Yang Jun, Singleton David W, Shaughnessy Elizabeth A, Khan Sohaib A
Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, United States.
Mol Cell Endocrinol. 2008 Nov 25;295(1-2):94-100. doi: 10.1016/j.mce.2008.08.001. Epub 2008 Aug 13.
The role of the carboxyl terminal F-domain of estrogen receptor (ERalpha) is uncertain, but evidence suggests that this region may impart internal restraint on ER dimerization in the presence of 17beta-estradiol (E2). To identify the C-terminal residues affecting human ERalpha activation, we created a series of deletions and examined E2 induced receptor dimerization and transactivation. Deletion of the final 24 C-terminal amino acids of the F-domain (Delta7b) yielded a fivefold increase in dimerization, when compared to wild type (wt) ERalpha in the presence of 2nM E2, utilizing a yeast two-hybrid assay. This increase in dimerization is similar to that observed when the entire F-domain was deleted. Measurement of mutant:mutant homodimer formation yielded similar increases compared to mutant:wt interactions. Interestingly, a point mutation at the C-terminus (mut 3) showed increases in dimerization comparable to that of Delta7b in the presence of nanomolar amounts of E2. However, at sub-nanomolar levels of E2, mut 3 behaved similarly to wt ERalpha, whereas Delta7b maintained striking increases in dimerization. Determination of E2 binding affinity (Kd) constants revealed only marginal differences for wt and F-domain mutants, suggesting that the F-domain affects dimerization directly. We also observed enhanced interaction of F domain mutants with p160 family coactivator SRC1. Finally, transcriptional regulation of estrogen responsive reporters, 2XERE-LacZ and 3XERE-Luc in yeast and mammalian cells, respectively, reflected the increased propensity for dimerization by F domain mutants. Together, these data indicate that the C-terminal amino acids of ERalpha are critical for attenuation of E2 induced receptor dimerization and transcriptional activity.
雌激素受体（ERα）羧基末端F结构域的作用尚不确定，但有证据表明，在17β-雌二醇（E2）存在的情况下，该区域可能对ER二聚化产生内在抑制作用。为了确定影响人ERα激活的C末端残基，我们创建了一系列缺失突变，并检测了E2诱导的受体二聚化和反式激活。利用酵母双杂交试验，在2 nM E2存在的情况下，与野生型（wt）ERα相比，F结构域最后24个C末端氨基酸的缺失（Delta7b）使二聚化增加了五倍。这种二聚化的增加与整个F结构域缺失时观察到的情况相似。与突变体：wt相互作用相比，突变体：突变体同源二聚体形成的测量结果显示出类似的增加。有趣的是，在纳摩尔量的E2存在下，C末端的一个点突变（mut 3）显示出与Delta7b相当的二聚化增加。然而，在亚纳摩尔水平的E2下，mut 3的行为与wt ERα相似，而Delta7b的二聚化则持续显著增加。E2结合亲和力（Kd）常数的测定表明，wt和F结构域突变体之间只有微小差异，这表明F结构域直接影响二聚化。我们还观察到F结构域突变体与p160家族共激活因子SRC1的相互作用增强。最后，雌激素反应性报告基因2XERE-LacZ和3XERE-Luc在酵母和哺乳动物细胞中的转录调控，分别反映了F结构域突变体二聚化倾向的增加。总之，这些数据表明，ERα的C末端氨基酸对于减弱E2诱导的受体二聚化和转录活性至关重要。
