REPRODUCTIVE FUNCTION OF MEMBRANE ESTROGEN RECEPTORS

阐明雌激素和其他类固醇的作用机制 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，以及雌激素类环境因子的作用。尽管它 人们普遍认为雌激素和许多其他类固醇激素 触发依赖于RNA的蛋白质合成，类固醇也是已知的 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，其机制 这些快速的、非基因组的影响还没有得到充分的解释。我们的 假说是雌激素受体(ER)的一个亚群驻留在 质膜介导雌激素的细胞致病作用。我们的 提案定义了一个检验这一假说的模型：雌激素- 诱导垂体瘤细胞释放催乳素。具体目标 用来检验这一假说的方法是：1)将 GH/B6垂体瘤细胞膜ER(Mer)的表达 (1-5分钟)催乳素对雌激素(包括 膜不通透性的时间进程、剂量响应特性 类固醇结合物，以及含血清与限定生长的影响 介质)，并明确将影响定义为非基因组效应(不改变 催乳素基因短时间表达)；2)使用ER抗体(包括 我们自己的)不同的表位来研究它们对催乳素释放的影响 从而映射表位的功能与响应的关系；3) 用免疫学方法将Mer定位于GH/B6细胞质膜 技术(如免疫细胞化学、免疫沉淀和 伴随的放射性激素亲和标记和胰酶去除 来自全细胞的抗原)，并使用与其他可用的 用于确认MER作为ER的身份并检查其暴露情况的抗体 膜上的抗原；分离浓缩的分离细胞群 并通过免疫选择耗尽Mer；以及4)表征Mer(in 与细胞内ER的关系)与类固醇结合有关， 附着在膜上，由于或引起蛋白质的修饰 它驻留在膜上，以及聚合量的调节剂。未来 本研究的目的包括：1)筛选大豆杉的cDNA文库。 以丰富细胞，询问这种特殊形式的受体是否 在核酸水平上差异转录和/或处理，或 仅由翻译后修改产生的结果；2)应用 这些技术和试剂在其他正常和 癌生殖组织；3)天然和合成的筛选 环境雌激素对非基因组毒性的影响；以及4) 该受体的存在与膜生理学的关系 激发分泌和其他快速反应的机制(如离子 通道功能)在各种生殖组织和细胞系中 是从它们衍生出来的。

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.