Identification of a novel C-terminal domain involved in the negative function of the rainbow trout Ah receptor nuclear translocator protein isoform a (rtARNTa) in Ah receptor-mediated signaling.
Identification of a novel C-terminal domain involved in the negative function of the rainbow trout Ah receptor nuclear translocator protein isoform a (rtARNTa) in Ah receptor-mediated signaling.
复制标题
鉴定了一个新的 C 末端结构域，该结构域参与虹鳟鱼 Ah 受体核易位蛋白亚型 a (rtARNTa) 在 Ah 受体介导的信号传导中的负功能。
DOI：
10.1016/s0006-2952(01)00671-2
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发表时间：
2001
影响因子：
5.8
通讯作者：
Pollenz,RS
中科院分区：
文献类型：
--
作者：
Necela,B;Pollenz,RS
虹鳟芳烃受体（AHR）核转位异构体a（rtARNTa）在AHR介导的信号转导中具有负性功能。先前的分析表明，负功能是在DNA结合的水平，可能是由于存在57个C-末端氨基酸，是强疏水性的。为了在分子水平上评估rtARNTa的负活性，分析了对应于氨基酸601-637、601-631和616-631的富含疏水性的结构域在互补和凝胶移位测定中影响截短的rtARNT蛋白的功能的能力。在小鼠肝癌细胞中，向这些蛋白质中添加富含疏水结构域使其补充AHR介导的信号转导的能力降低了65- 95%。功能的降低与AHR · rtARNT复合物结合DNA的能力降低有关，而不是由于缺乏与AHR的二聚化。Gal 4蛋白上的疏水性丰富的结构域的表达表明，rtARNTa的C-末端结构域不太可能包含反式激活功能;然而，疏水性结构域降低了Gal 4蛋白结合DNA的能力。免疫沉淀和突变实验表明，富含疏水性的结构域不与AHR的bHLH基序相互作用。有趣的是，免疫沉淀实验还显示，C-末端的rtARNTa的疏水丰富的区域可以寡聚体在体外嵌合体的Gal 4 DNA结合域。这些发现表明，C-末端疏水性氨基酸是至关重要的负功能的rtARNTa在AHR介导的信号，并表明，多种机制可能参与抑制DNA结合。
Rainbow trout aryl hydrocarbon receptor (AHR) nuclear translocator isoform a (rtARNTa) has a negative function in AHR-mediated signal transduction. Previous analyses suggest that the negative function is at the level of DNA binding and may be due to the presence of 57 C-terminal amino acids that are strongly hydrophobic. To assess the negative activity of rtARNTa at the molecular level, hydrophobic-rich domains corresponding to amino acids 601–637, 601–631, and 616–631 were analyzed for the ability to affect the function of truncated rtARNT proteins in complementation and gel shift assays. Addition of the hydrophobic-rich domains to these proteins reduced their ability to complement AHR-mediated signal transduction in mouse hepatoma cells by 65–95%. The decrease in function was related to a reduced ability of the AHR · rtARNT complex to bind DNA and not due to a lack of dimerization with AHR. Expression of the hydrophobic-rich domains on Gal4 proteins showed that the C-terminal domain of rtARNTa was unlikely to contain transactivation function; however, the hydrophobic domains reduced the ability of the Gal4 proteins to bind DNA. Immunoprecipitation and mutational experiments indicate that the hydrophobic-rich domains do not interact with the bHLH motif of AHR. Interestingly, immunoprecipitation experiments also revealed that the C-terminal hydrophobic-rich region of rtARNTa could oligomerize in vitro in a chimera with the Gal4 DNA binding domain. These findings indicate that the C-terminal hydrophobic amino acids are critical for the negative function of rtARNTa in AHR-mediated signaling and suggest that multiple mechanisms may be involved in the repression of DNA binding.