McGuire J, Whitelaw M L, Pongratz I, Gustafsson J A, Poellinger L
Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital, Novum, Sweden.
Mol Cell Biol. 1994 Apr;14(4):2438-46. doi: 10.1128/mcb.14.4.2438-2446.1994.
In response to dioxin, the nuclear basic helix-loop-helix (bHLH) dioxin receptor forms a complex with the bHLH partner factor Arnt that regulates target gene transcription by binding to dioxin-responsive sequence motifs. Previously, we have demonstrated that the latent form of dioxin receptor present in extracts from untreated cells is stably associated with molecular chaperone protein hsp90, and Arnt is not a component of this complex. Here, we used a coimmunoprecipitation assay to demonstrate that the in vitro-translated dioxin receptor, but not Arnt, is stably associated with hsp90. Although it showed ligand-binding activity, the in vitro-translated dioxin receptor failed to dissociate from hsp90 upon exposure to ligand. Addition of a specific fraction from wild-type hepatoma cells, however, to the in vitro-expressed receptor promoted dioxin-dependent release of hsp90. This stimulatory effect was mediated via the bHLH dimerization and DNA-binding motif of the receptor. Moreover, ligand-dependent release of hsp90 from the receptor was not promoted by fractionated cytosolic extracts from mutant hepatoma cells which are deficient in the function of bHLH dioxin receptor partner factor Arnt. Thus, our results provide a novel model for regulation of bHLH factor activity and suggest that derepression of the dioxin receptor by ligand-induced release of hsp90 may require bHLH-mediated concomitant recruitment of an additional cellular factor, possibly the structurally related bHLH dimerization partner factor Arnt. In support of this model, addition of in vitro-expressed wild-type Arnt, but not a mutated form of Arnt lacking the bHLH motif, promoted release of hsp90 from the dioxin receptor in the presence of dioxin.
作为对二噁英的响应，核碱性螺旋-环-螺旋（bHLH）二噁英受体与bHLH伴侣因子芳烃受体核转运蛋白（Arnt）形成复合物，该复合物通过与二噁英反应性序列基序结合来调节靶基因转录。此前，我们已经证明，未处理细胞提取物中存在的潜伏形式的二噁英受体与分子伴侣蛋白热休克蛋白90（hsp90）稳定结合，而Arnt不是该复合物的组成部分。在此，我们使用共免疫沉淀试验证明，体外翻译的二噁英受体而非Arnt与hsp90稳定结合。尽管体外翻译的二噁英受体显示出配体结合活性，但在暴露于配体时未能从hsp90上解离。然而，向体外表达的受体中添加来自野生型肝癌细胞的特定组分可促进二噁英依赖性的hsp90释放。这种刺激作用是通过受体的bHLH二聚化和DNA结合基序介导的。此外，来自缺乏bHLH二噁英受体伴侣因子Arnt功能的突变肝癌细胞的分级胞质提取物不能促进hsp90从受体上的配体依赖性释放。因此，我们的结果提供了一种调节bHLH因子活性的新模型，并表明配体诱导的hsp90释放导致二噁英受体的去抑制可能需要bHLH介导的额外细胞因子的伴随募集，可能是结构相关的bHLH二聚化伴侣因子Arnt。为支持该模型，在存在二噁英的情况下，添加体外表达的野生型Arnt而非缺乏bHLH基序的Arnt突变形式可促进hsp90从二噁英受体上释放。
