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:1.14.14.3 (luciferase)
38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

By binding to agonist-activated G protein-coupled receptors (GPCRs), beta-arrestins mediate homologous receptor desensitization and endocytosis via clathrin-coated pits. Recent data suggest that beta-arrestins also contribute to GPCR signaling by acting as scaffolds for components of the ERK mitogen-activated protein kinase cascade. Because of these dual functions, we hypothesized that the stability of the receptor-beta-arrestin interaction might affect the mechanism and functional consequences of GPCR-stimulated ERK activation. In transfected COS-7 cells, we found that angiotensin AT1a and vasopressin V2 receptors, which form stable receptor-beta-arrestin complexes, activated a beta-arrestin-bound pool of ERK2 more efficiently than alpha 1b and beta2 adrenergic receptors, which form transient receptor-beta-arrestin complexes. We next studied chimeric receptors in which the pattern of beta-arrestin binding was reversed by exchanging the C-terminal tails of the beta2 and V2 receptors. The ability of the V2 beta 2 and beta 2V2 chimeras to activate beta-arrestin-bound ERK2 corresponded to the pattern of beta-arrestin binding, suggesting that the stability of the receptor-beta-arrestin complex determined the mechanism of ERK2 activation. Analysis of covalently cross-linked detergent lysates and cellular fractionation revealed that wild type V2 receptors generated a larger pool of cytosolic phospho-ERK1/2 and less nuclear phospho-ERK1/2 than the chimeric V2 beta 2 receptor, consistent with the cytosolic retention of beta-arrestin-bound ERK. In stably transfected HEK-293 cells, the V2 beta 2 receptor increased ERK1/2-mediated, Elk-1-driven transcription of a luciferase reporter to a greater extent than the wild type V2 receptor. Furthermore, the V2 beta 2, but not the V2 receptor, was capable of eliciting a mitogenic response. These data suggest that the C-terminal tail of a GPCR, by determining the stability of the receptor-beta-arrestin complex, controls the extent of beta-arrestin-bound ERK activation, and influences both the subcellular localization of activated ERK and the physiologic consequences of ERK activation.
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PMID:The stability of the G protein-coupled receptor-beta-arrestin interaction determines the mechanism and functional consequence of ERK activation. 1247 60

In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395nm) using appropriate dual channel luminometry instruments (e.g., Fusion Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein-Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of beta-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2:beta-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2:beta-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R:RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand-GPCR interactions which can be applied to ligand identification for orphan receptors.
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PMID:The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS). 1250 39

The proglucagon gene encodes pancreatic glucagon and the glucagon-like peptides, which exert diverse effects on nutrient absorption and assimilation. The therapeutic potential of glucagon-like peptide-1 (GLP-1) has fostered interest in development of cellular engineering approaches to augment endogenous intestinal-derived GLP-1 for the treatment of type 2 diabetes. We have used adenovirus technology to examine the potential roles of the transcription factors Cdx-2/3 and Pax-6 as activators of endogenous proglucagon gene expression in enteroendocrine cell lines and in nontransformed rat intestinal cells. Adenoviral-expressed Cdx-2/3 and Pax-6 activated proglucagon promoter-luciferase activity in baby hamster kidney (BHK) fibroblasts, HEK 293 cells, and enteroendocrine cell lines. Pax-6, but not Cdx-2/3, induced expression of the endogenous proglucagon gene in enteroendocrine cell lines, but not in heterologous fibroblasts. Furthermore, transduction of primary rat intestinal cell cultures in vitro, or the rat colonic epithelium in vivo, with Ad-Pax-6 activated endogenous proglucagon gene expression. These data demonstrate that Pax-6, but not Cdx-2/3, is capable of activating the endogenous proglucagon gene in both immortalized enteroendocrine cells and the nontransformed intestinal epithelium in vivo.
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PMID:Pax-6 activates endogenous proglucagon gene expression in the rodent gastrointestinal epithelium. 1254 Jun 17

We report a novel activating mutation (E604K) of the calcium-sensing receptor in a family with autosomal dominant hypocalcemia. Whereas all affected individuals exhibited marked hypocalcemia, some cases with untreated hypocalcemia exhibited seizures in infancy, whereas others were largely asymptomatic from birth into adulthood. The missense mutation E604K (G2182A; GenBank accession no. U20759), which affects an amino acid residue in the C terminus of the cysteine-rich domain of the extracellular head, cosegregated with hypocalcemia in all seven individuals for whom DNA was available. Two unaffected, normocalcemic members of the family did not exhibit the mutation. The molecular impact of the mutation on two key components of the signaling response was assessed in HEK-293 cells transiently transfected with cDNA corresponding to either the wild-type calcium-sensing receptor or the E604K mutation derived by site-directed mutagenesis. There was a significant leftward shift in the concentration response curves for the effects of extracellular Ca(2+) on both intracellular Ca(2+) mobilization (determined by aequorin luminescence) and MAPK activity (determined by luciferase expression). The C terminus of the cysteine-rich domain of the extracellular head may normally act to suppress receptor activity in the presence of low extracellular Ca(2+) concentrations.
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PMID:Autosomal dominant hypocalcemia: a novel activating mutation (E604K) in the cysteine-rich domain of the calcium-sensing receptor. 1257 88

