Definition of a novel ligand binding domain of a nuclear bHLH receptor: co‐localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor.
Definition of a novel ligand binding domain of a nuclear bHLH receptor: co‐localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor.
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DOI：
10.1002/j.1460-2075.1993.tb06101.x
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发表时间：
1993-11
期刊：
影响因子：
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通讯作者：
M. Whitelaw;M. Göttlicher;J. Gustafsson;L. Poellinger
中科院分区：
文献类型：
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作者：
M. Whitelaw;M. Göttlicher;J. Gustafsson;L. Poellinger
二恶英受体介导二恶英（2，3，7，8-四氯二苯并对二恶英）的信号转导，并作为约100 kDa配体结合受体和约85 kDa辅助因子Arnt的异二聚体与DNA靶序列结合。这两种因子都包含DNA结合和二聚化所需的N末端碱性螺旋-环-螺旋（bHLH）基序。在这项研究中，我们描述了糖皮质激素/二恶英受体融合蛋白的构建，该蛋白允许在CHO和肝癌细胞的瞬时转染中通过二恶英调节糖皮质激素受体活性。因此，在不存在二恶英的情况下，含有大的500-720个氨基酸C末端二恶英受体片段但缺乏N末端bHLH基序的嵌合受体构建体对糖皮质激素受体衍生物Tau DBD的转录活性产生抑制，Tau DBD含有其N末端强反式激活信号（tau）及其DNA结合结构域（DBD）。在二恶英的存在下，这种抑制被逆转。重要的是，这些嵌合受体不需要bHLH Arnt辅因子来发挥功能。二恶英受体的一个相当小的区域，位于氨基酸230和421之间，在体外表现出特异性二恶英结合活性。此外，二恶英结合在体外与受体片段的能力，形成稳定的复合物在体外与分子伴侣热休克蛋白90。这些研究结果支持的概念，热休克蛋白90可能是重要的折叠二恶英结合构型的受体。最后，tau DBD活性以二恶英非响应性方式被二恶英受体片段组成性抑制，该片段未能结合配体，但也未能在体外结合hsp 90，表明除了hsp 90结合之外的替代机制可能有助于灭活功能。总之，二恶英受体系统提供了一种新的和复杂的bHLH因子调节模型，也可能对配体激活的核受体的作用机制提供重要的见解。
The dioxin receptor mediates signal transduction by dioxin (2,3,7,8‐tetrachlorodibenzo‐p‐dioxin) and binds to DNA target sequences as a heterodimer of the approximately 100 kDa ligand binding receptor and the approximately 85 kDa auxiliary factor, Arnt. Both of these factors encompass an N‐terminal basic helix‐loop‐helix (bHLH) motif required for DNA binding and dimerization. In this study we describe the construction of glucocorticoid/dioxin receptor fusion proteins which allow the regulation of glucocorticoid receptor activity by dioxin in transient transfections of CHO and hepatoma cells. Thus, in the absence of dioxin, chimeric receptor constructs which contain large 500–720 amino acid C‐terminal dioxin receptor fragments, but lack the N‐terminal bHLH motif, confer repression upon the transcriptional activity of a glucocorticoid receptor derivative, tau DBD, containing its N‐terminal strong transactivating signal (tau) and its DNA binding domain (DBD). In the presence of dioxin, this repression is reversed. Importantly, these chimeric receptors did not require the bHLH Arnt co‐factor for function. A considerably smaller region of the dioxin receptor, located between amino acids 230 and 421, showed specific dioxin binding activity in vitro. Moreover, dioxin binding in vitro correlated with the ability of receptor fragments to form stable complexes in vitro with the molecular chaperone hsp90. These findings support the notion that hsp90 may be important for folding of a dioxin binding configuration of the receptor. Finally, tau DBD activity was constitutively repressed in a dioxin non‐responsive manner by dioxin receptor fragments which failed to bind ligand but also failed to bind hsp90 in vitro, indicating that alternative mechanisms in addition to hsp90 binding may contribute to the inactivation function. In summary, the dioxin receptor system provides a novel and complex model of regulation of bHLH factors that may also give important insights into the mechanism of action of ligand‐activated nuclear receptors.