Reese J C, Katzenellenbogen B S
Department of Physiology and Biophysics, University of Illinois, Urbana 61801.
Nucleic Acids Res. 1991 Dec 11;19(23):6595-602. doi: 10.1093/nar/19.23.6595.
We have developed a transient transfection system using the Cytomegalovirus (CMV) promoter to express the human estrogen receptor (ER) at very high levels in COS-1 cells and have used it to study the interaction of agonist and antagonist receptor complexes with estrogen response element (ERE) DNA. ER can be expressed to levels of 20-40 pmol/mg or 0.2-0.3% of total soluble protein and all of the soluble receptor is capable of binding hormone. The ER binds estradiol with high affinity (Kd 0.2 nM), and is indistinguishable from native ER in that the receptor is capable of recognizing its cognate DNA response element with high affinity, and of transactivating a transgene in an estradiol-dependent manner. Gel mobility shift assays reveal interesting ligand-dependent differences in the binding of receptor complexes to ERE DNA. Receptors occupied by estradiol or the type I antiestrogen transhydroxytamoxifen bind to DNA response elements when exposed to the ligand in vitro or in vivo. Likewise, receptors exposed to the type II antiestrogen ICI 164,384 in vitro bind to ERE DNA. However, when receptor exposure to ICI 164,384 is carried out in vivo, the ER-ICI 164,384 complexes do not bind to ERE DNA, or do so only weakly. This effect is not reversed by subsequent incubation with estradiol in vitro, but is rapidly reversible by in vivo estradiol exposure of intact COS-1 cells. This suggests there may be some cellular process involved in the mechanism of antagonism by the pure antiestrogen ICI 164,384, which is not observed in cell-free extracts.
我们开发了一种瞬时转染系统，该系统利用巨细胞病毒（CMV）启动子在COS-1细胞中高水平表达人雌激素受体（ER），并利用它来研究激动剂和拮抗剂受体复合物与雌激素反应元件（ERE）DNA的相互作用。ER的表达水平可达20 - 40 pmol/mg或占总可溶性蛋白的0.2 - 0.3%，并且所有可溶性受体都能够结合激素。ER以高亲和力（Kd 0.2 nM）结合雌二醇，并且与天然ER没有区别，因为该受体能够以高亲和力识别其同源DNA反应元件，并以雌二醇依赖的方式反式激活转基因。凝胶迁移率变动分析揭示了受体复合物与ERE DNA结合中有趣的配体依赖性差异。被雌二醇或I型抗雌激素他莫昔芬转羟基物占据的受体，在体外或体内暴露于配体时会与DNA反应元件结合。同样，在体外暴露于II型抗雌激素ICI 164,384的受体也会与ERE DNA结合。然而，当在体内使受体暴露于ICI 164,384时，ER - ICI 164,384复合物不与ERE DNA结合，或仅微弱结合。这种效应在体外随后与雌二醇孵育时不会逆转，但通过完整COS-1细胞的体内雌二醇暴露可迅速逆转。这表明在纯抗雌激素ICI 164,384的拮抗机制中可能涉及一些细胞过程，而在无细胞提取物中未观察到这种过程。
