A MODIFIED ESTROGEN-RECEPTOR LIGAND-BINDING DOMAIN AS AN IMPROVED SWITCH FOR THE REGULATION OF HETEROLOGOUS PROTEINS
A MODIFIED ESTROGEN-RECEPTOR LIGAND-BINDING DOMAIN AS AN IMPROVED SWITCH FOR THE REGULATION OF HETEROLOGOUS PROTEINS
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DOI：
10.1093/nar/23.10.1686
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发表时间：
1995-05-25
影响因子：
14.9
通讯作者：
EVEN, GI
中科院分区：
文献类型：
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作者：
LITTLEWOOD, TD;HANCOCK, DC;EVEN, GI
通过与雌激素受体的激素结合结构域融合，许多蛋白质在功能上具有雌激素依赖性。然而，这种融合蛋白有几个显著的缺点。首先，它们在体外细胞中的使用需要无酚红培养基和从血清中费力地剥离类固醇激素，以避免结构性激活。其次，体内雌激素受体融合蛋白的控制受阻于内源性高水平的循环雌激素。第三，雌激素受体的激素结合结构域作为激素依赖的转录激活结构域发挥作用，这使得解释与转录因子的融合存在问题。为了克服这些缺点，我们使用了一种转录不活跃的小鼠雌激素受体突变体，它不能结合雌激素，但仍保持对合成配体4-羟基他莫昔芬的正常亲和力。当这种突变的雌激素受体的激素结合域与c-Myc蛋白的C末端融合时，Myc诱导的成纤维细胞的增殖和凋亡依赖于4-羟基三苯氧胺，但对17β-雌二醇仍然是无效的。
A number of proteins have been rendered functionally oestrogen-dependent by fusion with the hormone-binding domain of the oestrogen receptor. There are, however, several significant disadvantages with such fusion proteins. First, their use in cells in vitro requires phenol red-free medium and laborious stripping of steroid hormones from serum in order to avoid constitutive activation. Secondly, control of oestrogen receptor fusion proteins in vivo is precluded by high endogenous levels of circulating oestrogens. Thirdly, the hormone-binding domain of the oestrogen receptor functions as a hormone-dependent transcriptional activation domain making interpretation of fusions with transcription factors problematical. In order to overcome these drawbacks we have used a transcriptionally inactive mutant of the murine oestrogen receptor which is unable to bind oestrogen yet retains normal affinity for the synthetic ligand, 4-hydroxytamoxifen. When the hormone-binding domain of this mutant oestrogen receptor is fused to the C-terminus of the c-Myc protein, Myc-induced proliferation and apoptosis in fibroblasts becomes dependent on 4-hydroxytamoxifen, but remains refractory to 17 beta-oestradiol.