INITIAL INTRACELLULAR EVENTS OF STEROID HORMONE ACTION

我们的目标是定义初始的、细胞内事件 糖皮质激素作用和类固醇激素作用一般。 这类研究具有当前的相关性，因为类固醇激素受体 可以说是对真核基因最了解的调节者 抄写。类固醇与细胞内结合的第一步 受体之后是受体-类固醇复合体的激活以 一种与这些核受体相关的DNA结合物种 参与调控所选基因转录的位点 特定的细胞。我们已经集中讨论了配体与 糖皮质激素受体(GR)及其激活受体--类固醇 复合体影响基因转录。一个由来已久的难题 GRS一直是类固醇结合结构域在嵌合体中活跃的原因 蛋白质，但不是孤立的。出于这个原因， 类固醇结合结构域尚未被描述。使用各种不同的 与β-半乳糖苷酶融合的受体缺失构建体 二氢叶酸还原酶，我们鉴定了550-795氨基酸是 大鼠GR类固醇结合域的边界。 然而，令人惊讶的是，仅编码类固醇结合的质粒 DOMAIN没有产生稳定的蛋白质，尽管DNA正在 转录，如转录竞争分析所示。因此， 与普遍接受的观点相反，类固醇结合结构域 GR不是独立运行的。这些结果还揭示了 构建时蛋白质稳定性和蛋白质折叠的重要性 突变的受体。这种突变蛋白已被广泛应用于 瞬时转染法构建激素类药物的动物模型 荷尔蒙作用。我们还发现，几个诱导性质 类固醇不是恒定的，而是随着药物浓度的不同而变化。 瞬时转染型GRs。因此，最大诱发率的百分比 糖皮质激素的浓度显著低于饱和浓度 增加，抗糖皮质激素转化为部分 糖皮质激素，简单地通过增加细胞内 GRS。这一现象是特定的，因为A型雏鸟 孕激素受体在相同条件下不起作用。 这些变化表明，在 仅根据进行的实验得出机械性结论 瞬时转染型GR。总而言之，我们的发现 为我们定义类固醇作用的长期目标做出贡献 分子水平上的激素及其在人类中的作用 生理学。

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.