INITIAL INTRACELLULAR EVENTS OF STEROID HORMONE ACTION

类固醇激素作用的初始细胞内事件

• 批准号: 2573679
• 负责人: S S SIMONS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2573679
• 关键词: DNA binding protein; chickens; corticosteroid receptors; genetic regulation; genetic transcription; glucocorticoids; hormone regulation /control mechanism; laboratory rat; progesterone receptors; receptor binding; steroid hormone; transfection

Our objective is define the initial, intracellular events of glucocorticoid hormone action and steroid hormone action in general. Such studies are of current relevance since steroid hormone receptors are arguably the best understood regulators of eukaryotic gene transcription. The first step of steroid binding to the intracellular receptor is followed by activation of the receptor-steroid complex to a DNA-binding species that associates with those nuclear acceptor sites involved in the regulation of transcription of selected genes in specific cells. We have concentrated on the binding of ligands to glucocorticoid receptors (GR) and how the activated receptor-steroid complexes influence gene transcription. A long standing conundrum of GRs has been why the steroid binding domain is active in chimeric proteins but not in isolation. For this reason, the boundaries of the steroid binding domain have not been delineated. Using a variety of receptor deletion constructs that were fused to beta-galactosidase or dihydrofolate reductase, we identified amino acids 550-795 as being the boundaries of the steroid binding domain of the rat GR. Surprisingly, however, plasmids encoding just the steroid binding domain did not yield stable proteins although the DNA was being transcribed, as shown by a transcription competition assay. Thus, contrary to the generally accepted view, the steroid binding domain of GR is not independently functional. These results also revealed the importance of protein stability and protein folding when constructing mutant receptors. Such mutant proteins have been widely used in transient transfections to construct the current models of steroid hormone action. We also found that several induction properties of steroids were not constant but varied with the concentration of transiently transfected GRs. Thus, the percent of maximal induction for subsaturating concentrations of glucocorticoid was dramatically increased, and antiglucocorticoids were converted into partial glucocorticoids, simply by increasing the cellular concentration of GRs. This phenomenon was specific in that the A form of the chick progesterone receptor was without effect under the same conditions. These variations demonstrate that caution should be exercised in making mechanistic conclusions based solely on experiments conducted with transiently transfected GR. Collectively, our findings contribute to our long term goal of defining the action of steriod hormones at a molecular level and of understanding their role in human physiology.

我们的目标是确定最初的，细胞内事件， 糖皮质激素作用和类固醇激素作用。 这些研究是目前的相关性，因为类固醇激素受体 可以说是真核生物基因的最佳调节因子 转录。 类固醇结合细胞内的第一步 受体之后是受体-类固醇复合物的活化， 一种与核受体结合的DNA结合物质， 参与选择基因转录调控的位点， 特定细胞 我们已经集中在配体的结合， 糖皮质激素受体（GR）和如何激活受体类固醇 复合物影响基因转录。 一个长期存在的难题 GR一直是类固醇结合结构域在嵌合体中具有活性的原因。 蛋白质，但不是孤立的。 因此， 类固醇结合结构域尚未被描述。 使用各种 与β-半乳糖苷酶融合的受体缺失构建体，或 二氢叶酸还原酶，我们鉴定氨基酸550-795为 大鼠GR的类固醇结合域的边界。 然而，令人惊讶的是， 结构域没有产生稳定的蛋白质，尽管DNA被 转录，如转录竞争测定所示。 因此，在本发明中， 与普遍接受的观点相反， GR不是独立运作的。 这些结果也揭示了 蛋白质稳定性和蛋白质折叠的重要性 突变受体 这种突变蛋白已被广泛用于 瞬时转染来构建类固醇激素的当前模型 激素作用。 我们还发现，几个诱导性质， 类固醇不是恒定的，而是随着浓度的变化而变化 瞬时转染的GR。 因此，最大诱导的百分比 对于糖皮质激素的亚饱和浓度， 增加，抗糖皮质激素转化为部分 糖皮质激素，只是通过增加细胞浓度， 格令 这种现象的特殊之处在于， 孕酮受体在相同条件下无作用。 这些差异表明，在进行以下操作时应谨慎行事： 仅仅根据实验得出机械结论 瞬时转染GR。总的来说，我们的发现 有助于我们确定类固醇作用的长期目标 在分子水平上了解它们在人类 physiology.