Wilhelmsson A, Whitelaw M L, Gustafsson J A, Poellinger L
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Sweden.
J Biol Chem. 1994 Jul 22;269(29):19028-33.
The dioxin receptor is a ligand-dependent transcription factor that binds to target DNA sequences (xenobiotic responsive elements, XREs) following ligand-dependent dimerization with its partner factor, Arnt (aryl hydrocarbon receptor nuclear translocator). Both factors contain an N-terminal basic region helix-loop-helix motif mediating dimerization and subsequent DNA binding. In this study we investigate the possible role of Arnt in agonistic and antagonistic effects of the dioxin receptor ligand alpha-naphthoflavone (ANF). Using specific antisera for the ligand binding dioxin receptor and Arnt, respectively, we show that exposure of the dioxin receptor to ANF in vitro induced recruitment of Arnt, thus stimulating binding of the heteromeric complex to XRE. In transient transfection assays, ANF at high concentrations stimulated expression of an XRE-driven reporter gene. This agonistic effect of ANF is, therefore, most likely attributable to ANF stimulation of dioxin receptor-Arnt heterodimerization and subsequent binding of the complex to XRE. Using a minimal XRE-driven reporter gene construct, we could further confirm earlier studies showing that ANF antagonizes the effect of a dioxin receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Next we employed chimeric receptor constructs containing amino acids 1-500 of the human glucocorticoid receptor fused to dioxin receptor fragments lacking the very N-terminal basic region helix-loop-helix dimerization and DNA binding motif. These chimeric receptor constructs show dioxin responsiveness upon transient transfection into mutant Arnt-deficient hepatoma cells and are, thus, functionally uncoupled from Arnt. Importantly, dioxin-dependent activation of the chimeric receptors was inhibited in the presence of ANF, demonstrating that dimerization of dioxin receptor with Arnt was not necessary for manifestation of the antagonistic effect of ANF. Rather, dioxin receptor sequences, which confer dioxin regulation upon a heterologous DNA binding and transactivating domain, also mediated the antagonistic effects of ANF.
二噁英受体是一种配体依赖性转录因子，在与伴侣因子芳烃受体核转运蛋白（Arnt）进行配体依赖性二聚化后，它会与靶DNA序列（外源性反应元件，XREs）结合。这两个因子都含有一个N端碱性区域螺旋-环-螺旋基序，介导二聚化以及随后的DNA结合。在本研究中，我们探究了Arnt在二噁英受体配体α-萘黄酮（ANF）的激动和拮抗作用中可能发挥的作用。分别使用针对配体结合二噁英受体和Arnt的特异性抗血清，我们发现体外将二噁英受体暴露于ANF会诱导Arnt的募集，从而刺激异源复合物与XRE的结合。在瞬时转染实验中，高浓度的ANF刺激了XRE驱动的报告基因的表达。因此，ANF的这种激动作用很可能归因于ANF对二噁英受体-Arnt异源二聚化的刺激以及随后复合物与XRE的结合。使用最小的XRE驱动的报告基因构建体，我们可以进一步证实早期的研究，即ANF拮抗二噁英受体激动剂2,3,7,8-四氯二苯并对二噁英的作用。接下来，我们采用了嵌合受体构建体，其包含人糖皮质激素受体的1-500个氨基酸，与缺乏最N端碱性区域螺旋-环-螺旋二聚化和DNA结合基序的二噁英受体片段融合。这些嵌合受体构建体在瞬时转染到突变的Arnt缺陷型肝癌细胞中时表现出二噁英反应性，因此在功能上与Arnt解偶联。重要的是，在存在ANF的情况下，嵌合受体的二噁英依赖性激活受到抑制，这表明二噁英受体与Arnt的二聚化对于ANF拮抗作用的表现并非必要。相反，赋予异源DNA结合和反式激活结构域二噁英调节作用的二噁英受体序列也介导了ANF的拮抗作用。
