REPRODUCTIVE FUNCTION OF MEMBRANE ESTROGEN RECEPTORS

阐明雌激素和其他类固醇发挥作用的机制 它们对类固醇敏感细胞的影响对于理解 基本生殖功能，生殖组织肿瘤的行为 起源和雌激素环境因子的作用。 虽然 普遍认为雌激素和许多其他类固醇激素 触发RNA依赖性蛋白质合成，类固醇也被认为发挥 各种短期影响（需要几秒钟到几分钟）， 靶器官。与类固醇的基因组作用相比， 这些快速的、非基因组的影响还没有得到充分的解释。我们 一种假说认为，雌激素受体（ER）的亚群， 质膜介导雌激素的细胞狂暴作用。 我们 该提案定义了一个模型来测试这一假设：雌激素- 诱导垂体瘤细胞分泌催乳素。具体目标 旨在检验这一假设是：1）相关的富集 膜ER（mER）在GH/B6垂体瘤细胞中随时间的增加而迅速增加 (1-5催乳素释放对雌激素的反应（包括 时程、剂量响应和膜不可渗透性的表征 类固醇结合物，以及含血清与确定生长的影响 介质），并明确定义为非基因组效应（不改变 催乳素基因表达在短时间内）; 2）使用ER抗体（包括 我们自己的）不同的表位，以研究它们对催乳素释放的影响 并因此映射与应答相关的表位的功能; 3） 使用免疫组织化学方法将mER定位于GH/B6细胞的质膜上， 技术（如免疫细胞化学，免疫沉淀， 伴随的放射性激素亲和标记和胰蛋白酶去除 来自全细胞的抗原），并使用表位作图与其他可用的抗原结合。 抗体，以确认mER作为ER的身份，并检查 膜中的抗原;分离富集的单独细胞群 并通过免疫选择耗尽mER;和4）表征mER（在 与细胞内ER的关系）关于类固醇结合， 附着在膜上，由于或引起蛋白质的修饰 其在膜中的驻留，以及mER量的调节剂。未来 本研究的目的包括：1）筛选mER-1 cDNA文库， 富集细胞，以询问这种特殊形式的受体是否是 在核酸水平上差异转录和/或加工，或 结果完全来自翻译后修饰; 2）应用 这些技术和试剂，以证明在其他正常和 癌生殖组织; 3）筛选天然和合成的 非基因组毒性效应的环境雌激素;以及4） 该受体的存在与膜生理学特性的相关性 引起分泌和其他快速反应的机制（例如离子 通道功能）在多种生殖组织和细胞系中 源自于他们。

Elucidating the mechanisms by which estrogens and other steroids exert their effect upon steroid-sensitive cells is critical for understanding basic reproductive function, the behavior of tumors of reproductive tissue origin, and the actions of estrogenic environmental agents. Although it is generally accepted that estrogens and many other steroid hormones trigger RNA-dependent protein synthesis, steroids are also known to exert a variety of short-term effects (taking seconds to minutes) on their target organs. Compared to the genomic actions of steroids, the mechanism of these fast, non-genomic effects have not been adequately explained. Our hypothesis is that a subpopulation of estrogen receptors (ER) residing in the plasma membrane mediate cellular rabid actions of estrogens. Our proposal defines a model in which to test this hypothesis: Estrogen- induced prolactin release from pituitary tumor cells. Specific aims designed to test this hypothesis are: 1) Correlate the enrichment of membrane ER (mER) in GH/B6 pituitary tumor cells with the increased rapid (1-5 minute) prolactin release response to estrogen (including characterization of time course, dose response to membrane-impermeable steroid conjugates, and the effect of serum-containing vs. defined growth media) and specifically define the effect as non-genomic (not altering prolactin gene expression at short times); 2) Use ER antibodies (including our own) to different epitopes to study their effect on prolactin release and thus map functions of the epitopes in relationship to the response; 3) Localize mER to the plasma membrane in GH/B6 cells using immunological techniques (such as immunocytochemistry, immunoprecipitation with concomitant radioactive hormone affinity labeling, and trypsin removal of antigen from whole cells) and use epitope mapping with other available antibodies to confirm the identity of mER as an ER and examine exposure of the antigen in the membrane; isolate separate cell populations enriched and depleted for mER by immunoselection; and 4) Characterize mER (in relationship to intracellular ER) with respect to steroid binding, attachment to the membrane, modifications of the protein due to or causing its residence in the membrane, and modulators of mER quantity. Future goals of this study include: 1) Screening of a cDNA library made from mER- enriched cells to ask whether this special form of the receptor is differentially transcribed and/or processed at the nucleic acid level, or results solely from post-translational modifications; 2) Application of these techniques and reagents to demonstrate an mER in other normal and cancerous reproductive tissues; 3) Screening of both natural and synthetic environmental estrogens for non-genomic toxicity effects; and 4) Correlation of the presence of this receptor to membrane physiological mechanisms for eliciting secretion and other rapid responses (such as ion channel function) in a variety of reproductive tissues and cell lines derived from them.