NONGENOMIC STIMULATION OF ESTROGEN

描述（改编自申请人摘要）：长期目标 是为了了解E对CNS的非基因组作用，这是一个 相关性，因为已知类固醇激素会影响各种大脑 功能协调发展的 我们建议，根据我们的初步工作和 其他人，这种类固醇可以影响细胞活性不仅通过行动 对经典的核受体，但也通过对质膜的作用， 通过特定的膜雌激素受体（mER） 导致细胞内负责一个特定的 神经化学激活 这个修订项目的核心假设是 推测E对大鼠纹状体DA释放的快速刺激， 组织的生长取决于组织的特定化学基团之间的相互作用。 类固醇和纹状体（CS）mER中的特异性结合域 细胞 这取决于发情周期，导致 最终负责DA释放的细胞内信使。 的 具体目标是：（1）确定E在体内的快速效应， 大鼠纹状体碎片的体外DA释放是膜介导的事件 发生在大鼠发情周期的特定阶段;（2） 确定E立体特异性结合，并以高亲和力结合于 从大鼠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）为了分离， 从大鼠CS中纯化和化学表征mER，并产生 针对该蛋白质的多克隆和单克隆抗体;和（4） 启动克隆研究以分离E-6-125-IBSA cDNA克隆， 筛选用于选择性噬菌体噬斑的大鼠脑λ ZAP-II文库 产生对E-6-IBSA具有亲和力的蛋白质。 为了实现这些目标，我 将利用新的配体来检查类固醇膜 相互作用，即所谓的类固醇-BSA复合物。 该激素 共价结合牛血清白蛋白（BSA），一种蛋白质， 放射性碘标记的（用作结合研究中的配体）或偶联到 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 类固醇激素在神经元膜水平的作用以及 E的膜受体可能导致新的和有用的药理学药物。

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.