Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes

The insulin-responsive aminopeptidase (IRAP/VP165/gp160) was identified ori ginally in GLUT4-containing vesicles and shown to translocate in response t o insulin, much like the glucose transporter 4 (GLUT4), This study characte rizes the trafficking and kinetics of IRAP in exocytosis, endocytosis, and recycling to the membrane in 3T3-L1 adipocytes. After exposure of 3T3-L1 ad ipocytes to insulin, IRAP translocated to the plasma membrane as assessed b y either cell fractionation, surface biotinylation, or the plasma membrane sheet assay. The rate of exocytosis closely paralleled that of GLUT4. In th e continuous presence of insulin, IRAP was endocytosed with a half-time of about 3-5 min. IRAP endocytosis is inhibited by cytosol acidification, a pr operty of clathrin-mediated endocytosis, but not by the expression of a con stitutively active Akt/PKB, Arrival in an LDM fraction derived via subcellu lar fractionation exhibited a slower time course than disappearance from th e cell surface, suggesting additional endocytic intermediates. As assayed b y membrane "sheets," GLUT4 and IRAP showed similar internalization rates th at are wortmannin-insensitive and occur with a half-time of roughly 5 min. IRAP remaining on the cell surface 10 min following insulin removal was bot h biotin- and avidin-accessible, implying the absence of thin-necked invagi nations, Finally, endocytosed IRAP quickly recycled back to the plasma memb rane in a wortmannin-sensitive process. These results demonstrate rapid end ocytosis and recycling of IRAP in the presence of insulin and trafficking t hat matches GLUT4 in rate.