PHYSIOLOGY OF PITUITARY CELL GLUCOCORTICOID BINDING

垂体细胞糖皮质激素结合的生理学

• 批准号: 3182912
• 负责人: ROBERT W HARRISON
• 金额: $ 12.78万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-01 至 1988-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3182912
• 关键词: cell membrane; clone cells; corticosterone; exo alpha sialidase; genetic manipulation; glucocorticoids; hormone regulation /control mechanism; membrane permeability; pituitary neoplasms

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)

该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。 朝这个方向 最后，我们研究了糖皮质激素受体的几个方面 生理学，包括受体结合、激活和动力学 核易位。 本次会议的三个目标均取得了进展 项目：ACTH mRNA 的分离和 ACTH cDNA 的生产、分析 体内受体激活和糖皮质激素的核结合 受体，以及糖皮质激素受体本身的表征。 我们 已将大约 1200 个核苷酸的 cDNA 克隆至 ACTH mRNA 进入p8R322。 该克隆已被测序，用于鉴定 ACTH mRNA 和从 AtT-20 细胞中提取的多聚体 RNA，并由另一个细胞使用 范德比尔特实验室鉴定和表征 ACTH mRNA 人类产生异位激素的肿瘤。 体内的程度 AtT-20 细胞中四种物质产生的激活和核转位 糖皮质激素激动剂已确定。 这标志着第一次 受体激活和核易位相关 体内。 发现了地塞米松、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， 尽管每种类固醇的激活程度各不相同，但比例 被易位的激活受体的数量是相似的（大约 60%）在每种情况下都支持核能的概念 易位是一种简单的分配现象。 最后是老鼠 糖皮质激素受体已纯化，单克隆抗体已研制成功 它也能识别小鼠糖皮质激素 受体。 这些试剂将对小鼠进行进一步分析 糖皮质激素受体可能。 截至撰写本文时，稳定的杂交瘤 产生针对糖皮质激素受体的单克隆抗体尚未 被描述。 过去的一年里，多项工作取得了进展 领域包括：（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； (2)\隔离 小鼠糖皮质激素受体基因；和 (3)\ 的识别 天然糖皮质激素受体的亚基组成。 具体的 来年的目标是：（1）隔离小鼠 糖皮质激素受体基因，使用该受体的单克隆抗体 本实验室开发的； (2) 表征之间的关系 完整的细胞摄取和各种效力类固醇的生物效力； (3)\纯化小鼠和大鼠糖皮质激素受体 免疫亲和技术。 (四)