NONGENOMIC STIMULATION OF ESTROGEN

雌激素的非基因组刺激

• 批准号: 6186163
• 负责人: VICTOR D. RAMIREZ
• 金额: $ 20.97万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6186163
• 关键词: dopamine; estrogen receptors; estrogens; hormone regulation /control mechanism; laboratory rat; molecular cloning; neuroendocrine system; protein purification; receptor binding

DESCRIPTION (Adapted from applicant's abstract): The long-term objectives are to understand the non-genomic actions of E upon the CNS, an issue of relevance since steroid hormones are known to affect a variety of brain functions. We propose, on the grounds of our preliminary work and the work of others, that this steroid can influence cell activity not only by actions on the classical nuclear receptors but also by actions on the plasmalemma of neurons or glia cells through a specific membrane estrogen receptor (mER) leading to changes in intracellular messengers responsible for a particular neurochemical activation. The central hypothesis of this revised project postulates that the rapid stimulation of E upon DA release from rat striatal tissue depends on an interaction between a particular chemical group of the steroid and a specific binding domain in the mER of the corpus striatum (CS) cells. This, depending on the estrous cycle, leads to changes in intracellular messengers ultimately responsible for DA release. The specific aims are: (1) to establish that the rapid effect of E upon in vitro DA release from rat striatal fragments is a membrane mediated event taking place during a particular phase of the rat estrous cycle; (2) to establish that E binds stereospecifically and with high affinity to sites in the plasmalemma fraction from the rat CS and that the specific binding sites for E correspond to mERs and not to "acceptor" molecules; (3) to isolate, purify and chemically characterize the mER from the rat CS and generate polyclonal and monoclonal antibodies against this protein; and (4) to initiate cloning studies to isolate a cDNA clone for E-6-125-IBSA through screening a rat brain lambda ZAP-II library for selective phage plaques producing proteins with affinity for E-6-IBSA. To address these goals, I will take advantage of novel ligands to examine steroid membrane interactions, the so called steroid-BSA complexes. The hormone is covalently bound to bovine serum albumin (BSA), a protein that can either be radioiodinated (for use as a ligand in binding studies) or coupled to an agarose matrix (for use as an affinity chromatography column to isolate and purify the receptor). The work will lead to a better understanding of the role of steroids at the neuronal membrane level and the characterization of a membrane receptor for E may lead to new and useful pharmacological drugs.

描述（改编自申请人的摘要）：长期目标 是为了了解 E 对 CNS 的非基因组作用，这是一个问题 相关性，因为已知类固醇激素会影响多种大脑 功能。 基于我们的前期工作和工作，我们建议 其他人认为，这种类固醇不仅可以通过作用来影响细胞活性 对经典核受体的作用，而且还通过对质膜的作用 神经元或神经胶质细胞通过特定的膜雌激素受体（mER） 导致负责特定功能的细胞内信使发生变化 神经化学激活。 这个修订项目的中心假设 假设大鼠纹状体释放 DA 后 E 的快速刺激 组织取决于特定化学基团之间的相互作用 类固醇和纹状体 (CS) mER 中的特定结合域 细胞。 根据发情周期，这会导致 最终负责 DA 释放的细胞内信使。 这 具体目标是： (1) 确定 E 对 大鼠纹状体碎片的体外 DA 释放是膜介导的事件 发生在大鼠动情周期的特定阶段； (2) 至 确定 E 与位点立体特异性结合并具有高亲和力 大鼠 CS 的质膜部分和特异性结合位点 对于 E 对应于 mER，而不是“受体”分子； (3) 隔离， 从大鼠 CS 中纯化并化学表征 mER，并生成 针对该蛋白的多克隆和单克隆抗体； (4) 至 启动克隆研究，通过以下方式分离 E-6-125-IBSA 的 cDNA 克隆 筛选大鼠脑 lambda ZAP-II 文库中的选择性噬菌斑 产生对 E-6-IBSA 具有亲和力的蛋白质。 为了实现这些目标，我 将利用新型配体来检查类固醇膜 相互作用，即所谓的类固醇-BSA 复合物。 激素是 与牛血清白蛋白 (BSA) 共价结合，牛血清白蛋白是一种可以 放射性碘标记（用作结合研究中的配体）或与 琼脂糖基质（用作亲和层析柱以分离和 纯化受体）。 这项工作将使人们更好地了解 类固醇在神经元膜水平的作用及其特征 E的膜受体可能会产生新的有用的药物。