Functional analysis of membrane TNF-α

表达于CD_4~+T细胞表面的膜肿瘤坏死因子-α是一种在T细胞和B细胞之间传递信号的新候选分子。在活化的人CD_4~+淋巴细胞上诱导26 kDa的膜肿瘤坏死因子-α。由于膜型肿瘤坏死因子-α的生物学功能尚不清楚，我们对膜型肿瘤坏死因子-α传递的反向信号进行了研究。抗肿瘤坏死因子-α抗体(抗肿瘤坏死因子-α抗体)可诱导T细胞表面黏附分子E-选择素(CD62E)的产生，高峰时间为12~24 h，48h后黏附分子完全消失。这些结果不仅表明膜型肿瘤坏死因子-α将反向信号传递到表面表达该分子的细胞内，而且首次表明E-选择素在不同于内皮细胞的细胞类型中是可诱导的。然后，我们研究了膜肿瘤坏死因子-α胞浆结构域中的功能关键基序，以了解其反向信号转导。由于细胞质丝氨酸残基已被证明在一些单核细胞系中被磷酸化，丝氨酸残基被认为是反向信号转导的重要因素。我们通过定点突变构建了一系列携带丝氨酸到丙氨酸替代的突变膜肿瘤坏死因子-α，并将其导入Jurkat细胞。用抗肿瘤坏死因子-α抗体刺激S细胞膜上的肿瘤坏死因子-α，但不改变E-选择素的表达。因此，细胞质结构域中的三个丝氨酸残基都不是负责反向信号传递的。我们正在尝试用酵母双杂交系统寻找膜肿瘤坏死因子-α胞浆结构域的结合蛋白(S)。

The membrane TNF-α expressed on the cell surface od CD4+ T cells is a novel candidate that transmit signals from T cells to B cells and vice versa. The 26-kDa membrane TNF-α was induced on activated human CD4+ lymphocytes. As the biological functions of the membrane TNF-α is still not well understood, we studied the reverse signal transmitted by membrane TNF-α. Activation by anti-TNF-α antibody (Ab) against membrane TNF-αresulted in the induction of an adhesion molecule, E-selectin (CD62E), on the CD4+ T cells with the peak of 12 to 24 h, which was completely disappeared at 48 h. When wild-type or mutant membrane TNF-α (R78T/S79T) resistant to proteolytic cleavage was introduced into Jurkat or HeLa ceils, E-selectin was introduced upon activation of membrane TNF-α with the similar kinetics. These results not only indicates that membrane TNF-αtransmits reverse signals into the cells expressing this molecule on the surface, but also presented for the first time that E-selectin was inducible in cell types different from endothelial cells. We then studied the functionally critical motif in the cytoplasmic domain of the membrane TNF-α for its reverse signaling. As cytoplasmic serine residues have been shown to be phosphorylated in several monocyte cell lines, the serine residues were supposed to be important for reverse signaling. We constructed a series of mutant membrane TNF-α that carries serine to alanine replacement by sitedirected mutagenesis and transfected into Jurkat cells. Then the membrane TNF-α on the s transfectants were stimulated by anti-TNF-α Ab, however E-selectin expression was not altered in these transfectants. It is thus concluded that none of the three serine residues in the cytoplasmic domain were responsible for the reverse signaling. We are now trying to find the binding protein(s) to the cytoplasmic domain of membrane TNF-α by using yeast two-hybrid system.