SIGNALING BY NON-CLASSICAL LIGANDS OF ESTROGEN RECEPTOR NOVEL APPROACHES TO DET

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。雌激素影响大量的生理功能和疾病状态。除了内源性激素，人类还会从饮食、环境和药物中接触到各种各样的雌激素化合物。这些雌激素受体（ER）的非经典配体可以对人体健康产生重大影响。目的1：有效的雌激素活性检测方法对于药物开发、环境检测以及受体功能和机制的研究非常重要。基于酵母的外源表达内质网和报告基因的检测成本低廉，应用广泛，但存在对一些选择性雌激素受体调节剂（SERMS）和植物雌激素的反应与在哺乳动物细胞中观察到的非常不同的缺陷。该项目将在线虫C. elegans中开发和测试报告系统，该系统很容易在培养中生长。据推测，人类和秀丽隐杆线虫更密切的进化关系将使该系统产生比基于酵母的分析更相关的结果，但保留了简单和低成本的优势。目的2：尽管人类大量接触非经典内质网配体，但其作用方式尚不完全清楚。serm如他莫昔芬通常具有双相剂量-反应曲线，低浓度时具有内质网激动作用，高浓度时具有拮抗作用。有人提出，这些化合物除了占据内质网的激素结合口袋外，还以较低的亲和力结合到介导拮抗的第二个位点。一些植物雌激素具有类似的双相作用，表明它们是该位点的天然配体。该项目将确定植物雌激素和ANGELS（非基因致型雌激素样信号激活剂，一类新的内质网靶向药物）是否与SERMS竞争结合第二位点，通过靶向诱变识别和切除该位点，并通过报告基因分析、微阵列分析和实时PCR表征该位点在内质网功能中的重要性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Estrogens affect a large number of physiological functions and disease states. In addition to endogenous hormones, humans are exposed to a wide variety of estrogenic compounds in the diet, environment, and pharmaceuticals. These non-classical ligands for the estrogen receptor (ER) can have major effects on human health. Aim 1: Efficient methods of testing for estrogenic activity are important for drug development, environmental testing, and research on receptor functions and mechanisms. Yeast-based assays using exogenously expressed ERs and reporter genes are inexpensive and widely used, but have the defect that the response to some SERMS (selective estrogen receptor modulators) and phytoestrogens is very different from that seen in mammalian cells. This project will develop and test a reporter system in the nematode C. elegans, which is easily grown in culture. It is hypothesized that the closer evolutionary relationship of humans and C. elegans will enable this system to produce more relevant results than yeast-based assays, but retain the advantages of simplicity and low cost. Aim 2: Although there is substantial human exposure to non-classical ER ligands, their modes of action are incompletely understood. SERMs such as tamoxifen often have biphasic dose-response curves, with ER agonism at low concentrations and antagonism at high concentrations. It has been proposed that in addition to occupying the hormone-binding pocket of ER, these compounds bind (with lower affinity) to a 2nd site that mediates antagonism. Some phytoestrogens have a similar biphasic effect, suggesting that they are natural ligands for this site. This project will determine if phytoestrogens and ANGELS (activators of non-genotropic estrogen-like signaling, a new class of ER-targeted drugs) compete with SERMS for binding to the 2nd site, identify and ablate the site via targeted mutagenesis, and characterize the significance of the site in ER function by reporter gene assays, microarray analysis and real-time PCR.