NONGENOMIC STIMULATION OF ESTROGEN

雌激素的非基因组刺激

• 批准号: 2034856
• 负责人: VICTOR D. RAMIREZ
• 金额: $ 20.19万
• 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/2034856
• 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(摘自申请者的摘要)：长期目标 是为了了解E对中枢神经系统的非基因组作用，这是一个问题 与此相关，因为类固醇激素已知会影响多种大脑 功能。我们建议，根据我们的前期工作和工作 在其他方面，这种类固醇不仅可以通过行动影响细胞活动 在经典的核受体上，也通过对质膜的作用 神经元或神经胶质细胞通过特定的膜雌激素受体(Mer) 导致负责特定事件的细胞内信使的变化 神经化学激活。这个修订后的项目的中心假设 推测E对大鼠纹状体DA释放的快速刺激 组织依赖于特定化学基团之间的相互作用 类固醇与纹状体(CS)聚合体中的一个特定结合域 细胞。根据发情周期的不同，这会导致 细胞内信使最终负责DA的释放。这个 具体目标是：(1)确定E对中国经济的快速影响 大鼠纹状体碎片体外释放DA是一种膜介导的事件 发生在大鼠发情周期的特定阶段； 确定E以立体特异性结合，并与 大鼠海绵体质膜组分及其特异性结合部位 E对应于MERS而不对应于受体分子；(3)分离， 大鼠CS聚合体的纯化及化学特性研究 抗该蛋白的多克隆和单抗；以及(4) 开始克隆研究以分离E-6-125-IBSA的cDNA克隆 大鼠脑内lambda ZAP-II噬菌体噬菌体斑块的筛选 产生与E-6-IBSA有亲和力的蛋白质。为了实现这些目标，我 将利用新的配体来检测类固醇膜 相互作用，即所谓的类固醇-BSA复合体。荷尔蒙是 与牛血清白蛋白(BSA)共价结合，这种蛋白质可以是 放射性碘化的(在结合研究中用作配体)或偶联到一个 琼脂糖基(用作亲和层析柱以分离和 纯化受体)。这项工作将使我们更好地理解 类固醇在神经细胞膜水平上的作用及其特征 E的膜受体可能导致新的有用的药理药物。