Katzenellenbogen B S, Bhardwaj B, Fang H, Ince B A, Pakdel F, Reese J C, Schodin D, Wrenn C K
Department of Physiology and Biophysics, University of Illinois, Urbana 61801.
J Steroid Biochem Mol Biol. 1993 Dec;47(1-6):39-48. doi: 10.1016/0960-0760(93)90055-2.
We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the human estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labeling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. C530 mutant), C381 becomes the site of estrogen-compatible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed mutagenesis of selected residues in the ER and region-specific chemical mutagenesis of the ER hormone binding domain with initial phenotypic screening in yeast have enabled the identification of a region near C530 important in discrimination between estrogens and antiestrogens and of other residues important in hormone-dependent transcriptional activation. Some ER mutants with alterations in the carboxy-terminal portion of the hormone binding domain are transcriptionally inactive yet bind hormone and also function as potent dominant negative ERs, suppressing the activity of wild-type ER at low concentrations. These studies reveal a separation of the hormone binding and transcription activation functions of the ER. They are also beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer. In addition, some dominant negative receptors may prove useful in examining ER mechanisms of action and in suppressing the estrogen-dependent growth of breast cancer cells.
我们运用了亲和标记、定点诱变和区域化学诱变方法，以确定人雌激素受体（ER）中对激素结合、雌激素与抗雌激素之间的配体区分以及转录激活至关重要的区域。用抗雌激素他莫昔芬氮丙啶和雌激素酮诺司特罗氮丙啶进行的亲和标记研究已确定ER激素结合域中的半胱氨酸530是标记的主要位点。在530位不存在半胱氨酸时（即C530突变体），C381成为与雌激素兼容的他莫昔芬氮丙啶标记位点。因此，尽管这两个残基在ER蛋白的一级线性序列中相距甚远，但在蛋白的三维结构中，即在ER配体结合口袋中，它们必定彼此靠近，以便配体能够到达任一位点。通过对ER中选定残基的定点诱变以及对ER激素结合域进行区域特异性化学诱变并在酵母中进行初步表型筛选，已能够鉴定出C530附近对雌激素与抗雌激素区分至关重要的区域以及对激素依赖性转录激活重要的其他残基。激素结合域羧基末端部分发生改变的一些ER突变体在转录上无活性，但能结合激素，并且还作为强效显性负性ER发挥作用，在低浓度下抑制野生型ER的活性。这些研究揭示了ER的激素结合和转录激活功能的分离。它们也开始提供关于ER激素结合域以及对配体结合和不同类别激动剂和拮抗剂配体之间的区分重要的氨基酸的更详细情况。此类信息在设计最有效的抗雌激素药物方面将很重要。此外，由于现在有大量证据表明乳腺癌中存在野生型和变异型ER的混合物，我们的研究应能深入了解这些变异型受体的生物活性及其在调节野生型ER活性中的作用，并应有助于更好地理解变异型受体在乳腺癌对抗雌激素和内分泌治疗反应改变或耐药中可能发挥的作用。此外，一些显性负性受体可能在研究ER的作用机制以及抑制乳腺癌细胞的雌激素依赖性生长方面证明有用。
