Functional Analysis of Nuclear Receptor Variants

描述（由申请人提供）：组成型雄甾烷受体（CAR，NR1I3）是核受体超家族的成员，主要在肝脏中表达。 CAR 在调节生物转化的所有三个阶段中发挥着关键作用，这些途径决定了与药物、其他外源性物质和内源性物质（如胆汁酸、甲状腺激素、血红素和类固醇）暴露相关的代谢和毒性的命运。由于该受体系统作为人类异生素处置决定因素的重要性，以及 CAR 作为生理调节剂的作用日益得到认可，因此必须充分表征人类 CAR 蛋白质组指导的生物过程。前期研究项目的进展包括发现CAR、CAR2和CAR3的新形式；通过使用选择性剪接机制产生的受体，分别导致 4- 和 5- 氨基酸插入受体的配体结合域内。这些受体与人类肝脏中的 CAR 同时表达，但具有高度独特的功能属性。与 CAR 本身不同，变体 CAR 是配体激活受体，表现出选择性化学激活特征，并且似乎能够差异调节靶基因表达。我们假设选择性剪接增强了 CAR 分子的功能多样性，导致作为外源传感器的重叠但有区别的作用，驱动肝脏基因表达网络的相互作用，进而提供关键的整合信号，指导人类对化学环境的生理反应。为了检验我们的假设，我们将部署三个具体目标，包括：1）结晶 CAR 蛋白的结构分析； 2) 使用原代人肝细胞培养模型来实现受体驱动的转录分析、靶基因相互作用以及受体细胞内定位动态的评估； 3) 人源化转基因小鼠研究，以分析体内受体调节的转录控制，以及一系列血清标志物中受体表达的表型影响。我们预测，基因组程序的各自调节以及由变体 CAR 控制的信号通路对人类健康具有重要的功能意义，决定着药物相互作用、药物和胆汁酸诱导的肝毒性、致癌物和类固醇代谢以及脂质和能量稳态的调节等结果。拟议研究的结果将有助于关于 CAR 核受体作为动态基因调控网络的直接调节剂的作用的重要新进展和见解，该动态基因调控网络决定对外源性物质和内源性物质的毒理学和生理反应。 公共健康相关性：在本研究项目中，我们的研究将描述一系列新型受体蛋白的作用，这些蛋白与药物和环境化学物质相互作用，并调节肝脏代谢物质的能力。这些受体被称为组成型雄甾烷受体，是肝细胞中存在的一系列传感蛋白的一部分，有助于处理细胞化学环境的性质，从而更好地调整其对我们所接触的化学物质进行解毒的能力。

DESCRIPTION (provided by applicant): The constitutive androstane receptor (CAR, NR1I3) is a member of the nuclear receptor superfamily that is expressed primarily in the liver. CAR plays a key role in regulating all three phases of biotransformation, pathways that determine the fates of metabolism and toxicity associated with exposures to drugs, other xenobiotics, and endogenous substances such as bile acids, thyroid hormone, heme, and steroids. Due to the importance of this receptor system as a determinant of xenobiotic disposition in man, together with CAR's increasingly recognized role as a physiological regulator, it is imperative to fully characterize the biological processes directed by the human CAR proteome. Progress in the previous research program included the discovery of novel forms of CAR, CAR2 and CAR3; receptors generated through the use of alternative splicing mechanisms that result in the insertion of 4- and 5- amino acids, respectively, within the receptors' ligand binding domain. These receptors are expressed simultaneously with CAR in human livers, but possess highly unique functional attributes. Unlike CAR itself, the variant CARs are ligand-activated receptors, exhibit selective chemical activation profiles, and appear to differentially modulate target gene expression. We hypothesize that alternative splicing enhances the functional diversity of CAR molecules, resulting in overlapping, yet discriminating roles as xenobiotic sensors, driving the interplay of hepatic gene expression networks that in turn, provide critical integration signals directing human physiological responses to the chemical milieu. To test our hypotheses, a progression of three specific aims will be deployed that include: 1) the structural analysis of crystallized CAR proteins; 2) use of primary human hepatocyte culture models to enable receptor-driven transcriptional profiling, target gene interactions, and assessment of the dynamics of receptor intracellular localization; and, 3) humanized transgenic mouse investigations to analyze the transcriptional controls modulated by the receptors in vivo, and the phenotypic impact of receptor expression within a series of serum markers. We predict that the respective modulation of genomic programs and the resulting signaling circuitry controlled by the variant CARs have critical functional implications for human health, determining outcomes such as drug-drug interactions, drug and bile acid induced hepatotoxicity, carcinogen and steroid metabolism, and the regulation of lipid and energy homeostasis. The results of the proposed research will contribute important new advances and insights regarding the role of the CAR nuclear receptors as direct modulators of the dynamic gene regulatory networks that determine toxicological and physiological responses to both xenobiotic and endogenous substances. PUBLIC HEALTH RELEVANCE: In this research program, our studies will characterize the roles of a novel series of receptor proteins that interact with pharmaceuticals and environmental chemicals and function to regulate the liver's capacity to metabolize substances. These receptors are termed constitutive androstane receptors and are part of a battery of sensing proteins that exist with liver cells to assist in processing the nature of the cell's chemical environment so that it can better tune its ability to detoxify chemicals we are exposed to.