Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The melanocortin receptors, MC3R and MC4R, are G protein-coupled receptors that are involved in regulating energy homeostasis. Using a luciferase reporter gene under the transcriptional control of a cAMP- responsive element (CRE), the coupling efficiency of the MC4R and MC3R to G-proteins was previously shown to be different. MC4R exhibited only 30-50% of the maximum activity induced by MC3R. To assess the role of the different MC3R and MC4R domains in G-protein coupling, several chimeric MC3R/MC4R receptors were constructed. The relative luciferase activities, which were assessed after transfecting the chimeric receptors into HEK 293T cells, showed that the i3 (3rd intracellular) loop domain has an essential role in the differential signaling of MC3R and MC4R. To reveal which amino acid residue was involved in the MC4R-specific signaling in the i3 loop, a series of mutant MC4Rs was constructed. Reporter gene analysis showed that single mutations of Arg(220) to Ala and Thr(232) to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg(220) and Thr(232), in the i3 loop of MC4R play crucial roles in G-protein coupling and the subtype-specific signaling pathways. An examination of the inositol phosphate (IP) levels in the cells transfected with either MC3R or MC4R after being exposed to the melanocortin peptides revealed significant stimulation of IP production by MC3R but no detectable increase in IP production was observed by MC4R. Furthermore, none of the MC4R mutants displayed melanocortin peptide-stimulated IP production. Overall, this study demonstrated that MC3R and MC4R have distinct signaling in either the cAMP- or the inositol phospholipid-mediated pathway with different conformational requirements.
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PMID:Identification of domains directing specificity of coupling to G-proteins for the melanocortin MC3 and MC4 receptors. 1204 90

Protein kinase Cdelta (PKCdelta) is a member of the PKC family of phospholipid-dependent serine/threonine kinases and is involved in cell proliferation, apoptosis, and differentiation. Previous studies have suggested that different PKC isoforms might be translationally regulated. We report here that the 395-nt-long 5' untranslated region (5' UTR) of PKCdelta is predicted to form very stable secondary structures with free energies (deltaG values) of around -170 kcal/mol. The 5' UTR of PKCdelta can significantly repress luciferase translation in rabbit reticulocyte lysate but does not repress luciferase translation in a number of transiently transfected cell lines. By using a bicistronic luciferase reporter, we show that the 5' UTR of PKCdelta contains a functional internal ribosome entry segment (IRES). The activity of the PKCdelta IRES is greatest in densely growing cells and during apoptosis, when total protein synthesis and levels of full-length eukaryotic initiation factor 4G are reduced. However, the IRES activity of the 5' UTR of PKCdelta is not enhanced during serum starvation, another condition shown to inhibit cap-dependent translation, suggesting that its potency is dependent on specific cellular conditions. Accumulating data suggest that PKCdelta has a function as proliferating cells reach high density and in early and later events of apoptosis. Our studies suggest a mechanism whereby PKCdelta synthesis can be maintained under these conditions when cap-dependent translation is inhibited.
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PMID:The 5' untranslated region of protein kinase Cdelta directs translation by an internal ribosome entry segment that is most active in densely growing cells and during apoptosis. 1216 3

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) activates the aryl hydrocarbon receptor (AhR) to mediate transcriptional activity of dioxin-responsive genes. The transactivation domain (TAD) of human AhR (hAhR) has potentially distinct acidic, glutamine-rich, and proline/serine/threonine-rich subdomains. Cotransfection of exogenous hAhR into BP8 cells with isolated subdomains of hAhR TAD fused to glutathione S-transferase exhibited squelching of TCDD-dependent dioxin-response element (DRE)-driven luciferase reporter-gene activity with each subdomain. To study the potential cross talk between AhR- and estrogen receptor (ER)-mediated activities, BP8 cells were cotransfected with hAhR TAD subdomain constructs and ERalpha. The three hAhR TAD subdomains inhibited the 17beta-estradiol-induced estrogen-response element-mediated reporter-gene transactivation. Cotransfection of hAhR with the ligand-binding domain (LBD) of ERalpha also squelched TCDD-dependent DRE-driven reporter-gene activity in the presence of 17beta-estradiol. Similar results were observed in T47D cells that express functional AhR and ERalpha. These results indicate that the isolated subdomains of hAhR's TAD and LBD of ERalpha are capable of squelching ligand-dependent transactivation of either the AhR or the ER, by titrating crucial proteins from an existing common pool of cofactors.
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PMID:The subdomains of the transactivation domain of the aryl hydrocarbon receptor (AhR) inhibit AhR and estrogen receptor transcriptional activity. 1248 7

