Analysis of subcellular region-specific activation of Ras and Rho family GTPases and its application

1.rac1在骨骼肌细胞胰岛素依赖的葡萄糖摄取中的作用分析：利用L6成肌细胞系，观察到当组成性激活的rac1异位表达时，GLUT4移位到质膜上，而当siRNA下调内源性rac1的表达时，GLUT4移位到胰岛素的反应中。通过腺病毒介导的基因转移和靶向基因破坏，在小鼠身上对这些效应进行了评估。2.rac1对依赖于转铁蛋白的受体内吞的负性调节分析：异位表达成分激活的rac1可有效抑制转铁蛋白受体内吞。鸟嘌呤核苷酸交换因子Ost-III，而不是其剪接变异体Ost-I和Ost-II，也取消了转铁蛋白受体内吞作用，提示Ost-III可能是参与受体内吞作用负调控的rac1的特异性激活剂。此外，我们还分离到γ-氨基丁酸A受体结合蛋白(GABARAP)作为OST-III的特异性结合伙伴，它抑制OST-III的活性。3.分析磷脂酶Cε在小鼠体内的生理作用。4.鸟嘌呤核苷酸交换因子RAP1、RA-GEM-1和RA-GEF-2在小鼠体内的生理作用分析：我们建立了RA-GEM-1 KO和RA-GEM-2 KO小鼠。由于血管形成不足，Ra-gef-1 KO小鼠在E9.5之前死亡。RA-gef-2小鼠看起来很健康，但其脾细胞中肿瘤坏死因子-a依赖的整合素的激活和细胞黏附严重受损，提示RA-gef-2在依赖Rap1的整合素激活中起作用。

1. Analysis of the role for Rac1 in insulin-dependent glucose uptake in skeletal muscle cells: By using the L6 myoblast cell line, translocation of GLUT4 to the plasma membrane upon ectopic expression of constitutively activated Rac1 and lowered GLUT4 translocation in response to insulin upon downregulation of the expression of endogenous Rac1 by siRNA were observed. These effects were tried to be evaluated in mice by adenovirus-mediated gene transfer and targeted gene disruption. 2. Analysis of the negative regulation of clathrin-dependent receptor endocytosis by Rac1: Ectopic expression of constitutively activated Rac1 potently inhibited transferrin receptor endocytosis. The guanine nucleotide exchange factor Ost-III, but not its splice variants Ost-I and Ost-II, also abolished transferrin receptor endocytosis, suggesting that Ost-III may act as a specific activator for Rac1 that is involved in the negative regulation of receptor endocytosis. Furthermore, we isolated γ-aminobutylic acid A receptor-binding protein (GABARAP) as a specific binding partner for Ost-III, which inhibits the activity of Ost-III. 3. Analysis of physiological roles of phospholipase Cε in the mouse. 4. Analysis of physiological roles of the guanine nucleotide exchange factos for Rap1, RA-GEF-1 and RA-GEF-2, in the mouse: We generated RA-GEF-1 KO and RA-GEF-2 KO mice. RA-GEF-1 KO mice died by E9.5 due to deficiencies in vascular formation. RA-GEF-2 mice appeared healthy, but tumor necrosis factor-a-dependent integrin activation and cell adhesion was severely impaired in their splenocytes, suggesting a role for RA-GEF-2 in Rap1-dependent integrin activation.