Initial entry of IRAP into the insulin-responsive storage compartment occurs prior to basal or insulin-stimulated plasma membrane recycling.
To examine the acquisition of insulin sensitivity after the initial biosynthesis of the insulin-responsive aminopeptidase (IRAP), 3T3-L1 adipocytes were transfected with an enhanced green fluorescent protein-IRAP (EGFP-IRAP) fusion protein. In the absence of insulin, IRAP was rapidly localized (1-3 h) to secretory membranes and retained in these intracellular membrane compartments with little accumulation at the plasma membrane. However, insulin was unable to induce translocation to the plasma membrane until 6-9 h after biosynthesis. This was in marked contrast to another type II membrane protein (syntaxin 3) that rapidly defaulted to the plasma membrane 3 h after expression. In parallel with the time-dependent acquisition of insulin responsiveness, the newly synthesized IRAP protein converted from a brefeldin A-sensitive to a brefeldin A-insensitive state. The initial trafficking of IRAP to the insulin-responsive compartment was independent of plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis but had no effect on the insulin-stimulated translocation of the newly synthesized IRAP protein.
Liu
Gang
G
Dept. of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota, USA.
Hou
June Chunqiu
JC
Watson
Robert T
RT
Pessin
Jeffrey E
JE
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism; D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D010122:Cystinyl Aminopeptidase; D034281:Dynamins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000172:deficiency / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D007328:Insulin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000626:Aminopeptidases; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase; D034281:Dynamins