PA gene expression as it was inhibited by dexamethasone and promoted by dibutyryl cyclic AMP. Inhibition of PA or plasmin using anti-u-PA IgG or aprotinin prevented ECM degradation and invasion. These results demonstrate that u-PA expression alone is sufficient to confer to a cell an experimental invasive phenotype.
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PMID:Mouse L cells expressing human prourokinase-type plasminogen activator: effects on extracellular matrix degradation and invasion. 250 27

Heterologous gene expression in yeast can be increased up to several hundred-fold by expressing a foreign gene as a fusion to the ubiquitin gene. An endogenous yeast endoprotease (Ub-Xase) removes the ubiquitin from the fusion product to produce the authentic protein. The utility of this technique has been demonstrated by expression of three different proteins in yeast as both unfused and ubiquitin-fused forms: 1) the alpha subunit of the mammalian stimulating G-protein of the adenylate cyclase complex (Gs alpha); 2) a soluble fragment of the T cell receptor protein (sCD4); and 3) the protease domain of human urokinase (UKP). The sequence specificity of the Ub-Xase was demonstrated by mutagenesis of the carboxyl-terminal glycine of ubiquitin to an alanine, which inhibited ubiquitin removal in vivo. Processing of the ubiquitin-Gs alpha fusion protein (ub-Gs alpha) in vivo resulted in Gs alpha which could be reconstituted in mammalian membrane preparations and had the same specific activity as the authentic Gs alpha expressed in yeast. The yeast Ub-Xase has also been shown to work in vitro by the processing of a ub-sCD4 fusion protein synthesized in Escherichia coli. This technology should greatly enhance the utility of yeast for heterologous protein production.
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PMID:Increasing gene expression in yeast by fusion to ubiquitin. 254 Feb 2

The activation of cyclic AMP-dependent protein kinase (cAMP-PK) in vivo was studied in LLC-PK1 pig kidney cells and the mutant cell lines M18 and FIB5, which have total levels of cAMP-PK catalytic-subunit and regulatory-subunit activities comparable with those of parental cells. The extent of cAMP-PK activation (release of active catalytic subunit from the holoenzyme) was directly correlated with the cellular cyclic AMP concentration in LLC-PK1 cells. In LLC-PK1 cells, as well as in the mutants M18 and FIB5, the extent of the induction of urokinase-type plasminogen activator (uPA) by the cyclic AMP-mediated effectors calcitonin, vasopressin and forskolin was directly correlated with the levels of activated catalytic subunit. The 'receptorless' mutant M18, which is impaired in calcitonin- and vasopressin-receptor function, did not show any activation of cAMP-PK or uPA production in response to either hormone, whereas cAMP-PK and uPA responses to forskolin were about 35% higher than in parental cells. Analysis of the FIB5-cell line revealed a lesion affecting the regulation of adenylate cyclase activity, whereby basal and stimulated (both receptor- and non-receptor-mediated) adenylate cyclase levels were less than 36% of those in parental cells. The activation of cAMP-PK in response to cyclic AMP effectors was similarly reduced, and uPA induction was concomitantly lower than that in parental cells. The results demonstrate the dependence of uPA induction by cyclic AMP effectors on dissociation of the cAMP-PK holoenzyme, implying the importance of activated free cAMP-PK catalytic subunit in this process. Thus it is concluded that the mutations in the cellular cyclic AMP-generating apparatus of the M18 and FIB5 cell lines impair uPA induction by preventing cAMP-PK activation.
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PMID:Dependence of urokinase-type-plasminogen-activator induction on cyclic AMP-dependent protein kinase activation in LLC-PK1 cells. 282 Mar 80

A mutant LLC-PK1 cell line, M18, was isolated after a single treatment of the parent culture with N-methyl-N'-nitro-N-nitroso-guanidine. In contrast to LLC-PK1 cells, the mutant did not exhibit production of urokinase-type plasminogen activator (uPA) in response to the hormones calcitonin and vasopressin, but produced the expected levels of uPA upon stimulation by the receptor-independent adenylate cyclase activators forskolin and cholera toxin, as well as by the phosphodiesterase inhibitor isobutylmethylxanthine and the 8-bromo analogue of adenosine cyclic monophosphate, Br8cAMP. The patterns of activation of cAMP-dependent protein kinase were identical to those of uPA induction: calcitonin and vasopressin were without effect, but the response to all other agents was normal. In similar fashion, mutant cell homogenates displayed normal activation of adenylate cyclase upon treatment with sodium fluoride, forskolin, or the non-hydrolyzable GTP analogue guanosine 5'-[beta, gamma-imino]triphosphate, but were unresponsive to calcitonin or vasopressin. The ability of M18 cells to bind radioactively labelled calcitonin and vasopressin was measured. The mutant possessed less than 4% of the normal levels of the receptor binding activity for both hormones. Somatic cell hybrids formed between M18 and LLC-PK1 cells were found to retain normal hormone binding activity and responsiveness to hormones, indicating that the defect in M18 cells was recessive. M18 was concluded most probably to contain a single mutation impairing the function of two distinct polypeptide hormone receptors.
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PMID:Isolation of a mutant LLC-PK1 cell line defective in hormonal responsiveness. A pleiotropic lesion in receptor function. 302 58


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