Katzenellenbogen B S, Fang H, Ince B A, Pakdel F, Reese J C, Wooge C H, Wrenn C K
Department of Physiology and Biophysics, University of Illinois, Urbana 61801.
Breast Cancer Res Treat. 1993;27(1-2):17-26. doi: 10.1007/BF00683190.
We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labelling 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. Cys530A1a 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 and region-specific chemical mutagenesis have identified a region around C530 important in discrimination between estrogens and antiestrogens, and other mutants have allowed identification of residues important in hormone-dependent transcriptional activation. Some transcriptionally inactive ER mutants also function as potent dominant negative ERs, suppressing the activity of wild-type ERs at low concentrations. These studies are 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.
我们运用了亲和标记、定点诱变和区域化学诱变技术，以确定雌激素受体（ER）中在激素结合、雌激素与抗雌激素之间的配体区分以及转录激活方面重要的区域。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Cys530Ala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。定点诱变和区域特异性化学诱变已确定C530周围的一个区域在雌激素与抗雌激素的区分中很重要，其他突变体则有助于确定在激素依赖性转录激活中重要的残基。一些转录无活性的ER突变体也作为有效的显性负性ER发挥作用，在低浓度下抑制野生型ER的活性。这些研究开始提供关于ER激素结合域以及在配体结合和不同类别激动剂与拮抗剂配体区分中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时将很重要。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的反应改变或耐药中可能发挥的作用。
