FUNCTIONAL ANALYSIS OF NUCLEAR RECEPTOR VARIANTS

描述（由申请人提供）：响应细胞外刺激、病原体、药物给药或环境因子的基因激活需要高度整合的信号转导过程，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（CYP450）基因以及其他生物转化功能是这些信号的靶点。与CYP一样，核激素受体（NHR）由基因超家族编码，并在很大程度上协调化学和激素效应物引发的反应。这项研究计划将特别关注人类组成型雄甾烷受体（CAR）和RXR α受体（RXR α），这些受体最近已被表征为介导“苯巴比妥样”诱导剂化合物的转录活性，其中包括各种药物和异生素制剂。将用该程序检验的主要假设是CAR和RXRa核受体在人群中具有遗传多态性和结构变异性。此外，我们将测试的假设，即各自的变体受体决定不同的生物反应。提出了一系列3个实验目标，以促进研究计划从最初的发现阶段到体外和体内方法的进展，从而实现CAR和RXRa结构变体的生物学表征。研究将包括部署新的敲除小鼠模型，该模型能够在原代肝细胞和肝脏组织中表达人NHR及其结构变体，否则这些组织将具有无效受体背景。将分析“人源化”小鼠与受体同种型表达相关的改变的生物活性，包括响应于药物暴露的扰动、使用扫描荧光显微镜的异常受体相互作用和使用DNA微阵列杂交的基因表达库的变化。人群中存在的变异NHR受体变异体可能决定了化学暴露引起的药物反应和毒性的个体间差异。此外，结构变异受体的存在可能导致人类疾病（包括某些癌症）风险的个体差异。从这些调查中获得的数据将产生重要的新的遗传和生物学信息的NHR超家族的突出成员的结构多样性。

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.