Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulin-regulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.

The challenges of abundance: epithelial junctions and small GTPase signalling

Small GTPases of the Ras superfamily play critical roles in epithelial biogenesis. Many key morphogenetic functions occur when small GTPases act at epithelial junctions, where they mediate an increasingly complex interplay between cell-cell adhesion molecules and fundamental cellular processes, such as cytoskeletal activity, polarity and trafficking. Important recent advances in this field include the role of additional members of the Ras superfamily in cell-cell contact stability and the capacity for polarity determinants to regulate small GTPase signalling. Interestingly, small GTPases may participate in the cross-talk between different adhesive receptors: in tissues classical cadherins can selectively regulate other junctions through cell signalling rather than through a global influence on cell-cell cohesion.