REGULATION OF THE AH RECEPTOR

AH 受体的调节

• 批准号: 2153801
• 负责人: Gary H. Perdew
• 金额: $ 20.98万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2153801
• 关键词: DNA binding protein; chemical binding; computer assisted sequence analysis; confocal scanning microscopy; dioxins; environmental toxicology; gel electrophoresis; genetic mapping; high performance liquid chromatography; immunoprecipitation; nicotinic receptors; phosphorylation; posttranslational modifications; protein sequence; protein structure; receptor binding; receptor expression; site directed mutagenesis; stress proteins; tissue /cell culture; toxicant interaction; western blottings

The Ah receptor (AhR) has been shown to be largely responsible for the toxic and tumor promotional properties of 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) seen in rodents. The human population is constantly exposed to low levels of TCDD, and related compounds, the actual long term health effects remain to be elucidated. Little is known about the biochemical processes involved in the activation and regulation of this ligand-activated helix-loop-helix/basic region transcriptional factor. The multiple mechanisms of AhR regulation will be examined along with elucidation of the biochemical events from initial synthesis and assembly to translocation into the nucleus. Using an in vitro translation system the 90 kDa heat shock protein (hsp90) binding site on the AhR will be determined using a series of truncation and deletion cDNA mutants. The location of the binding site(s) will be compared with the DNA binding, dimerization, ligand binding, and transactivation domains to gain insight into the functional significance of this protein/protein interaction. Synthetic peptides will be used in an in vitro reconstitution system to precisely define the actual site of interaction The protein composition of the 9S cytosolic form of the AhR in Hepa 1 cells will be determined by immunoprecipitation with antibodies raised against specific AhR peptide sequences. Both chemically crosslinked and non-crosslinked AhR will be characterized on western blots using antibodies to the AhR, Arnt, and hsp90. The intracellular location of the unliganded and liganded. AhR in Hepa 1 cells will be determined using immunocytochemical techniques and one- and two-dimensional confocal microscopy. The protein and cofactor requirements (e.g. ATP, hsp70, Arnt) for nuclear localization/translocation will be determined using a permeabilized cell system. The role of phosphorylation in AhR function will be examined by first mapping the location of the phosphoamino acid residues, followed by examination of the functional significance of individual phosphoamino acids using site-directed mutagenesis and cell transfection techniques. Another aspect of regulating the level of toxicity caused by exposure to a given amount of TCDD is how each cell type maintains a specific AhR concentration. The role of the cellular protease m-calpain in AhR turnover and ligand dependent down regulation will be examined by exposing Hep G2 cells to a calpain or calpastatin antisense oligonucleotides and determining their effect on AhR levels, both in the presence and absence of TCDD. Collectively, these studies will develop an understanding of the pathway of AhR action and the various points of regulation.

AH受体（AHR）已被证明对 2,3,7,8-四氯迪本佐-P-的有毒和肿瘤促进特性 在啮齿动物中看到的二恶英（TCDD）。 人口一直在 暴露于低水平的TCDD和相关化合物，实际的长 术语健康效应尚待阐明。 关于 激活和调节涉及的生化过程 配体激活的螺旋环螺旋/基本区域转录因子。 将检查AHR调节的多种机制 从初始合成和组装中阐明生化事件 易位到核。 使用体外翻译系统 AHR上的90 kDa热休克蛋白（HSP90）结合位点将是 使用一系列截断和缺失cDNA突变体确定。 这 结合位点的位置将与DNA结合进行比较 二聚化，配体结合和反式激活结构域以获得洞察力 进入该蛋白/蛋白质相互作用的功能意义。 合成肽将在体外重构系统中使用 精确定义相互作用的实际位点蛋白质成分 将确定HEPA 1细胞中AHR的9s胞质形式中 通过用针对特定AHR提出的抗体免疫沉淀 肽序列。 化学交联和非交联的AHR 将使用抗Ahr的抗体在蛋白质印迹上表征，arnt， 和HSP90。 非配体和配体的细胞内位置。 HEPA 1细胞中的AHR将使用免疫细胞化学确定 技术以及一维共聚焦显微镜。 核的蛋白质和辅因子要求（例如ATP，HSP70，ARNT） 将使用通透性细胞确定定位/易位 系统。 磷酸化在AHR功能中的作用将通过 首先绘制磷酸氨基酸残基的位置，然后 通过检查单个磷酸氨基的功能意义 使用位置定向的诱变和细胞转染技术的酸。 调节暴露于暴露引起的毒性水平的另一个方面 给定数量的TCDD是每种细胞类型如何维护特定的AHR 专注。 细胞蛋白酶M-钙蛋白酶在AHR中的作用 营业额和依赖配体调节法规将由 将HEP G2细胞暴露于钙蛋白酶或calpastatin反义 寡核苷酸并确定其对AHR水平的影响 TCDD的存在和不存在。 总的来说，这些研究将对途径有所了解 AHR动作和各种监管点。