Grf40, A Novel Grb2 Family Member, Is Involved in T Cell Signaling

我们分子克隆了一个新的Grb2家族成员，命名为Grf40。Grf40主要表达于造血细胞，尤其是T细胞。Grf40与tp SLP-76的结合比Grb2更紧密。顺便说一句，Grf40可能通过其SH2结构域与接头结合以激活T细胞(LAT)。野生型Grf40在Jurkat细胞中的过表达可诱导T细胞受体(TCR)刺激下SLP-76依赖的IL-2启动子和活化T细胞核因子(NF-AT)活性显著增加，而C端SH3缺失的Grf40突变体对IL-2启动子活性无明显影响。此外，SH2缺失的Grf40突变体(Grf40-dSH2)对这些调节活性有明显的抑制作用，其作用明显强于SH2缺失的Grb2突变体。我们的数据表明Grf40是一种接头分子，通过比Grb2与SLP-76和LAT更有效的相互作用参与TCR介导的信号转导。为了研究Grf40的体内功能，我们建立了由lck近端启动子驱动的表达Grf40-dSH2的转基因小鼠。在转基因小鼠中，胸腺细胞总数显著减少，而在双阴性(CD4-CD8-)胸腺细胞亚群中，CD25+CD44-前T细胞数量显著增加。然而，由TCR前刺激介导的CD5表达在转基因小鼠的CD4-CD8-胸腺细胞上受到显著抑制。此外，依赖SLP-76的信号也被显著抑制。这些数据表明，Grf40在胸腺细胞的TCR前信号转导和TCR信号转导中起重要作用。

We molecularly cloned a new Grb2 family member, named Grf40. Expression of Grf40 is predominant in hematopoietic cells, particularly T cells. Grf40 binds tp SLP-76 more tightly than Grb2. Incidentally, Grf40 binds to linker for activation of T cells (LAT) possibly via its SH2 domain. Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76-dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the C-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity. Furthermore, the SH2-deleted Grf40 mutant (Grf40-dSH2) led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant. Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT.To investigate an in vivo function of Grf40, We generated transgenic mice expressing Grf40-dSH2, which is driven by the lck proximal promoter. The total number of thymocytes was profoundly reduced in the transgenic mice, whereas in the double-negative (CD4-CD8-) thymocyte subset, in particular, the CD25+CD44-pre-T cell population was significantly increased. However, CD5 expression, which is mediated by pre-TCR stimulation, was significantly suppressed on the CD4-CD8-thymocytes of the transgenic mice. Furthermore, the SLP-76-dependent signaling was markedly suppressed as well. These data suggest that Grf40 plays an important role in the pre-TCR as well as TCR signaling in thymocytes.