Littlewood T D, Hancock D C, Danielian P S, Parker M G, Evan G I
Molecular Endocrinology Laboratories, Imperial Cancer Research Fund, London, UK.
Nucleic Acids Res. 1995 May 25;23(10):1686-90. doi: 10.1093/nar/23.10.1686.
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.
通过与雌激素受体的激素结合结构域融合，许多蛋白质已被赋予功能性雌激素依赖性。然而，这类融合蛋白存在几个显著缺点。首先，在体外细胞中使用它们需要无酚红培养基，并且要费力地从血清中去除类固醇激素，以避免组成性激活。其次，由于循环雌激素的内源性水平较高，体内对雌激素受体融合蛋白的控制受到限制。第三，雌激素受体的激素结合结构域作为激素依赖性转录激活结构域，使得与转录因子的融合的解释存在问题。为了克服这些缺点，我们使用了小鼠雌激素受体的转录失活突变体，它不能结合雌激素，但对合成配体4-羟基他莫昔芬仍保持正常亲和力。当这种突变雌激素受体的激素结合结构域与c-Myc蛋白的C末端融合时，Myc诱导的成纤维细胞增殖和凋亡变得依赖于4-羟基他莫昔芬，但对17β-雌二醇仍无反应。
