Ah receptor nuclear translocator protein heterogeneity is altered after heterodimerization with the Ah receptor.
Ah receptor nuclear translocator protein heterogeneity is altered after heterodimerization with the Ah receptor.
复制标题
Ah 受体核转位蛋白异质性在与 Ah 受体异二聚化后发生改变。
DOI：
10.1021/bi970891w
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Perdew,GH
中科院分区：
文献类型：
--
作者：
Tsai,JC;Perdew,GH
AhR和AhR核转运子（ARNT）能够形成具有转录活性的异二聚体复合物。二聚化和反式激活所需的生物化学事件尚未完全了解。本研究的目的是确定ARNT的共价修饰是否发生在以单体形式存在的ARNT和与AhR异源二聚化后以及随后与DNA结合之间。对小鼠肝癌细胞系1c 1c 7（Hepa 1）胞质溶胶和ARNT免疫沉淀进行二维凝胶电泳。ARNT可视化与两种抗体，具有不同的表位特异性，每个检测到相当程度的电荷异质性。观察到的pI范围为5.7 - 6.4，主要形式的pI为6.2。AhR/ARNT异源二聚体从用β-萘甲酮处理的Hepa 1细胞获得的高盐核提取物中使用抗AhR多克隆抗体免疫沉淀。将该免疫沉淀物进行二维凝胶电泳，并将共免疫沉淀的ARNT可视化。结果表明，在细胞核中与AhR复合的ARNT具有向基端移动的同种型模式，主要同种型具有6.8的pI。因此，在二聚化过程中和/或与DNA结合后，PI发生显著变化。在细胞质中，2，3，7，8-四氯二苯并-p-二恶英与AhR的体外转化导致与ARNT的异源二聚化。双向凝胶电泳的ARNT coimmunoprecipitated与AhR揭示了相同的亚型模式中所看到的细胞质。这将表明ARNT的每种同种型能够与AhRinvitro异二聚化。ARNT是一种磷蛋白，酸性越强的同种型磷酸化水平越高。
TheAhreceptor (AhR) and theAhreceptor nuclear translocator (ARNT) are capable of forming a transcriptionally active heterodimeric complex. The biochemical events that are required for dimerization and transactivation are not fully understood. The purpose of this study was to determine whether covalent modifications of ARNT occur between ARNT existing in the monomeric form and after heterodimerization with the AhR and subsequent binding to DNA. Mouse hepatoma cell line 1c1c7 (Hepa 1) cytosol and ARNT immunoprecipitations were subjected to two-dimensional gel electrophoresis. ARNT was visualized with two antibodies, with distinct epitope specificity, and each detected a considerable level of charge heterogeneity. The pIrange observed was 5.7−6.4, with the predominant form at a pIof 6.2. The AhR/ARNT heterodimer was immunoprecipitated from high-salt nuclear extract obtained from Hepa 1 cells treated with β-naphthoflavone using an anti-AhR polyclonal antibody. This immunoprecipitate was subjected to two-dimensional gel electrophoresis, and coimmunoprecipitated ARNT was visualized. The results indicated that ARNT complexed with the AhR in the nucleus has an isoform pattern shifted toward the basic end, with the predominant isoform having a pIof 6.8. Thus, a significant shift in pIoccurs during the dimerization and/or after binding to DNA.In vitrotransformation of the AhR with 2,3,7,8-tetrachlorodibenzo-p-dioxin in cytosol leads to heterodimerization with ARNT. Two-dimensional gel electrophoresis of ARNT coimmunoprecipitated with the AhR revealed the same isoform pattern as seen in cytosol. This would indicate that each isoform of ARNT is capable of heterodimerizing with the AhRinvitro. ARNT is a phosphoprotein, and the more acidic isoforms appear to have a higher level of phosphorylation.