| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
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.
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