Identification of amino acids in the hormone binding domain of the human estrogen receptor important in estrogen binding
Identification of amino acids in the hormone binding domain of the human estrogen receptor important in estrogen binding
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DOI：
10.1074/jbc.271.33.20053
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发表时间：
1996-08-16
影响因子：
4.8
通讯作者：
Katzenellenbogen, BS
中科院分区：
文献类型：
--
作者：
Ekena, K;Weis, KE;Katzenellenbogen, BS
雌激素受体（ER）转录活性调节的第一步是与激素的结合。已有研究表明，人ER C端515 - 535位氨基酸之间的区域可能是配体结合的重要区域。为了明确定义该区域中的哪些氨基酸对于配体识别和结合是关键的，我们已经在整个515-535区域上利用丙氨酸扫描诱变。在与雌激素响应报告基因的细胞共转染测定中评估了这21个突变体响应天然雌激素17 β-雌二醇（E（2））激活转录的能力。此外，还测试了它们结合E（2）的能力。在521-528区域的四个位点上的突变对E（2）诱导的转录具有最大的影响，其中L525 A使反应性降低250倍，G521 A和H524 A降低35倍，M528 A降低11倍。其他位点的突变对E（2）（M517 A，Y 526 A，N532 A和P535 A）的敏感性没有影响或降低4倍或更少。三个在其转录反应中受影响最大的突变体，G521 A，H524 A和M528 A，显示E（2）结合亲和力的协同降低，不能检测到受影响最大的突变体L525 A的E（2）结合。因此，这些ER突变体的改变的转录反应似乎仅仅来自于它们对配体E（2）的亲和力的改变。受丙氨酸取代影响最大的四个位点521、524、525和528遵循α-螺旋周期性，使得它们将位于α-螺旋的一个面上。此外，它们精确地对应于最近描述的核激素受体超家族的两个其他成员的X射线晶体结构中显示与配体接触的α-螺旋中的残基，即视黄酸受体-和甲状腺激素受体-配体复合物。我们的研究结果，扩大观察核受体超家族内的类固醇受体家族，表明雌激素受体的这一区域是在与其同源配体以类似的方式接触。
The initial step in the regulation of the transcriptional activity of the estrogen receptor (ER) is the binding of hormone, Previous studies have suggested that the region between amino acids 515 and 535 near the C terminus of the human ER is likely to be important in ligand binding. In order to explicitly define which amino acids in this region are critical for ligand recognition and binding, we have utilized alanine-scanning mutagenesis over the complete 515-535 region, The ability of these 21 mutants to activate transcription in response to the natural estrogen, 17 beta-estradiol (E(2)), was evaluated in cell co-transfection assays with estrogen-responsive reporter genes, In addition, their ability to bind E(2) was also tested. Mutations at four sites in the 521-528 region had the greatest effects on E(2)-induced transcription, with L525A reducing responsiveness 250-fold, G521A and H524A 35-fold, and M528A 11-fold. Mutations at other sites had either no effect or a 4-fold or lesser reduction in sensitivity to E(2) (M517A, Y526A, N532A, and P535A). Three of the mutants most affected in their transcriptional response, G521A, H524A, and M528A, showed a coordinate reduction in E(2) binding affinity, E(2) binding by the most affected mutant, L525A, could not be detected, Thus, the altered transcriptional response of these ER mutants appears to derive solely from an alteration in their affinity for the ligand E(2). The four sites most affected by alanine substitution, 521, 524, 525, and 528, follow an alpha-helical periodicity, such that they would be positioned on one face of an alpha-helix, Furthermore, they correspond precisely to residues in an alpha-helix shown to be in contact with ligand in the recently described x-ray crystal structures of two other members of the nuclear hormone receptor superfamily, namely the retinoic acid receptor- and thyroid hormone receptor-ligand complexes. Our findings, which broaden observations to the steroid receptor family within the superfamily of nuclear receptors, suggest that this region of the estrogen receptor is in contact with its cognate ligand in a similar fashion.