Blood levels of the satiety hormone leptin are directly correlated to fat stores in obese and lean people. Therefore, leptin resistance is the logical explanation for the phenomenon of common obesity. However, the important question of whether or not the intrinsic leptin activity could differ between obese and lean people has not been examined before. In the present study, serum leptin activity was measured by an in vitro assay of leptin signaling in a modified culture of HEK-293 cells. The system is based on activation of a luciferase reporter gene through a leptin receptor-dependent activation of the signal transducer and activator of transcription (STAT3). Serum samples from 20 obese and 20 non-obese individuals with leptin levels ranging from 3 to 75 ng/ml, as determined by radioimmunoassay (RIA), were used. A high correlation was observed for each serum sample between leptin RIA values and leptin activity in the bioassay. The results indicate that obesity in the 20 obese patients among the 40 individuals examined cannot be accounted for by alterations in leptin activity in our assay. The assay system provides a tool to screen for possible rare cases exhibiting alteration in leptin activity either due to a change in leptin itself or through interaction with other serum factors.
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PMID:Serum leptin activity in obese and lean patients. 1260 52

A number of G protein-coupled receptors (GPCRs) have been shown to stimulate signal transducers and activators of transcription (STAT) activities while STAT3 activation by G alpha(o) can lead to neoplastic transformation in fibroblasts. In the present study we examined the ability of GPCRs to activate STAT3 via G alpha(16), a G alpha subunit which is primarily expressed in hematopoietic cells. In HEK 293 cells expressing a STAT3-driven luciferase reporter, the G alpha(16)-coupled ORL(1) and fMLP receptors stimulated luciferase activity upon activation by their agonists. Agonist-induced STAT3 activity required coexpression of G alpha(16) and was resistant to PTX treatment. Upon activation of the ORL(1) and fMLP receptors, phosphorylation of STAT3 at Tyr(705) was detected by immunoblot analysis. Additional experiments indicated that GPCR-mediated STAT3 activation was dependent on JAK and Raf1 signaling, but did not require phosphatidylinositol 3-kinase. This is the first study that demonstrates the stimulatory effect of ORL(1) and fMLP receptors on STAT3 activity.
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PMID:Regulation of STAT3 activity by G16-coupled receptors. 1267 Apr 99

Regulated expression systems are invaluable for studying gene function, offer advantages of dosage-dependent and temporally defined gene expression, and limit possible clonal variation when toxic or pleiotropic genes are overexpressed. Previously, establishment of inducible expression systems, such as tetracycline- and ecdysone-inducible systems, required assessment of the inducible characteristics of individual clones by tedious luciferase assays. Taking advantage of a green fluorescent protein (GFP) reporter controlled by tetracycline- or ecdysone-responsive element and fluorescence-activated cell sorting, we propose a simple and efficient strategy to select highly inducible cell lines according to their fluorescence profiles after transiently transfecting the candidate cell pools with a surrogate GFP reporter. We have demonstrated that tetracycline- and ecdysone-inducible systems could be set up in Madin-Darby canine kidney and HEK-293 cells by employing this selection scheme. Importantly, this dual regulatory expression system is applied in studying the complex interplay between two Ras-related small GTPases, Cdc42 and Rac1, on detachment-induced apoptosis. Furthermore, establishment of two tightly regulated expression systems in one target cell line could be of great advantage for dissecting small GTPase Rac1-transduced signaling pathways by using global gene expression approaches such as proteomic assays.
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PMID:An ecdysone and tetracycline dual regulatory expression system for studies on Rac1 small GTPase-mediated signaling. 1273 35