The two forms (DTyr8 and LTyr8) of the putative somatostatin sst2 receptor antagonist CYN 154806 (Ac-4NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-D/LTyr-NH2) were investigated on recombinant human somatostatin receptors and endogenous guinea-pig ileum receptors. In radioligand binding studies using the agonist radioligands [125I]LTT-SRIF-28, [125I][Tyr10]cortistatin-14, [125I]CGP 23996 and [125I][Tyr3]octreotide in Chinese hamster lung fibroblast (CCL39) and Chinese hamster ovary (CHO) cells expressing human somatostatin receptors (hsst1-5), CYN 154806 binds to sst2 receptors with nanomolar affinity (pKD=8.14-8.89), 40- to 4500-fold higher than for sst1, sst3 or sst4. High affinity was also demonstrated for sst5 receptors, particularly for LTyr8CYN 154806 where the sst5 affinity was higher than for sst2 receptors when using [125I]CGP 23996 and [125I][Tyr3]octreotide. Functional properties of the compounds were examined in Chinese hamster ovary (CHO) cells expressing human sst2 receptors, in (1) inhibition of forskolin-stimulated adenylate cyclase, (2) stimulation of serum response element-driven luciferase expression and (3) [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPS) binding. L- and DTyr8CYN 154806 showed full agonism at inhibition of forskolin-stimulated cAMP accumulation (pEC50=7.73 for both, Emax 104% and 78%, respectively), partial agonism at luciferase expression (pEC50=7.85 and 8.16, Emax=50% and 29%, respectively) and behaved as apparently silent antagonists at [35S]GTPS binding (no agonism observed, pKB=6.88 and 7.50, respectively). The agonist potential was confirmed in isolated guinea-pig ileum preparations via measurement of SRIF-induced inhibition of neurotransmission, where the L-isoform had marked agonism (pEC50=8.23, Emax=32%) whereas the D-isoform was apparently devoid of agonism. The present data suggest that CYN 154806 should be used with caution as an sst2 receptor antagonist tool, since it possesses intrinsic activity at sst2, and high affinity for both sst2 and sst5 receptors. The DTyr form, having lower intrinsic activity, especially in natural tissues, and greater selectivity for sst2 receptors, may be more reliable than LTyr CYN 154806.
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PMID:Functional characterisation of the putative somatostatin sst2 receptor antagonist CYN 154806. 1261 35

Tyrosine phosphorylation of the aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix/PER-ARNT-SIM transcription factor family, has been shown to regulate its dioxin response elements (DRE) binding ability, although no specific residues have been directly demonstrated to be phosphorylated. Of the 23 tyrosines in the mouse AhR, 19 are conserved across all mammalian species sequenced thus far. The studies presented here were conducted to examine tyrosine residue(s) that are both likely candidates of phosphorylation and necessary for DNA binding and/or transcriptional activity of the AhR. Two-dimensional gel electrophoresis of phosphatase-treated AhR indicated that the receptor is phosphorylated on serine/threonine and tyrosine residues. Computational analysis predicted several highly conserved tyrosine residues to be phosphorylated. Both the N terminus (amino acids 1-399) and the C terminus (amino acids 399-805) of the mouse receptor synthesized in vitro using a rabbit reticulocyte lysate system are tyrosine phosphorylated as detected by antiphosphotyrosine antibodies. Furthermore, the N-terminal AhR bound DRE in a ligand-dependent manner similar to that by the full-length receptor, suggesting that phosphorylated tyrosines involved in DNA binding are likely located in the region between residues 1 and 399. Mouse AhR tyrosine (Y) residues were evaluated by phenylalanine (F) mutational analysis for both DNA binding (electrophoretic mobility shift assays; EMSAs) and ability to induce a DRE-driven reporter gene in transiently transfected AhR-deficient cells. Of the 12 tyrosine residues in the N-terminal AhR, only a tyrosine 9 mutant (AhRY9F) significantly decreased DRE binding as determined by EMSA. Similarly, only the AhRY9F mutant decreased the DRE-driven luciferase expression in AhR-deficient cells. Overall, these data strongly suggest that the putative posttranslational modification at, or mediated by, tyrosine 9, and not any other individual mouse AhR tyrosine residue, is necessary for AhR DRE binding and transcriptional activity.
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PMID:Mutational analysis of the mouse aryl hydrocarbon receptor tyrosine residues necessary for recognition of dioxin response elements. 1264 72

