Ince B A, Zhuang Y, Wrenn C K, Shapiro D J, Katzenellenbogen B S
Department of Cell and Structural Biology, University of Illinois, Urbana, 61801.
J Biol Chem. 1993 Jul 5;268(19):14026-32.
We have identified and characterized three human estrogen receptor (ER) mutants, which, at low concentrations, are capable of blocking the intracellular activity of wild type ER. The mutants, a truncated ER (ER1-530), a point mutant (L540Q), and a frameshift (S554fs), were generated by random chemical mutagenesis of the ER hormone binding domain and screened first for low activity in a yeast selection system. In transient co-transfection assays using ER-deficient Chinese hamster ovary cells, all three mutants exhibited less than 10% of the transcription activation activity of wild type ER, and when co-expressed with wild type ER, each of the mutants effectively suppressed the ability of wild type ER to activate transcription of an estrogen-regulated reporter plasmid. When equal amounts of plasmid encoding the ER mutants and wild type ER were used, S554fs, ER1-530, and L540Q suppressed the activity of wild type ER by 80, 55, and 75%, respectively. At a ratio of 1 part S554fs to 10 parts wild type ER, transcription was still inhibited by 40%. Western blot analysis showed that all three mutants were expressed at approximately the same level as wild type ER. Suppression of transcription was specific for ER, since the mutants did not inhibit progesterone receptor-mediated transcription. Not all mutations leading to inactive ER confer the dominant negative phenotype, as five ER mutants rendered transcriptionally inactive by point mutations between residues 516 and 524 of the ER hormone binding domain were poor inhibitors of wild type ER activity. Binding studies showed that the L540Q and S554fs dominant negative mutants bound 17 beta-estradiol with wild type affinity (Kd = 0.3-0.5 nM), whereas ER1-530 exhibited a 15-fold reduction in affinity for estradiol. The three dominant negative ERs showed significant ability to interact with the estrogen response element (ERE) in promoter interference assays, but ER1-530 and S554fs displayed little or no binding to the ERE in gel mobility shift assays where higher affinity for the DNA may be required for the receptor-ERE complex to remain associated during the electrophoresis. These data support the idea that, in all three mutants, it is loss of function of the COOH-terminal transactivation domain which leads to the dominant negative phenotype. S554fs, a powerful dominant negative mutant, is a good candidate for further studies aimed at suppressing the estrogen-dependent growth of human breast cancer cells.
我们已经鉴定并表征了三种人类雌激素受体（ER）突变体，它们在低浓度时能够阻断野生型ER的细胞内活性。这些突变体，一个截短的ER（ER1-530）、一个点突变体（L540Q）和一个移码突变体（S554fs），是通过对ER激素结合域进行随机化学诱变产生的，并首先在酵母选择系统中筛选低活性突变体。在使用ER缺陷的中国仓鼠卵巢细胞进行的瞬时共转染实验中，所有这三种突变体的转录激活活性均不到野生型ER的10%，并且当与野生型ER共表达时，每个突变体都能有效抑制野生型ER激活雌激素调节报告质粒转录的能力。当使用等量的编码ER突变体和野生型ER的质粒时，S554fs、ER1-530和L540Q分别抑制野生型ER活性的80%、55%和75%。当S554fs与野生型ER的比例为1:10时，转录仍被抑制40%。蛋白质免疫印迹分析表明，所有这三种突变体的表达水平与野生型ER大致相同。转录抑制对ER具有特异性，因为这些突变体不抑制孕酮受体介导的转录。并非所有导致无活性ER的突变都会产生显性负性表型，因为在ER激素结合域第516至524位残基之间通过点突变导致转录无活性的五个ER突变体对野生型ER活性的抑制作用较弱。结合研究表明，L540Q和S554fs显性负性突变体以野生型亲和力（Kd = 0.3 - 0.5 nM）结合17β-雌二醇，而ER1-530对雌二醇的亲和力降低了15倍。在启动子干扰实验中，这三种显性负性ER显示出与雌激素反应元件（ERE）相互作用的显著能力，但在凝胶迁移率变动分析中，ER1-530和S554fs与ERE的结合很少或没有结合，在该分析中，受体-ERE复合物在电泳过程中保持结合可能需要对DNA有更高的亲和力。这些数据支持这样一种观点，即在所有这三种突变体中，是COOH末端反式激活域的功能丧失导致了显性负性表型。S554fs是一种强大的显性负性突变体，是旨在抑制人乳腺癌细胞雌激素依赖性生长研究的良好候选对象。
