Wang Y, Miksicek R J
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
Mol Endocrinol. 1991 Nov;5(11):1707-15. doi: 10.1210/mend-5-11-1707.
Experiments were undertaken to characterize mRNAs coding for the estrogen receptor (ER) in the human breast cancer cell line T47D. We report here the isolation of cDNAs corresponding to three isoforms of this receptor in addition to a majority of wild-type clones. Sequence analysis showed that these isoforms are generated through alternative splicing. None of the alternatively spliced isoforms of ER is able to bind to an estrogen-responsive element (ERE) in a gel mobility shift assay in vitro or to activate transcription of a reporter gene containing an ERE in vivo. One isoform, ER delta E3, which harbors a deletion of exon 3 encoding the second zinc finger of the DNA-binding domain, inhibits estrogen-dependent transcription activation in a dominant negative fashion when it is cotransfected with the wild-type ER and reporter plasmid. It also inhibits DNA binding of wild-type ER in a gel mobility shift assay in vitro. Since ER delta E3 is not able to bind to its response element, the observed inhibitory effect probably occurs through protein-protein interactions. This could involve the formation of a heterodimer between mutant and wild-type receptors, competition for a limiting transcription factor, or both. These results may have implications for understanding the loss of estrogen responsiveness that frequently occurs in breast cancer.
开展实验以表征人类乳腺癌细胞系T47D中编码雌激素受体（ER）的mRNA。我们在此报告，除了大多数野生型克隆外，还分离出了与该受体三种同工型相对应的cDNA。序列分析表明，这些同工型是通过可变剪接产生的。在体外凝胶迁移率变动分析中，ER的可变剪接同工型均不能与雌激素反应元件（ERE）结合，在体内也不能激活含有ERE的报告基因的转录。一种同工型ER delta E3缺失编码DNA结合域第二个锌指的外显子3，当它与野生型ER和报告质粒共转染时，以显性负性方式抑制雌激素依赖性转录激活。它还在体外凝胶迁移率变动分析中抑制野生型ER的DNA结合。由于ER delta E3不能与其反应元件结合，观察到的抑制作用可能是通过蛋白质-蛋白质相互作用发生的。这可能涉及突变体和野生型受体之间异二聚体的形成、对有限转录因子的竞争，或两者兼有。这些结果可能对理解乳腺癌中频繁发生的雌激素反应丧失具有重要意义。
