FUNCTIONAL ANALYSIS OF NUCLEAR RECEPTOR VARIANTS

描述（由申请人提供）：基因在响应细胞外刺激、病原体、药物管理或环境因子时的激活，需要一个高度集成的信号转导过程，该过程指导转录机制调节适当的基因网络。一系列细胞色素P450 （CYP）基因以及其他生物转化功能是这些信号的靶标。与CYPs一样，核激素受体（nhr）由一个基因超家族编码，并在很大程度上协调由化学和激素效应物引发的反应。本研究计划将特别关注人类组成型雄甾受体（CAR）和rxrα受体（RXRa），这两种nhr最近被描述为介导“类苯巴比妥”诱导剂化合物的转录活性，包括各种药物和外源药物。该计划将测试的主要假设是，CAR和RXRa核受体在人类群体中具有遗传多态性和结构变异。此外，我们将测试这一假设，即各自的变异受体决定不同的生物反应。一系列的3个实验目标被推进，以促进研究计划的进展，从最初的发现阶段到体外和体内方法，以实现CAR和RXRa结构变异的生物学表征。研究将包括部署新的敲除小鼠模型，使人类nhr及其结构变异能够在原代肝细胞和具有零受体背景的肝组织中表达。将分析“人源化”小鼠与受体同种异构体表达相关的生物活性变化，包括药物暴露引起的扰动、扫描荧光显微镜下的受体异常相互作用以及DNA微阵列杂交技术下基因表达库的变化。人群中NHR受体变异的存在可能表明药物反应和化学品暴露引起的毒性存在实质性的个体差异。此外，结构变异受体的存在可能导致人类疾病风险的个体差异，包括某些癌症。从这些调查中获得的数据将产生关于NHR超家族突出成员的结构多样性的重要的新的遗传和生物学信息。

DESCRIPTION (provided by applicant): Activation of genes in response to extracellular stimuli, pathogens, pharmaceutical administration, or environmental agents, requires a highly integrated signal transduction process that directs the transcriptional machinery to modulate the appropriate network of genes. An array of cytochrome P450 (CYP) genes, as well as other biotransformation functions, are targets of these signals. Like the CYPs, the nuclear hormone receptors (NHRs) are encoded by a superfamily of genes and largely orchestrate the responses triggered by chemical and hormonal effectors. This research proposal will focus in particular on the human constitutive androstane receptor (CAR) and the RXRalpha receptor (RXRa), NHRs that have been characterized recently to mediate the transcriptional activity of 'phenobarbital-like' inducer compounds that include a variety of pharmaceuticals and xenobiotic agents. The primary hypothesis that will be tested with this program is that the CAR and RXRa nuclear receptors are genetically polymorphic and structurally variant in human populations. Furthermore, we will test the hypothesis that the respective variant receptors dictate differential biological responses. A series of 3 experimental aims are advanced to facilitate the progression of the research program through initial discovery phases to in vitro and in vivo approaches to enable biological characterization of CAR and RXRa structural variants. Investigations will include the deployment of novel knockout mouse models that enable expression of the human NHRs, and structural variants thereof, in primary hepatocytes and in liver tissues that otherwise possess null receptor backgrounds. The "humanized" mice will be analyzed for altered biological activities associated with expression of the receptor isoforms, including perturbations in response to drug exposures, aberrant receptor interactions using scanning fluorescence microscopy, and changes in the repertoire of gene expression using DNA microarray hybridizations. The presence of variant NHR receptor variants in the population likely dictates substantial interindividual differences in drug response and toxicities resulting from chemical exposures. In addition, the existence of structurally variant receptors may contribute to individual differences in the risk for human diseases, including certain cancers. The data obtained from these investigations will generate important new genetic and biological information regarding the structural diversity of prominent members of the NHR superfamily.