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

1. 分析Rac1在骨骼肌细胞胰岛素依赖性葡萄糖摄取中的作用：通过L6成肌细胞系，观察到本构激活的Rac1异位表达后GLUT4向质膜的易位，以及内源性Rac1通过siRNA表达下调后GLUT4易位的降低，以响应胰岛素。这些影响试图通过腺病毒介导的基因转移和靶向基因破坏在小鼠中进行评估。2. Rac1对网格蛋白依赖受体内吞的负调控分析：组成型激活的Rac1异位表达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，而不是其剪接变体Ost-I和Ost-II，也能消除转铁蛋白受体内吞作用，这表明Ost-III可能是参与受体内吞作用负调控的Rac1的特异性激活因子。此外，我们分离出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，抑制Ost-III的活性。3. 磷脂酶Cε在小鼠体内的生理作用分析。4. Rap1、RA-GEF-1和RA-GEF-2的鸟嘌呤核苷酸交换因子在小鼠中的生理作用分析：我们生成了RA-GEF-1 KO和RA-GEF-2 KO小鼠。RA-GEF-1 KO小鼠因血管形成不足而死于E9.5。RA-GEF-2小鼠看起来健康，但其脾细胞中肿瘤坏死因子-a依赖性整合素激活和细胞粘附严重受损，提示RA-GEF-2在rap1依赖性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.