共查询到20条相似文献,搜索用时 15 毫秒
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Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres 总被引:3,自引:0,他引:3
BACKGROUND: We describe a novel microsphere-based system to identify and characterize multiplexed interactions of nuclear receptors with peptides that represent the LXXLL binding region of coactivator proteins. METHODS: In this system, individual microsphere populations with unique red and orange fluorescent profiles are coupled to specific coactivator peptides. The coactivator peptide-coupled microsphere populations are combined and incubated with a nuclear receptor that has been coupled to a green fluorochrome. Flow cytometric analysis of the microspheres simultaneously decodes each population and detects the binding of receptor to respective coactivator peptides by the acquisition of green fluorescence. RESULTS: We have used this system to determine the binding affinities of human estrogen receptor beta ligand binding domain (ERbeta LBD) and human peroxisome proliferator activated receptor gamma ligand binding domain (PPARgamma LBD) to a set of 34 coactivator peptides. Binding of ERbeta LBD to a coactivator peptide sequence containing the second LXXLL motif of steroid receptor coactivator-1 (SRC-1(2) (676-700) is shown to be specific and saturable. Analysis of receptor binding to a multiplexed set of coactivator peptides shows PPARgamma LBD binds with high affinity to cAMP response element binding protein (CBP) peptides and to the related P300 peptide while ERbeta LBD exibits little binding to these peptides. Using the microsphere-based assay we demonstrate that ERbeta LBD and PPARgamma LBD binding affinities for the coactivator peptides are increased in the presence of agonist (estradiol or GW1929, respectively) and that ERbeta LBD binding is decreased in the presence of antagonist (raloxifene or tamoxifen). CONCLUSIONS: This unique microsphere-based system is a sensitive and efficient method to simultaneously evaluate many receptor-coactivator interactions in a single assay volume. In addition, the system offers a powerful approach to study small molecule modulation of nuclear receptor binding. 相似文献
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DNA binding and dimerization determinants for thyroid hormone receptor alpha and its interaction with a nuclear protein. 总被引:7,自引:0,他引:7
The gel retardation assay was used to analyze the role of the thyroid hormone receptor alpha (TR alpha) ligand-binding domain (LBD) in controlling receptor interaction with a thyroid hormone responsive element (TRE). While wild type receptor TR alpha binds to the TRE mainly as monomer, deletion of 85 amino acids from its C-terminus results in a mutant receptor with enhanced DNA binding that forms several slow mobility complexes as revealed by gel retardation assay. Receptor deletion mutants that lack most of the LBD show significantly elevated DNA binding and are still able to bind to DNA as two complexes. Thus, the C-terminal end of TR alpha appears to interfere with the dimerization/oligomerization function and DNA binding of TR alpha. All C-terminal deletion mutants have lost their T3-responsive activator function, but some show constitutive activity. Nuclear factor from several cell lines, including CV-1, F9, and GC cells, interacts with TR alpha receptor to form a larger molecular weight complex as determined by gel retardation assay. This factor could not be detected in HeLatk- cells, where TR alpha does not activate a TRE-containing reporter gene. The nuclear factor is heat sensitive and does not bind to TRE itself but can interact with TR alpha in the absence of DNA. Deletion analysis demonstrates that the leucine zipper-like sequence located in the LBD of TR alpha is involved in this interaction. Together, our data suggest that TR alpha contains a dimerization function outside the LBD which is inhibited by the carboxy-terminal region, while the leucine zipper-like sequence in the LBD is required for interaction with a nuclear factor. 相似文献
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Ligand binding and activation of the Ah receptor 总被引:2,自引:0,他引:2
Denison MS Pandini A Nagy SR Baldwin EP Bonati L 《Chemico-biological interactions》2002,141(1-2):3-24
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雌激素或类雌激素活性物质通过细胞核雌激素受体(nuclear estrogen receptor, nER)通路发挥相应的生理性作用。当这些配体被nER的配体结合域(ligand binding domain, LBD)识别后进入疏水性配体结合空腔内并引起受体构象发生改变,使得原先处于高度活动性的helix 12(H12)被固定从而进一步稳定空腔结构|同时nER也能通过招募一系列辅助调节因子及其他共调节蛋白质,最终调控基因转录。但是,由于不同的配体和受体结合形成的晶体结构并不完全相同,导致这些复合体具有不同的性质,从而影响基因的转录活性。本文综述了nER配体结合域及结合配体后形成的相应晶体结构与活性以及不同配体对受体结构和基因转录的影响。 相似文献
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Przibilla S Hitchcock WW Szécsi M Grebe M Beatty J Henrich VC Spindler-Barth M 《Biological chemistry》2004,385(1):21-30
The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, 1323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein. 相似文献
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Sandowski Y Raver N Gussakovsky EE Shochat S Dym O Livnah O Rubinstein M Krishna R Gertler A 《The Journal of biological chemistry》2002,277(48):46304-46309
A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding. 相似文献
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Wang L Zuercher WJ Consler TG Lambert MH Miller AB Orband-Miller LA McKee DD Willson TM Nolte RT 《The Journal of biological chemistry》2006,281(49):37773-37781
X-ray crystal structures of the ligand binding domain (LBD) of the estrogen-related receptor-gamma (ERRgamma) were determined that describe this receptor in three distinct states: unliganded, inverse agonist bound, and agonist bound. Two structures were solved for the unliganded state, the ERRgamma LBD alone, and in complex with a coregulator peptide representing a portion of receptor interacting protein 140 (RIP140). No significant differences were seen between these structures that both exhibited the conformation of ERRgamma seen in studies with other coactivators. Two structures were obtained describing the inverse agonist-bound state, the ERRgamma LBD with 4-hydroxytamoxifen (4-OHT), and the ERRgamma LBD with 4-OHT and a peptide representing a portion of the silencing mediator of retinoid and thyroid hormone action protein (SMRT). The 4-OHT structure was similar to other reported inverse agonist bound structures, showing reorientation of phenylalanine 435 and a displacement of the AF-2 helix relative to the unliganded structures with little other rearrangement occurring. No significant changes to the LBD appear to be induced by peptide binding with the addition of the SMRT peptide to the ERRgamma plus 4-OHT complex. The observed agonist-bound state contains the ERRgamma LBD, a ligand (GSK4716), and the RIP140 peptide and reveals an unexpected rearrangement of the phenol-binding residues. Thermal stability studies show that agonist binding leads to global stabilization of the ligand binding domain. In contrast to the conventional mechanism of nuclear receptor ligand activation, activation of ERRgamma by GSK4716 does not appear to involve a major rearrangement or significant stabilization of the C-terminal helix. 相似文献
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We have developed a novel assembly assay to examine structural changes in the ligand binding domain (LBD) of the thyroid hormone receptor (TR). Fragments including the first helix of the TR LBD interact only weakly with the remainder of the LBD in the absence of hormone, but this interaction is strongly enhanced by the addition of either hormone or the corepressor NCoR. Since neither the ligand nor the corepressor shows direct interaction with this helix, we propose that both exert their effects by stabilizing the overall structure of the LBD. Current models of activation of nuclear hormone receptors focus on a ligand-induced allosteric shift in the position of the C-terminal helix 12 that generates the coactivator binding site. Our results suggest that ligand binding also has more global effects that dynamically alter the structure of the receptor LBD. 相似文献