AH RECEPTOR ASSOCIATED PROTEINS--ROLE IN DIOXIN ACTION

接触2，3，7，8，-四氯二苯并-对二恶英(TCDD，二恶英)和 相关的化合物导致了各种各样的物种和组织- 特定的毒性和生物影响，其中许多似乎是 由AH受体(AhR)介导，AhR是一种细胞内蛋白，对其 这些化学物质具有很高的亲和力。我们研究的总体目标是 是为了了解AhR复合体的分子机制 调节二恶英的作用。AhR是一种依赖于配体的转录 通过与特定DNA相互作用来调节基因表达的因子 增强元件，二恶英响应元件(DRE)，毗邻TCDD- 反应基因。TCDD：AHR：DRB络合物的形成导致激活 基因表达，并依赖于至少一种额外的蛋白质， Ah受体核转运体(ARNT)。AhR和ARNT基因的克隆 基因已经揭示了每个包含的结构域都与蛋白质有关 二聚化和DNA结合。最近，我们证明了 含有At的AhR复合体的多种异构体DNA结合形式 至少三个不同的蛋白质亚基。考虑到功能性活动 已知的蛋白反式激活剂依赖于特定的 与之相关的蛋白质(S)，我们假设 AHR和AhR复合体相关蛋白可能导致改变 AHR综合体的功能，因此将有助于 毒性和基因的物种和组织特异性差异 表情。在这里，我们建议描述多个DNA结合 AhR复合体的形式以及分离和表征AhR- 相关蛋白质。利用AhR和Arnt抗体我们将确定 结合多个蛋白AhR复合亚单位的同源性 (并且可以以TCDD依赖的方式交联化)到DRE。多重 将从豚鼠中分离出AhR复合体的DNA结合形式 结合常规和磁性DNA亲和结合的肝脏 技巧。每个复合体的功能活性将通过以下方式进行评估 它们在体外刺激转录和DNA的能力 每个络合物的结合将通过凝胶位移、UV- 交联、DNA突变和DNA足迹。在免费赠送的 方法，从小鼠、豚鼠中分离出AhR相关蛋白 和稳定表达重组AhR融合基因的人细胞系 蛋白表达载体。上存在亲和性标签 重组蛋白将允许快速和选择性地分离 重组AhR及其相关蛋白。生化和生物化学 分离的络合物及其蛋白质的功能性质 组件将在重建实验和身份验证中进行检查 通过序列分析确定的相关蛋白。CDNA克隆 编码AhR相关蛋白的基因将从cDNA文库中分离 使用酵母双杂交系统以及来自 纯化蛋白的氨基酸序列。我们的研究结果将 不仅加深了我们对二恶英分子机理的了解 行动，并提供对TCDD响应的多样性的洞察 人类和动物，但它们将有助于我们了解 哺乳动物基因表达的可诱导调节。

Exposure to 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related compounds results in a wide variety of species- and tissue- specific toxic and biological effects, many of which appear to be mediated by the Ah receptor (AhR), an intracellular protein to which these chemicals bind with high affinity. The overall goal of our research is to understand the molecular mechanisms by which the AhR complex modulates dioxin action. The AhR is a ligand-dependent transcription factor that regulates gene expression via interaction with a specific DNA enhancer element, the dioxin responsive element (DRE), adjacent to TCDD- responsive genes. TCDD:AhR:DRB complex formation leads to activation of gene expression and is dependent upon at least one additional protein, the Ah receptor nuclear translocator (ARNT). Cloning of the AhR and ARNT genes has revealed that each contains domains are involved in protein dimerization and DNA binding. Recently, we demonstrated the existence of multiple heteromeric DNA binding forms of the AhR complex containing at least three distinct protein subunits. Given that the functional activity of protein transactivators is known to be dependent upon the specific protein(s) to which it is associated, we hypothesize that differences in AhR- and AhR complex-associated proteins could, result in an alteration of the functionality of the AhR complex which would therefore contribute to species- and tissue-specific differences in toxicity and gene expression. Here we propose to characterize the multiple DNA binding forms of the AhR complex as well as to isolate and characterize AhR- associated proteins. Utilizing AhR and ARNT antibodies we will determine the identity of the multiple proteins AhR complex subunits which bind (and can be crosslinked) to the DRE in a TCDD-dependent manner. Multiple DNA binding forms of the AhR complex will be isolated from guinea pig liver by a combination of conventional and magnetic DNA affinity binding techniques. The functional activity of each complex will he assessed by the ability of each to stimulate transcription in vitro and the DNA binding of each complex will be examined using gel shift, UV- crosslinking, DNA mutagenesis and DNA footprinting. In a complimentary approach, AhR-associated proteins will be isolated from mouse, guinea pig and human cell lines stably transfected with a recombinant AhR fusion protein expression vector. The presence of an affinity tag on the recombinant proteins will allow rapid and selective isolation of the recombinant AhR and its associated proteins. The biochemical and functional properties of the isolated complexes and their protein components will be examined in reconstitution experiments and identity of the associated proteins determined by sequence analysis. cDNA clones encoding AhR-associated proteins will be isolated from cDNA libraries using both the yeast two-hybrid system as well as probes derived from the amino acid sequence of purified proteins. The results of our studies will not only increase our understanding of the molecular mechanisms of dioxin action and provide insight into the diversity in TCDD-responsiveness in humans and animals but they will contribute to our knowledge of the inducible regulation of mammalian gene expression.