共查询到20条相似文献,搜索用时 20 毫秒
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De S Macara IG Lannigan DA 《The Journal of steroid biochemistry and molecular biology》2005,96(3-4):235-244
There exists a significant need for the detection of novel estrogen receptor (ER) ligands for pharmaceutical uses, especially for treating complications associated with menopause. We have developed fluorescence resonance energy transfer (FRET)-based biosensors that permit the direct in vitro detection of ER ligands. These biosensors contain an ER ligand-binding domain (LBD) flanked by the FRET donor fluorophore, cyan fluorescent protein (CFP), and the acceptor fluorophore, yellow fluorescent protein (YFP). The ER-LBD has been modified so that Ala 430 has been changed to Asp, which increases the magnitude of the FRET signal in response to ligand-binding by more than four-fold compared to the wild-type LBD. The binding of agonists can be distinguished from that of antagonists on the basis of the distinct ligand-induced conformations in the ER-LBD. The approach to binding equilibrium occurs within 30min, and the FRET signal is stable over 24h. The biosensor demonstrates a high signal-to-noise, with a Z' value (a statistical determinant of assay quality) of 0.72. The affinity of the ER for different ligands can be determined using a modified version of the biosensor in which a truncated YFP and an enhanced CFP are used. Thus, we have developed platforms for high-throughput screens for the identification of novel estrogen receptor ligands. Moreover, we have demonstrated that this FRET technology can be applied to other nuclear receptors, such as the androgen receptor. 相似文献
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Likhite VS Stossi F Kim K Katzenellenbogen BS Katzenellenbogen JA 《Molecular endocrinology (Baltimore, Md.)》2006,20(12):3120-3132
Posttranslational modifications of the estrogen receptor (ER) are emerging as important regulatory elements of cross talk between different signaling pathways. ER phosphorylation, in particular, has been implicated in the ligand-independent effects of ER and in tamoxifen resistance of breast tumors. In our studies, Western immunoblot analysis of endogenous ER in parental MCF-7 cells reveals specific, ligand-dependent phosphorylations at S118 and S167, with this ligand dependence being lost in tamoxifen-resistant, MCF-7 Her2/neu cells. Using highly purified components and sensitive fluorescence methods in an in vitro system, we show that phosphorylation by different kinases alters ER action through distinct mechanisms. Phosphorylation by Src and protein kinase A increases affinity for estradiol (E2), whereas ER phosphorylation by MAPK decreases trans-hydroxytamoxifen (TOT) binding. Affinity of ER for the consensus estrogen response element is also altered by phosphorylation in a ligand-specific manner, with decrease in affinity of MAPK- and Src-phosphorylated ER in the presence of TOT. ER phosphorylation by MAPK, AKT, or protein kinase A increases recruitment of steroid receptor coactivator 3 receptor interaction domain to the DNA-bound receptor in the presence of E2. Taken together, these results suggest that ER phosphorylation alters receptor functions (ligand, DNA, and coactivator binding), effecting changes that could lead to an increase in E2 agonism and a decrease in TOT antagonistic activity, reflecting changes encountered in tamoxifen resistance in endocrine therapy of breast cancer. 相似文献
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Estrogen receptor pathways to AP-1 总被引:21,自引:0,他引:21
Kushner PJ Agard DA Greene GL Scanlan TS Shiau AK Uht RM Webb P 《The Journal of steroid biochemistry and molecular biology》2000,74(5):311-317
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With one million new cases in the world each year, breast cancer is the most common malig- nancy in women and comprises 18% of all female cancers. The incidence and mortality of breast cancer in China have been significantly increased in the past years. It has been known that several risk factors are associated with breast cancer[1], including inherited mutation in the BRCA1 and BRCA2 genes, increasing age, early onset of menstruation, late menopause, never having had chil- dren or havin… 相似文献
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Yukitomo Arao Katherine J. Hamilton Laurel A. Coons Kenneth S. Korach 《The Journal of biological chemistry》2013,288(29):21105-21116
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