Song H Y, Dunbar J D, Donner D B
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202.
J Biol Chem. 1994 Sep 9;269(36):22492-5.
The yeast-based two hybrid system has been used to determine whether oligomerization of the intracellular domain of the 55-kDa type 1 tumor necrosis factor (TNF) receptor may occur during TNF action. This assay depends upon reconstitution of the function of the GAL4 transcriptional activator through interaction of a protein fused to the GAL4 DNA binding domain with a protein fused to the transcriptional activation domain of GAL4. Fusion of the type 1 TNF receptor intracellular domain with the DNA binding domain and the transactivation domain of GAL4 led to activation of the lacZ indicator gene, demonstrating interaction of the receptor intracellular domain with itself. A HeLa cell cDNA library was searched for proteins that interact with the intracellular domain of the type 1 TNF receptor. A protein corresponding to amino acids 329-426 in the type 1 TNF receptor intracellular domain was identified by this screen. The aggregation domain was further defined by testing the ability of deletion mutants of the type 1 TNF receptor intracellular region to interact with the complete intracellular domain. These experiments map the aggregation domain to a sequence of amino acids previously shown to be responsible for mediating TNF-induced cytotoxicity. These results suggest that aggregation of type 1 TNF receptor intracellular domains may be important in TNF signal transduction.
基于酵母的双杂交系统已被用于确定55 kDa 1型肿瘤坏死因子（TNF）受体的胞内结构域在TNF作用过程中是否会发生寡聚化。该检测方法依赖于通过与GAL4转录激活结构域融合的蛋白质与与GAL4 DNA结合结构域融合的蛋白质之间的相互作用来重建GAL4转录激活因子的功能。将1型TNF受体胞内结构域与GAL4的DNA结合结构域和反式激活结构域融合，导致lacZ报告基因激活，证明受体胞内结构域自身相互作用。在HeLa细胞cDNA文库中搜索与1型TNF受体胞内结构域相互作用的蛋白质。通过该筛选鉴定出一种与1型TNF受体胞内结构域中氨基酸329 - 426相对应的蛋白质。通过测试1型TNF受体胞内区域缺失突变体与完整胞内结构域相互作用的能力，进一步确定了聚集结构域。这些实验将聚集结构域定位到先前已证明负责介导TNF诱导的细胞毒性的氨基酸序列。这些结果表明1型TNF受体胞内结构域的聚集在TNF信号转导中可能很重要。
