Takahata S, Sogawa K, Kobayashi A, Ema M, Mimura J, Ozaki N, Fujii-Kuriyama Y
Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, Japan.
Biochem Biophys Res Commun. 1998 Jul 30;248(3):789-94. doi: 10.1006/bbrc.1998.9012.
We isolated a cDNA clone encoding a polypeptide of 626 amino acids containing basic helix-loop-helix (bHLH) and PAS domains from a mouse cDNA library of P19 cells. This protein, termed Arnt3, showed the highest similarity to Arnt and Arnt2 in the bHLH and PAS regions. Arnt3 mRNA was expressed in brain, skeletal muscle, 13.5-day embryos, and P19 cells treated with retinoic acid. The partner PAS proteins of Arnt3 were searched for by the two-hybrid system in yeast, and HIF-1 alpha, HLF, and Clock among various bHLH/PAS proteins were found. Gel mobility shift analysis using nuclear extracts from 293T cells cotransfected with Arnt3 and HIF-1 alpha (or HLF) expression plasmids revealed that these complexes specifically bound the hypoxia-response element (HRE). Coexpression of Arnt3 and HIF-1 alpha (or HLF) in Arnt-deficient c4 cells enhanced transcription of a reporter gene driven by the HRE sequences. We also showed that Arnt3 contained an activation domain at the C-terminal region and a repression domain between the PAS-A and PAS-B regions.
我们从P19细胞的小鼠cDNA文库中分离出一个编码626个氨基酸多肽的cDNA克隆，该多肽含有碱性螺旋-环-螺旋（bHLH）和PAS结构域。这种蛋白质被称为Arnt3，在bHLH和PAS区域与Arnt和Arnt2具有最高的相似性。Arnt3 mRNA在脑、骨骼肌、13.5天胚胎以及用视黄酸处理的P19细胞中表达。通过酵母双杂交系统在酵母中搜索Arnt3的伴侣PAS蛋白，在各种bHLH/PAS蛋白中发现了HIF-1α、HLF和Clock。使用共转染Arnt3和HIF-1α（或HLF）表达质粒的293T细胞核提取物进行凝胶迁移率变动分析，结果显示这些复合物特异性结合缺氧反应元件（HRE）。在Arnt缺陷的c4细胞中共表达Arnt3和HIF-1α（或HLF）可增强由HRE序列驱动的报告基因的转录。我们还表明，Arnt3在C末端区域含有一个激活结构域，在PAS-A和PAS-B区域之间含有一个抑制结构域。
