Swanson H I, Yang J h
Department of Pharmacology, University of Kentucky, University of Kentucky Medical Center, Lexington, Kentucky 40536, USA.
J Biol Chem. 1996 Dec 6;271(49):31657-65. doi: 10.1074/jbc.271.49.31657.
The Ah receptor (AHR) and its DNA binding partner, the Ah receptor nuclear translocator (ARNT), are basic helix-loop-helix proteins distinguished by their PER, AHR, ARNT, and SIM (PAS) homology regions. To identify the amino acids of the AHR.ARNT heterodimer that contact the TNGCGTG recognition sequence, we have performed deletion mapping and amino acid substitutions within the N termini of both the AHR and ARNT. The ability of the variant AHR and ARNT proteins to bind DNA and activate gene transcription was determined by the gel shift analysis and transient transfection assays. We have found that the amino acids of ARNT that contact DNA are similar to those of other basic/helix-loop-helix proteins and include glutamic acid residue 83 and arginine residues 86 and 87. Although our initial experiments indicated that DNA binding of the AHR may involve two regions that are bordered by amino acids 9-17 and amino acids 34-42, further analysis demonstrated that only amino acids 34-39 are critical for the AHR.TNGC interaction. These experiments indicate that while the structural features of the ARNT.GTG complex may closely resemble that deduced for proteins such as Max, E47, and USF, the AHR.TNGC complex may represent a unique DNA binding form of basic/helix-loop-helix proteins.
芳烃受体（AHR）及其DNA结合伴侣芳烃受体核转运蛋白（ARNT）是碱性螺旋-环-螺旋蛋白，其特点是具有PER、AHR、ARNT和SIM（PAS）同源区域。为了确定AHR-ARNT异二聚体中与TNGCGTG识别序列接触的氨基酸，我们对AHR和ARNT的N端进行了缺失作图和氨基酸替换。通过凝胶迁移分析和瞬时转染试验确定了变异的AHR和ARNT蛋白结合DNA并激活基因转录的能力。我们发现，与DNA接触的ARNT氨基酸与其他碱性/螺旋-环-螺旋蛋白的氨基酸相似，包括第83位谷氨酸残基以及第86和87位精氨酸残基。虽然我们最初的实验表明，AHR与DNA的结合可能涉及两个区域，其边界分别为第9至17位氨基酸和第34至42位氨基酸，但进一步分析表明，只有第34至39位氨基酸对AHR与TNGC的相互作用至关重要。这些实验表明，虽然ARNT与GTG复合物的结构特征可能与Max、E47和USF等蛋白推导的结构特征非常相似，但AHR与TNGC复合物可能代表碱性/螺旋-环-螺旋蛋白一种独特的DNA结合形式。
