PHYSIOLOGY OF PITUITARY CELL GLUCOCORTICOID BINDING

本项目的最终目标是了解 糖皮质激素结合和激素作用的生理学。 朝着这个 最后，我们对糖皮质激素受体的几个方面进行了研究， 生理学，包括受体结合、激活和 核转位 在三个目标方面取得了进展， 项目：ACTH mRNA的分离和ACTH cDNA的产生， 糖皮质激素的体内受体活化和核结合 受体和糖皮质激素受体本身的表征。 我们 已经鉴定了大约1200个核苷酸的cDNA到ACTH mRNA克隆 转化为p8 R322 该克隆已测序，用于鉴定ACTH mRNA 和从AtT-20细胞中提取的多核糖体RNA，并由另一个 实验室在范德比尔特，以确定和表征ACTH mRNA从 人类的异位肿瘤。 体内的程度 在AtT-20细胞中产生的激活和核转位 已经确定了糖皮质激素激动剂。 这是首次 受体激活和核转位在 vivo. 地塞米松，曲安奈德， 泼尼松龙和皮质酮之间存在严格的相关性 结合亲和力、活化和核结合之间的关系。 此外，委员会认为， 虽然激活的程度随每种类固醇而变化， 激活受体的易位是相似的（大约 在每种情况下，支持核概念， 易位是简单的分配现象。 最后，老鼠 糖皮质激素受体已经被纯化，单克隆抗体已经 也能识别老鼠的糖皮质激素 受体的 这些试剂将对小鼠进行进一步的分析 可能是糖皮质激素受体。 在撰写本文时，稳定的杂交瘤 产生糖皮质激素受体的单克隆抗体， 被描述。 在过去一年中，在若干领域取得了进展。 包括：（1）小鼠和大鼠的免疫纯化 糖皮质激素受体及其蛋白水解片段的分离 小鼠糖皮质激素受体基因;（3）小鼠糖皮质激素受体基因的鉴定 天然糖皮质激素受体的亚基组成。 具体 来年的目标是：（1）隔离老鼠 糖皮质激素受体基因，使用受体的单克隆抗体 （2）在本实验室开发的;（3）描述 各种效力类固醇的完整细胞摄取和生物效力; （3）纯化小鼠和大鼠糖皮质激素受体， 免疫亲和技术。 （丁）

The ultimate objective of this project is to understand the role of glucocorticoid binding and the physiology of hormone action. Toward this end, we have studied several aspects of glucocorticoid receptor physiology, including the kinetics of receptor bind, activation, and nuclear translocation. Progress has been made on three objectives of this project: isolation of ACTH mRNA and production of ACTH cDNA, analysis of in vivo receptor activation and nuclear binding of the glucocorticoid receptor, and characterization of the glucocorticoid receptor itself. We have characterized approximately 1200 nucleotide cDNA to ACTH mRNA cloned into p8R322. This clone has been sequenced, used to identify ACTH mRNA and polysomal RNA extracted from AtT-20 cells, and used by another laboratory at Vanderbilt to identify and characterize ACTH mRNA from ectopic hormone-producing tumors of humans. The extent of in vivo activation and nuclear translocation produced in AtT-20 cells by four glucocorticoid agonists has been determined. This marks the first time that receptor activation and nuclear translocation have been correlated in vivo. It was found for dexamethasone, triamcinolone acetonide, prednisolone, and corticosterone that there was a rigid correlation between binding affinity, activation, and nuclear bind. Furthermore, although the extent of activation varied with each steroid, the fraction of activated receptor that was translocated was similar (approximately 60%) in each case, lending support to the concept that nuclear translocation is a simple partitioning phenomenon. Last, the rat glucocorticoid receptor has been purified and monoclonal antibodies have been produced to it that also recognize the mouse glucocorticoid receptor. These reagents will make further analysis of the mouse glucocorticoid receptor possible. As of this writing, stable hybridomas producing monoclonal antibodies to the glucocorticoid receptor have not been described. During the past year progress has been made in several areas including: (1)\immunopurification of the mouse and rat glucocorticoid receptors and their proteolytic fragments; (2)\isolation of the mouse glucocorticoid receptor gene; and (3)\identification of the subunit composition of the native glucocorticoid receptor. Specific objectives for the coming year are: (1) to isolate the mouse glucocorticoid receptor gene, using a monoclonal antibody to the receptor developed in this laboratory; (2) to characterize the relationship between intact cell uptake and biological potency of steroids of various potency; and (3)\to purify the mouse and rat glucocorticoid receptor with immunoaffinity techniques. (D)