Xing H, Shapiro D J
Department of Biochemistry, University of Illinois, Urbana 61801-3792.
J Biol Chem. 1993 Nov 5;268(31):23227-33.
To study transactivation by the Xenopus laevis estrogen receptor (XER), we inserted one or two copies of a synthetic amphipathic helix at amino acid 276 of the XER. The XER mutants containing one or two copies of the amphipathic helix (XER/1AH and XER/2AH, respectively) and wild-type XER were expressed at similar levels. In transient transfection assays, XER/1AH exhibited only a modest, promoter-specific increase in transactivation. Constitutive (estrogen-independent) transcription of a synthetic promoter containing two estrogen response elements (EREs) was approximately 10-fold higher for the XER/2AH mutant than for wild-type XER. The XER/2AH mutant and wild-type XER exhibited similar 17 beta-estradiol dose-response curves for transactivation. In studies carried out over a broad range of DNA concentrations using the simple 2ERE-TATA promoter or a complex vitellogenin-derived promoter, the XER/2AH mutant exhibited an estrogen-dependent 2-3-fold increase in transactivation. A 2-3-fold increase in transactivation by XER/2AH was also observed using synthetic promoters in which the two EREs exhibit synergistic interactions with the NF1, AP1, or vitellogenin activator upstream activator sequences. Using a promoter interference assay to investigate intracellular interactions between the estrogen receptor and the ERE, we showed that binding of wild-type XER to the ERE was strongly estrogen-dependent. In the presence of 17 beta-estradiol, XER/2AH and wild-type XER exhibited similar promoter interference curves. In the absence of 17 beta-estradiol, the expression plasmid encoding the XER/2AH mutant achieved levels of promoter interference with 0.25-0.5 microgram of transfected DNA that were similar to those observed with 5-10 micrograms of the expression plasmid encoding wild-type XER. The ability of the XER/2AH mutant to activate transcription in the absence of estrogen therefore is likely to be related to the approximately 20-fold increase in its apparent ability to bind to the ERE. Since XER/2AH was unable to activate transcription from a glucocorticoid response element, enhanced binding of XER/2AH to the ERE did not result from a general increase in binding to DNA. The XER/2AH mutant appears to be the first nuclear receptor mutant to retain hormone-dependent transactivation and to exhibit enhanced hormone-independent binding to its hormone response element.
为了研究非洲爪蟾雌激素受体（XER）的反式激活作用，我们在XER的第276位氨基酸处插入了一或两个合成两亲性螺旋拷贝。含有一或两个两亲性螺旋拷贝的XER突变体（分别为XER/1AH和XER/2AH）与野生型XER以相似水平表达。在瞬时转染实验中，XER/1AH仅表现出适度的、启动子特异性的反式激活增加。对于含有两个雌激素反应元件（ERE）的合成启动子，XER/2AH突变体的组成型（雌激素非依赖性）转录比野生型XER高约10倍。XER/2AH突变体和野生型XER在反式激活方面表现出相似的17β-雌二醇剂量反应曲线。在使用简单的2ERE-TATA启动子或复杂的卵黄生成素衍生启动子进行的广泛DNA浓度研究中，XER/2AH突变体表现出雌激素依赖性的反式激活增加2 - 3倍。在两个ERE与NF1、AP1或卵黄生成素激活剂上游激活序列表现出协同相互作用的合成启动子中，也观察到XER/2AH的反式激活增加2 - 3倍。使用启动子干扰实验来研究雌激素受体与ERE之间的细胞内相互作用，我们发现野生型XER与ERE的结合强烈依赖雌激素。在17β-雌二醇存在下，XER/2AH和野生型XER表现出相似的启动子干扰曲线。在没有17β-雌二醇的情况下，编码XER/2AH突变体的表达质粒用0.25 - 0.5微克转染DNA达到的启动子干扰水平与用5 - 10微克编码野生型XER的表达质粒观察到的水平相似。因此，XER/2AH突变体在没有雌激素时激活转录的能力可能与其与ERE结合的表观能力增加约20倍有关。由于XER/2AH无法从糖皮质激素反应元件激活转录，XER/2AH与ERE结合的增强并非源于与DNA结合的普遍增加。XER/2AH突变体似乎是第一个保留激素依赖性反式激活并表现出增强的激素非依赖性与其激素反应元件结合的核受体突变体。