Insulin exerts its biological effects through a plasma membrane receptor that possesses a tyrosine-kinase activity. This tyrosine-kinase activity depends on the autophosphorylation of the receptor on tyrosine residues and on its dephosphorylation by protein tyrosine-phosphatases. The discovery of pharmacological agents that specifically stimulate the autophosphorylation of the insulin receptor or inhibit its dephosphorylation will be of great importance for the treatment of insulin resistant or insulin deficient patients. Bioluminescence Resonance Energy Transfer (BRET) has developed in recent years as a new technique to study protein-protein interactions. In the BRET technique, one partner is fused to Renilla luciferase, whereas the other partner is fused to a fluorescent protein (e.g. YFP, Yellow Fluorescent Protein). The luciferase is excited by addition of its substrate, coelenterazine. If the two partners interact, resonance energy transfer occurs between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, can be detected. Our work indicates that this methodology could be an important tool for the search of molecules that activate insulin receptor autophosphorylation or that inhibit its dephosphorylation. Indeed, we first showed that the activation of the insulin receptor by different ligands can be monitored using a chimeric receptor with one B-subunit fused to Renilla luciferase and the other B-subunit fused to YFP. The conformational changes induced by different ligands could be detected as an energy transfer (BRET signal) between the luciferase and the YFP, that reflects the activation state of the receptor. This methodology allows for rapid analysis of the effects of agonists on insulin receptor activity and may therefore be used in high-throughput screening for the discovery of molecules with insulin-like properties. More recently, we demonstrated that the BRET methodology could also be used to monitor the interaction of the insulin receptor with protein tyrosine-phosphatase 1B, one of the main tyrosine-phosphatase that controls its activity. HEK cells were co-transfected with the insulin receptor fused to Renilla luciferase and a substrate-trapping mutant of PTP1B (PTP1B-D181A) fused to YFP. Insulin-induced BRET signal could be followed in real time for more than 30 min. Therefore, this methodology can also be used in high-throughput screening for the search of molecules that will specifically disrupt the interaction between the insulin receptor and PTP1B.
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PMID:Looking for an insulin pill? Use the BRET methodology! 1274 30

The early response gene IEX-1 is involved in the regulation of cellular growth and survival, and its expression is related to stress-, growth- and death-inducing signals. Addressing the role of IEX-1 in the promotion of apoptosis, we investigated the effect of IEX-1 on nuclear factor-kappaB (NF-kappaB) activation. Stably transfected HEK-293 cells conditionally overexpressing IEX-1 exhibit decreased levels of NF-kappaB activity, either basal or TNFalpha induced, as shown by gel-shift and luciferase reporter gene assay. Furthermore, activated p65 accumulated in the nuclei of 293 cells to a lower degree, if IEX-1 expression was increased. This inhibited NF-kappaB activation was preceded by an altered turnover of IkappaBalpha and phospho-IkappaBalpha. In addition, IEX-1 expression also inhibited the activity of the 26S-proteasome, as shown by a fluorometric proteasome assay. Conversely, disruption of IEX-1 expression in 293 cells by stable transfection with specific anti-IEX-1 hammerhead ribozymes increased NF-kappaB activity, and accelerated the degradation of IkappaBalpha. Along with these opposite effects of IEX-1 expression and IEX-1 disruption on NF-kappaB activation, the sensitivity of 293 cells towards various apoptotic stimuli also changed. In contrast to ribozyme-transduced 293 cells that were significantly less sensitive to apoptosis, this sensitivity was enhanced if IEX-1 expression was increased. Our data suggest that IEX-1 - itself an NF-kappaB target gene - inhibits the activation of this transcription factor, and hereby may counteract the antiapoptotic potential of NF-kappaB.
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PMID:The early response gene IEX-1 attenuates NF-kappaB activation in 293 cells, a possible counter-regulatory process leading to enhanced cell death. 1276 4

The hyperprolificacy phenotype of Booroola ewes is due to the presence of the FecB(B) allele at the FecB locus, recently identified as a single amino acid substitution (Q249R) in the bone morphogenetic protein (BMP) type-IB receptor (BMPR1B), and is associated with a more precocious differentiation of ovarian granulosa cells (GCs). To evaluate the consequences of the Booroola mutation on BMPR1B functions, the action of ligands of the transforming growth factor-beta (TGFbeta)/BMP family that act through (growth and differentiation factor-5, BMP-4) or independently of (activin A, TGFbeta-1) BMPR1B were studied on primary cultures of GCs from homozygous FecB(+) and FecB(B) ewes. All the tested TGFbeta/BMP family ligands inhibited progesterone secretion by FecB(+) GCs. Those inhibitory effects were lower for GCs from preovulatory (5-7 mm diameter) than from small antral follicles (1-3 mm diameter). The presence of the Booroola mutation was associated with a 3- to 4-fold (P<0.001) decreased responsiveness of GCs from FecB(B) compared with FecB(+) small follicles to the action of BMPR1B ligands. In contrast, TGFbeta-1 and activin A had similar inhibitory effects on progesterone secretion by GCs from FecB(+) and FecB(B) small follicles. No difference between genotypes was observed with GCs from preovulatory follicles. In transfection experiments with HEK-293 cells, co-expression of FecB(+) BMPR1B and BMPR2 resulted in a 2.6-fold (P<0.01) induction of the activity of a BMP-specific luciferase reporter construct by BMP-4. Interestingly, no response to BMP-4 was observed when cells were transfected with the FecB(B) form of the BMPR1B receptor. Overall, these data strongly suggest that the Q249R mutation is associated with a specific alteration of BMPR1B signaling in hyperprolific Booroola ewes.
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PMID:The Booroola mutation in sheep is associated with an alteration of the bone morphogenetic protein receptor-IB functionality. 1277 24


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