AH RECEPTOR ANATOMY--IMPLICATIONS FOR DIOXIN TOXICITY

关于环境风险的非决定性流行病学研究 污染物，例如超过2，3，7，8的人类健康担忧- 四氯二苯并对二恶英(TCDD，二恶英)，强调需要 这些毒物学问题的机械论方法。大多数，如果不是全部的话 TCDD的生物效应是通过芳香烃介导的 (啊)受体。这项研究提案的中心是这样一个前提： 对TCDD毒性的机械理解，取决于识别 并对参与AH受体功能的所有成分进行了表征。 在被TCDD结合后，ah受体与特定的DNA增强子结合 调节基因表达的元素。靶基因包括编码 正常细胞中重要的药物代谢酶和生长因子 生长和分化。Ah受体与DNA的相互作用涉及 AH受体核转运体(ARNT)蛋白，DNA结合伙伴。我们 先前发现110 kDa是ah受体DNA结合的一部分 很复杂。在这里，我们的初步研究确定了一种独特的110 kDa DNA- 与大鼠Ah受体和Arnt结合的结合蛋白 蛋白。110 kDa蛋白质片段的肽序列显示NO 与已知蛋白质同源。我们的数据表明110 kDa的蛋白质 代表了ah受体DNA结合复合体的另一个组成部分。 来自其他人的功能、遗传和生化证据提供了进一步的 支持我们的假设，即AH受体DNA结合和功能 与Arnt蛋白以外的其他因素有关。因此， 这项提议试图定义110 kDa蛋白在Ah. 受体DNA结合和功能，在我们的长程背景下 目的了解TCDD诱导的细胞内信号转导。我们 提出四个具体目标来检验这一假设： 1.大鼠110 kDa蛋白基因的克隆CDNA克隆将被分离为 这是了解110 kDa蛋白质功能的第一步。氨基 纯化的110多个多肽片段的酸性显微测序 KDA蛋白允许设计用于简并的寡核苷酸 在两种cdna克隆策略中的任何一种。 2.大鼠110 kDa人同源基因克隆的分离 蛋白。为了研究110 kDa蛋白在人血液中的作用 受体功能，人类蛋白的cDNA克隆将被分离 使用大鼠克隆作为探针，并用于表达研究。 3.酵母双杂交系统的研究。双混合系统 代表了分离110 kDa蛋白质克隆的第三种选择，即 与使用简并寡核苷酸的两种策略不同。 双杂交系统还允许在体内检查功能 110 kDa蛋白质之间的重要蛋白质相互作用，啊 受体和Arnt蛋白。 4.检测110 kDa蛋白在AH中的作用的表达研究 受体DNA结合和功能。研究将集中在确定 110 kDa蛋白的重要蛋白质结构域及其对Ah的影响 受体与Arnt蛋白的DNA结合及功能。

Inconclusive epidemiological studies on the risks of environmental contaminants, exemplified by the human health concerns over 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD, dioxin), emphasizes the need for a mechanistic approach to these toxicological issues. Most, if not all the biological effects of TCDD are mediated through the aromatic hydrocarbon (Ah) receptor. This research proposal centers on the premise that a mechanistic understanding of TCDD toxicity, is contingent upon identifying and characterizing all the components involved in Ah receptor function. Following binding by TCDD, the Ah receptor binds to specific DNA enhancer elements to regulate gene expression. Target genes include those encoding drug metabolizing enzymes and growth factors important in normal cell growth and differentiation. The Ah receptor-DNA interaction involves the Ah receptor nuclear translocator (Arnt) protein, a DNA-binding partner. We previously identified a 110 kDa as part of the Ah receptor DNA-binding complex. Here, our preliminary research identifies a distinct 110 kDa DNA- binding protein that copurifies with both the rat Ah receptor and Arnt protein. Peptide sequencing of 110 kDa protein fragments reveals no homology to known proteins. Our data suggests that the 110 kDa protein represents an additional component of the Ah receptor DNA-binding complex. Functional, genetic and biochemical evidence from others provides further support for our hypothesis that Ah receptor DNA binding and function involves association with factors other than the Arnt protein. Therefore, this proposal seeks to define the role of the 110 kDa protein in Ah receptor DNA-binding and function, in the context of our long range objective of understanding TCDD-induced intracellular signaling. We propose four specific aims to test the hypothesis: 1. cDNA cloning the rat 110 kDa protein. cDNA clones will be isolated as a first step towards learning the function of the 110 kDa protein. Amino acid microsequencing on several peptide fragments from the purified 110 kDa protein permits design of degenerate oligonucleotides intended for use in either of two cDNA cloning strategies. 2. The isolation of cDNA clones for the human homolog of the rat 110 kDa protein. In order to study the role of the 110 kDa protein in human Ah receptor function, cDNA clones for the human protein will be isolated using the rat clones as probes, and used in expression studies. 3. Studies using the yeast two-hybrid system. The two-hybrid system represents a third alternative for isolating 110 kDa protein clones, that is distinct from the two strategies using the degenerate oligonucleotides. The two-hybrid system also permits an in vivo examination of functionally important protein-protein interactions between the 110 kDa protein, Ah receptor and Arnt protein. 4. Expression studies to examine the role of the 110 kDa protein in Ah receptor DNA binding and function. Studies will focus on identifying the important protein domains i the 110 kDa protein and their impact on Ah receptor and Arnt protein DNA binding and function.