The availability of antagonist ligands for somatostatin receptors is very limited, with those that are available often displaying agonist properties or limited receptor subtype selectivity. Hay et al. [Bioorg. Med. Chem. Lett. 11 (2001) 2731] recently described the development of small-molecule somatostatin receptor subtype 2 (sst(2)) selective compounds. This study investigates the binding affinity and functional characteristics of two of those antagonists (2 and 3) and the agonist compound, from which they were derived (1). In radioligand binding studies using the agonist radioligands [125I][Tyr(11)]SRIF-14 (Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Thr-Ser-Cys]-OH), [125I]LTT-SRIF-28 ([Leu(8),DTrp(22),125I-Tyr(25)]SRIF-28; Ser-Ala-Asn-Ser-Asn-Pro-Ala-Leu-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-DTrp-Lys-Thr-(125I-Tyr)-Thr-Ser-Cys]-OH), [125I]CGP 23996 (c[Lys-Asu-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Thr-Ser]), [125I][Tyr(3)]octreotide (DPhe-c[Cys-(125I-Tyr)-DTrp-Lys-Thr-Cys]-Thr-OH) and [125I][Tyr(10)]cortistatin-14 (Pro-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Ser-Ser-Cys]-Lys) at human recombinant somatostatin receptors expressed in Chinese hamster lung fibroblast (CCL39) cells and native rat cortex, the compounds bound with high affinity (pK(d) 6.8-9.7) and selectivity to human sst(2) receptors. Some affinity was also observed for sst(5) labelled by [125I][Tyr(3)]octreotide and [125I]CGP 23996. In functional studies at human sst(2) receptors expressed in Chinese hamster ovary (CHO) cells, both the agonist 1 and the two putative antagonists 2 and 3 concentration dependently inhibited forskolin-stimulated adenylate cyclase and stimulated luciferase reporter gene expression, with similar efficacy to the natural ligand somatotropin release inhibiting factor (SRIF)-14. Compound 1 had similar potency to SRIF-14, which was in the nanomolar range, whereas 2 and 3 were 10-100-fold less potent. The intrinsic activity of 2 and 3 was too high to allow antagonist studies to be carried out. In conclusion, in contrast to previous findings, all three compounds are potent agonists at recombinant human sst(2) receptors.
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PMID:Agonist properties of putative small-molecule somatostatin sst2 receptor-selective antagonists. 1268 32

Previous studies have demonstrated that the serine/threonine protein phosphatase 2A (PP2A) can modulate the transcriptional activity of several sequence-specific DNA-binding proteins. However, less is known about the effect of PP2A on the activities of general transcription factors and transcriptional coregulators. Here we describe that the activity of a general coactivator, the four-and-a-half-LIM-only protein 2 (FHL2), is regulated in a PP2A-dependent manner. Specific inhibition of PP2A by simian virus 40 (SV40) small t-antigen (st-ag) stimulated the intrinsic transcriptional activity of FHL2 more than 10-fold, while a st-ag mutant unable to bind PP2A had no effect. Overexpression of the B56 subunits alpha, beta, and gamma1 of PP2A impaired the induction of FHL2 by st-ag. FHL2 functioned as a coactivator for CREB-mediated transcription, and inactivation of PP2A further increased FHL2-induced CREB-directed transcription. Overexpression of FHL2 readily enhanced the transcription of the luciferase reporter gene driven by the c-fos promoter, and inhibition of PP2A further stimulated FHL2-induced transactivation of this promoter. These results suggest that dephosphorylation of the general coactivator FHL2 may represent a novel mechanism by which PP2A modulates the transcription of FHL2-responsive genes.
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PMID:Activation of the coactivator four-and-a-half-LIM-only protein FHL2 and the c-fos promoter through inhibition of protein phosphatase 2A. 1269 72

The robust induction of metallothionein-I and II (MT-I and MT-II) genes by several heavy metals such as zinc and cadmium requires the specific transcription factor metal-responsive transcription factor 1 (MTF1). Chromium (VI), a major environmental carcinogen, not only failed to activate these genes but also inhibited their induction by Zn2+ or Cd2+. The heavy metal-induced expression of another MTF1 target gene, zinc transporter 1 (ZnT-1), was also down-regulated by Cr6+. By contrast, the expression of two MTF1-independent Cd2+-inducible genes, heme oxygenase 1 (HO-1) and HSP-70, was not sensitive to Cr6+. Cr6+ did not also affect the expression of housekeeping genes such as GAPDH or beta-actin. Stable cell lines overexpressing variable levels of MTF1, the key transactivator of the MT genes, demonstrated differential resistance toward the inhibitory effect of Cr6+, indicating MTF1 as a target of chromium toxicity. The basal and inducible binding of MTF1 to metal response elements was not affected by treatment of cells with Cr6+. Transient transfection studies showed that the ability of MTF1 to transactivate the MT-I promoter was significantly compromised by Cr6+. The fusion protein consisting of a Gal-4 DNA binding domain and one or more of the three transactivation domains of MTF1, namely the acidic domain, proline-rich domain, and serine-threonine rich domain, activated the GAL-4-driven luciferase gene to different degrees, but all were sensitive to Cr6+. MTF1 null cells were prone to apoptosis after exposure to Zn2+ or Cd2+ that was augmented in presence Cr6+, whereas the onset of apoptosis was significantly delayed in cells overexpressing MTF1.
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PMID:Chromium(VI) down-regulates heavy metal-induced metallothionein gene transcription by modifying transactivation potential of the key transcription factor, metal-responsive transcription factor 1. 1271 93

