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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 雌激素影响许多生理功能和疾病状态。除了内源性激素外，人类还暴露于饮食、环境和药物中的各种雌激素化合物。雌激素受体（ER）的这些非经典配体可对人类健康产生重大影响。目标1：雌激素活性的有效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都是非常重要的。使用外源表达的ER和报告基因的基于酵母的测定是廉价且广泛使用的，但具有对某些SERMS（选择性雌激素受体调节剂）和植物雌激素的响应与在哺乳动物细胞中所见的非常不同的缺陷。本计画将在线虫C.这是一种很容易在培养中生长的线虫。推测人类与C. elegans将使该系统能够产生比基于酵母的测定更相关的结果，但保留了简单和低成本的优点。目的2：虽然有大量的人类暴露于非经典的ER配体，他们的作用模式是不完全了解。SERMs如他莫昔芬通常具有双相剂量反应曲线，低浓度时具有ER激动作用，高浓度时具有拮抗作用。已经提出，除了占据ER的酶结合口袋之外，这些化合物结合（以较低的亲和力）介导拮抗作用的第二位点。 一些植物雌激素具有类似的双相效应，表明它们是该位点的天然配体。本项目将确定植物雌激素和ANGELS（非遗传性雌激素样信号传导的激活剂，一类新的ER靶向药物）是否与SERMS竞争结合到第二个位点，通过靶向诱变鉴定和消除该位点，并通过报告基因测定，微阵列分析和实时PCR表征该位点在ER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.