title: Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes.
abstract: The insulin-responsive aminopeptidase (IRAP/VP165/gp160) was identified originally in GLUT4-containing vesicles and shown to translocate in response to insulin, much like the glucose transporter 4 (GLUT4). This study characterizes the trafficking and kinetics of IRAP in exocytosis, endocytosis, and recycling to the membrane in 3T3-L1 adipocytes. After exposure of 3T3-L1 adipocytes to insulin, IRAP translocated to the plasma membrane as assessed by either cell fractionation, surface biotinylation, or the plasma membrane sheet assay. The rate of exocytosis closely paralleled that of GLUT4. In the continuous presence of insulin, IRAP was endocytosed with a half-time of about 3-5 min. IRAP endocytosis is inhibited by cytosol acidification, a property of clathrin-mediated endocytosis, but not by the expression of a constitutively active Akt/PKB. Arrival in an LDM fraction derived via subcellular fractionation exhibited a slower time course than disappearance from the cell surface, suggesting additional endocytic intermediates. As assayed by membrane "sheets," GLUT4 and IRAP showed similar internalization rates that 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 both biotin- and avidin-accessible, implying the absence of thin-necked invaginations. Finally, endocytosed IRAP quickly recycled back to the plasma membrane in a wortmannin-sensitive process. These results demonstrate rapid endocytosis and recycling of IRAP in the presence of insulin and trafficking that matches GLUT4 in rate.
authors.affiliation: Howard Hughes Medical Institute, Philadelphia, Pennsylvania 19104, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: Garza
authors.forename: M J
authors.initials: MJ
authors.lastname: Birnbaum
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology*; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D010122:Cystinyl Aminopeptidase; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051275:Glucose Transporter Type 4; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D004791:Enzyme Inhibitors; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495693:Slc2a4 protein, mouse; D001360:Avidin; D001710:Biotin; D000626:Aminopeptidases; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase; D000077191:Wortmannin