Calcineurin is a calcium-regulated serine-threonine protein phosphatase that controls developmental and inducible biological responses in diverse cell types, in part through activation of the transcription factor nuclear factor of activated T cells (NFAT). In skeletal muscle, calcineurin has been implicated in the regulation of myoblast differentiation, hypertrophy of mature myofibers, and fiber type switching in response to alterations in intracellular calcium concentration. However, considerable disagreement persists about the functional role of calcineurin signaling in each of these processes. Here we evaluated the molecular phenotypes of skeletal muscle from both calcineurin Aalpha and calcineurin Abeta gene-targeted mice. Calcineurin Aalpha was observed to be the predominant catalytic isoform expressed in nearly all skeletal muscles examined. Neither calcineurin Aalpha or Abeta null mice showed any gross growth-related alterations in skeletal muscle, nor was fiber size or number altered in glycolytic/fast muscle types. In contrast, both calcineurin Aalpha and Abeta gene-targeted mice demonstrated an alteration in myofiber number in the soleus, an oxidative/slow-type muscle. More significantly, calcineurin Aalpha and Abeta gene-targeted mice showed a dramatic down-regulation in the oxidative/slow fiber type program in multiple muscles (both slow and fast). Associated with this observation, NFAT-luciferase reporter transgenic mice showed significantly greater activity in slow fiber-containing muscles than in fast. However, only calcineurin Aalpha null mice showed a defect in NFAT nuclear occupancy or NFAT-luciferase transgene activity in vivo. Collectively, our results suggest that calcineurin signaling plays a critical role in regulating skeletal muscle fiber type switching but not hypertrophy. Our results also suggest that fiber type switching occurs through an NFAT-independent mechanism.
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PMID:Altered skeletal muscle phenotypes in calcineurin Aalpha and Abeta gene-targeted mice. 1277 74

Occupational exposure to asphalt fumes may pose a health risk. Experimental studies using animal and in vitro models indicate that condensates from asphalt fumes are genotoxic and can promote skin tumorigenesis. Enhanced activity of activator protein-1 (AP-1) is frequently associated with the promotion of skin tumorigenesis. The current study investigated the effect of exposure to asphalt fumes on AP-1 activation in mouse JB6 P+ epidermal cells and the skin of transgenic mice expressing the AP-1 luciferase reporter gene. Asphalt fumes were generated from a dynamic generation system that simulated road-paving conditions. Exposure to asphalt fumes significantly increased AP-1 activity in JB6 P+ cells as well as in cultured keratinocytes isolated from transgenic mice expressing AP-1 reporter. In addition, topical application of asphalt fumes by painting the tail skin of mice increased AP-1 activity by 14-fold. Exposure to asphalt fumes promoted basal as well as epidermal growth factor-stimulated anchorage-independent growth of JB6 P+ cells in soft agar. It activated phosphatidylinositol 3-kinase and induced phosphorylation of Akt at Ser-473/Thr-308, and concurrently activated downstream p70 S6 kinase as well as glycogen synthase kinase-3beta. Asphalt fumes transiently activated c-Jun NH2-terminal kinases without affecting extracellular signal-regulated kinases and p38 mitogen-activated protein kinases. Further study indicated that blockage of phosphatidylinositol 3-kinase activation eliminated asphalt fume-stimulated AP-1 activation and formation of anchorage-independent colonies in soft agar. This is the first report showing that exposure to asphalt fumes can activate AP-1 and intracellular signaling that may promote skin tumorigenesis, thus providing important evidence on the potential involvement of exposure to asphalt fumes in skin carcinogenesis.
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PMID:Exposure to asphalt fumes activates activator protein-1 through the phosphatidylinositol 3-kinase/Akt signaling pathway in mouse epidermal cells. 1294


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