INITIAL INTRACELLULAR EVENTS OF STEROID HORMONE ACTION

本项目的目标是定义初始的，细胞内的 糖皮质激素作用和类固醇激素作用的事件， 将军 类固醇与细胞内受体结合的第一步 分子之后是受体-类固醇复合物活化为 DNA结合和核结合物种，然后结合活化的 复合物的那些核受体位点参与调控 特定基因的转录。 各种技术的结合 用于检测糖皮质激素类固醇的关键第一步。 生物化学、亲和标记和蛋白水解已被用于定义 受体的类固醇结合核心元件，其包含大多数，如果不是 所有的氨基酸都需要特异性类固醇结合沿着 结合热休克蛋白90的能力。 代谢标记和 免疫化学显示，翻译后糖基化不 似乎是受体的类固醇结合活性所必需的。 分子生物学，用于创建3个半胱氨酸的点突变， 类固醇结合核心元件，已经产生了“超级”受体， 对糖皮质激素的结合亲和力和特异性增加。 等 “超级”受体可能被证明在未来的基因工程中有用 受体和/或类固醇缺陷患者的治疗 生物合成最后，对类固醇结合的合理检查 结构域的所有类固醇受体的存在下，潜在的邻近 二硫醇使我们能够设计一个涉及预处理的协议， 亚砷酸钠，仅特异性灭活糖皮质激素 受体。 这一程序应有助于 孕酮和盐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，这将有助于了解 控制类固醇调节的基因转录和治疗 患者

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.