INITIAL INTRACELLULAR EVENTS OF STEROID HORMONE ACTION

这个项目的目标是定义初始的、细胞内的 糖皮质激素作用事件和类固醇激素作用事件 将军。类固醇与细胞内受体结合的第一步 分子之后，受体-类固醇复合体被激活为 DNA结合和核结合的物种，然后结合活化 与参与调节的核受体结合部位的复合体 特定基因的转录。一种技术的组合已经被 用于检查糖皮质激素的关键第一步。 生物化学、亲和标记和蛋白水解法已被用于确定 受体的类固醇结合核心元件，它包含大多数，如果不是 所有，特定类固醇结合所需的氨基酸以及 结合热休克蛋白90的能力。代谢物标记和 免疫化学显示翻译后糖基化不会 似乎是受体的类固醇结合活性所必需的。 分子生物学，用于制造3个半胱氨酸点突变 类固醇结合核心成分，已产生“超级”受体 增加糖皮质激素的结合亲和力和特异性。是这样的 “超级”受体可能被证明在未来的基因工程中有用 受体和/或类固醇受体缺陷患者的治疗 生物合成。最后，对类固醇结合的理性审视 所有类固醇受体的结构域是否存在潜在的邻近 二硫醇允许我们设计一种方案，包括用 仅能使糖皮质激素失活的亚砷酸钠 感受器。这一过程应该会对 黄体酮和矿物皮质激素受体的定量。未来 类固醇-受体相互作用的分子细节研究 应提供进一步的信息，以帮助理解 类固醇调节基因转录的调控与骨髓瘤的治疗 病人。

The objective of this project is to define the initial, intracellular events of glucocorticoid hormone action, and steroid hormone action in general. The first step of steroid binding to the intracellular receptor molecule is followed by activation of the receptor-steroid complex to a DNA-binding and nuclear-binding species and then binding of activated complexes to those nuclear acceptor sites involved in the regulation of transcription of specific genes. A combination of techniques have been used to examine the crucial first step for glucocorticoid steroids. Biochemistry, affinity labeling, and proteolysis have been used to define a steroid binding core element of the receptor which contains most, if not all, of the amino acids required for specific steroid binding along with the ability to bind heat shock protein 90. Metabolic labeling and immunochemistry revealed that posttranslational glycosylation does not appear to be required for the steroid binding activity of the receptor. Molecular biology, used to create point mutations of the 3 cysteines in the steroid binding core element, has yielded "super" receptors with increased binding affinity and specificity for glucocorticoids. Such "super" receptor may prove to be of use in future genetic engineering treatment of patients with defective receptors and/or steroid biosynthesis. Finally, a rational examination of the steroid binding domains of all the steroid receptors for the presence of potential vicinal dithiols permitted us to devise a protocol involving pretreatment with sodium arsenite that specifically inactivates only glucocorticoid receptors. This procedure should be of great assistance in the quantitation of progesterone and mineral-ocorticoid receptors. Future studies concerning the molecular details of steroid-receptor interactions should provide further information which will aid in understanding the control of steroid-regulated gene transcription and the treatment of patients.