Song H Y, Dunbar J D, Zhang Y X, Guo D, Donner D B
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202.
J Biol Chem. 1995 Feb 24;270(8):3574-81.
The yeast-based two hybrid has been used to identify a novel protein that binds to the intracellular domain of the type 1 receptor for tumor necrosis factor (TNFR-1IC). The TNF receptor-associated protein, TRAP-1, shows strong homology to members of the 90-kDa family of heat shock proteins. After in vitro transcription/translation and 35S labeling, TRAP-1 was precipitated using a fusion protein consisting of glutathione S-transferase and TNFR-1IC, showing that the two proteins directly interact. The ability of deletion mutants of TNFR-1 to interact with TRAP-1 was tested using the two hybrid system. This showed that the amino acid sequences that mediate binding are diffusely distributed outside of the domain in the C terminus of TNFR-1IC that signals cytotoxicity. The 2.4-kilobase TRAP-1 mRNA was variably expressed in skeletal muscle, liver, heart, brain, kidney, pancreas, lung, and placenta. TRAP-1 mRNA was also detected in each of eight different transformed cell lines. Identification of TRAP-1 may be an important step toward defining how TNFR-1, which does not contain protein tyrosine kinase activity, transmits its message to signal transduction pathways.
基于酵母的双杂交技术已被用于鉴定一种与肿瘤坏死因子1型受体细胞内结构域（TNFR-1IC）结合的新型蛋白质。肿瘤坏死因子受体相关蛋白TRAP-1与90 kDa热休克蛋白家族成员具有高度同源性。体外转录/翻译及35S标记后，使用由谷胱甘肽S-转移酶和TNFR-1IC组成的融合蛋白沉淀TRAP-1，表明这两种蛋白直接相互作用。使用双杂交系统测试TNFR-1缺失突变体与TRAP-1相互作用的能力。结果表明，介导结合的氨基酸序列分散分布在TNFR-1IC C末端中发出细胞毒性信号的结构域之外。2.4千碱基的TRAP-1 mRNA在骨骼肌、肝脏、心脏、大脑、肾脏、胰腺、肺和胎盘中有不同程度的表达。在8种不同的转化细胞系中也检测到了TRAP-1 mRNA。鉴定TRAP-1可能是确定不含有蛋白酪氨酸激酶活性的TNFR-1如何将其信息传递至信号转导途径的重要一步。
