Regulation of insulin-stimulated glucose transport by small GTP-binding proteins

Insulin stimulates glucose uptake into adipocytes and muscle cells by promoting translocation of the Glut4 glucose transporter from intracellular storage compartments to the plasma membrane. In the absence of insulin, Glut4 is retained intracellularly, although the mechanism underlying this process remains uncertain. A yeast two-hybrid screen using the TC10-interacting protein CIP4 as bait led to the identification of a novel RasGAP and VPS9 domain-containing protein called Gapex-5. Whereas the GAP domain promotes GTP hydrolysis on H-Ras and TC10, the VPS9 domain is a guanine nucleotide exchange factor for Rab31, a Rab5 subfamily GTPase implicated in trans-Golgi Network (TGN)-to-endosome trafficking. Overexpression of Rab31 blocks insulin-stimulated Glut4 translocation, and knockdown of Rab31 potentiates insulin-stimulated Glut4 translocation and glucose uptake. Gapex-5 is predominantly cytosolic in untreated cells; its overexpression promotes intracellular retention of Glut4 in adipocytes by maintaining Rab31 in an active state. Insulin recruits the CIP4/Gapex-5 complex to the plasma membrane, thus reducing Rab31 activity and permitting Glut4 vesicles to translocate to the cell surface, where Glut4 docks and fuses to transport glucose into the cell.; Following its insulin-dependent translocation to the plasma membrane, Gapex-5 activates a pool of Rab5 at the cell surface. Furthermore, the GAP domain of Gapex-5 forms a stable complex with TC10, and overexpression of constitutively active TC10 also promotes Rab5 activation. Disruption of Gapex-5 function by expression of a dominant-interfering mutant or siRNA-mediated knockdown abolishes this effect of TC10, suggesting that Gapex-5 is required for TC10-mediated Rab5 activation. Rab5 has been implicated in the production of phosphatidylinositol 3-phosphate (PI3P), a lipid signaling molecule involved in Glut4 vesicle trafficking. Furthermore, insulin has been shown to induce PI3P formation through TC10 activation, although the mechanism underlying this process remains unclear. Consistent with the role of Gapex-5 as a molecular link between TC10 and Rab5 signaling, overexpression of plasma membrane targeted Gapex-5 promotes insulin-stimulated PI3P production. Conversely, knockdown of Gapex-5 blocks insulin-stimulated PI3P synthesis, Glut4 translocation and glucose uptake. Together, these data suggest that Gapex-5 is a multi-functional regulator of small G protein function, and may link TC10 to Rab5 signaling.