Wang H, Peters G A, Zeng X, Tang M, Ip W, Khan S A
Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Ohio 45267-0521, USA.
J Biol Chem. 1995 Oct 6;270(40):23322-9. doi: 10.1074/jbc.270.40.23322.
Previous studies using in vitro procedures have not clearly established whether the estrogen receptor (ER) acts as a monomer or dimer in the cell. We have used the yeast two-hybrid system as an in vivo approach to investigate the dimerization of the estrogen receptor in the absence and presence of estrogen and anti-estrogens. This system is independent of ER binding to the estrogen response element. Two vectors, expressing GAL4 DNA binding domain-human ER and GAL4 transactivation domain-human ER, were constructed. Control experiments showed that each fusion protein had a high affinity binding site for estradiol-17 beta and could transactivate an ERE-LacZ reporter gene in yeast similar to the wild type ER. The two fusion proteins, GAL4 DB-hER and GAL 4 TA-hER, were expressed in the yeast strain, PCY2, which carries a GAL1 promoter-lacZ reporter. ER dimerization was measured via reconstitution of GAL4 through interaction of the fusion proteins, which transactivates LacZ through the GAL1 promoter. When both ER fusion proteins were expressed, beta-galactosidase activity was estradiol-17 beta-inducible. Furthermore, we showed that both tamoxifen and ICI 182,780 also induced beta-galactosidase activity, albeit lower than that induced by estradiol-17 beta. These results strongly argue that ER dimerization is ligand-dependent and the dimer can be induced by estradiol-17 beta, tamoxifen, or ICI 182,780. We also treated the yeast containing the two fusion proteins with estradiol-17 beta and tamoxifen or ICI 182,780 simultaneously to determine the effects on ER dimerization. beta-Galactosidase activity was lower when the yeast was treated with a higher ratio of tamoxifen or ICI 182,780 to estrogen than estradiol-17 beta alone. Taken together, we conclude that ER dimerization is ligand (estradiol-17 beta, tamoxifen, or ICI 182, 780)-dependent, and we suggest that estradiol-17 beta-induced dimers are destabilized when estradiol-17 beta is used with tamoxifen or ICI 182,780 simultaneously.
以往使用体外实验方法的研究尚未明确雌激素受体（ER）在细胞中是以单体还是二聚体形式发挥作用。我们利用酵母双杂交系统作为一种体内研究方法，来探究在有雌激素和抗雌激素存在及不存在的情况下雌激素受体的二聚化情况。该系统不依赖于ER与雌激素反应元件的结合。构建了两个载体，分别表达GAL4 DNA结合结构域-人ER和GAL4反式激活结构域-人ER。对照实验表明，每种融合蛋白都有一个对雌二醇-17β具有高亲和力的结合位点，并且能够在酵母中激活一个ERE-LacZ报告基因，这与野生型ER相似。两种融合蛋白，即GAL4 DB-hER和GAL 4 TA-hER，在携带GAL1启动子-lacZ报告基因的酵母菌株PCY2中表达。通过融合蛋白的相互作用使GAL4重组，从而通过GAL1启动子激活LacZ，以此来检测ER的二聚化。当两种ER融合蛋白都表达时，β-半乳糖苷酶活性是雌二醇-17β诱导型的。此外，我们还表明他莫昔芬和ICI 182,780也能诱导β-半乳糖苷酶活性，尽管低于雌二醇-17β诱导的活性。这些结果有力地表明ER二聚化是依赖配体的，并且二聚体可由雌二醇-17β、他莫昔芬或ICI 182,780诱导产生。我们还同时用雌二醇-17β和他莫昔芬或ICI 182,780处理含有这两种融合蛋白的酵母，以确定对ER二聚化的影响。当酵母用比单独使用雌二醇-17β更高比例的他莫昔芬或ICI 182,780与雌激素处理时，β-半乳糖苷酶活性较低。综上所述，我们得出结论，ER二聚化是依赖配体（雌二醇-17β、他莫昔芬或ICI 182,780）的，并且我们认为当雌二醇-17β与他莫昔芬或ICI 182,780同时使用时，雌二醇-17β诱导的二聚体会变得不稳定。
