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。朝向这个方向 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 核移位。在这方面的三个目标方面取得了进展 项目：ACTH信使核糖核酸的分离及ACTH基因的制备、分析 糖皮质激素的体内受体激活和核结合 受体，以及糖皮质激素受体本身的特性。我们 克隆了约1200个核苷酸的cDNAto ACTH mRNA. 转化到p8R322中。该克隆已测序，用于鉴定ACTH mRNA 和从AtT-20细胞中提取的多倍体RNA，并由另一个 范德比尔特实验室鉴定和鉴定ACTH mRNA 人类的异位荷尔蒙分泌肿瘤。在体内的程度 四种细胞因子对AtT-20细胞的激活和核转位 糖皮质激素激动剂已经确定。这标志着第一次 受体激活和核移位在 活着。地塞米松，曲安奈德， 强的松龙和皮质酮之间有严格的相关性 结合亲和力、激活和核结合之间的关系。此外， 尽管每种类固醇的激活程度都不同，但 被移位的激活受体的比例相似(大约 60%)，都支持核能的概念 易位是一种简单的分割现象。最后，老鼠 糖皮质激素受体已被提纯，并产生了单抗 对它产生了也能识别小鼠糖皮质激素 受体。这些试剂将对老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 产生抗糖皮质激素受体的单抗还没有 都被描述过。在过去的一年里，在几个方面取得了进展 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物片段； 小鼠糖皮质激素受体基因；(3)鉴定 天然糖皮质激素受体的亚基组成。特定的 来年的目标是：(1)隔离小鼠 糖皮质激素受体基因，使用针对该受体的单抗 在这个实验室中开发的；(2)描述 不同效力的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。(D)

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)