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

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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.