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Papers In Press, published online ahead of print June 13, 2005
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8
Corresponding Author: amira{at}sickkids.ca
Insulin increases glucose uptake into muscle via glucose transporter-4 (GLUT4) translocation to the cell membrane, but the regulated events in GLUT4 traffic are unknown. Here we focus on the role of class IA phosphatidylinositol (PI) 3-kinase and specific phosphoinositides in the steps of GLUT4 arrival and fusion with the membrane, using L6 muscle cells expressing GLUT4myc. To this end, we detected the availability of the myc epitope at the cell surface or intravesicular spaces and of the cytosol-facing C-terminal epitope, in cells and membrane lawns derived from them. We observed that: a) Wortmannin and LY294002 at concentrations that inhibit class IA PI 3-kinase reduced but did not abate the C-terminus gain, yet the myc epitope was unavailable for detection unless lawns or cells were permeabilized, suggesting the presence of GLUT4myc in docked, unfused vesicles. Accordingly, GLUT4myc-containing vesicles were detected by immunoelectron microscopy of membranes from cells pretreated with wortmannin and insulin, but not insulin or wortmannin alone. b) Insulin caused greater immunological availability of the C-terminus than myc epitopes, suggesting that C-terminus unmasking had occurred. Delivering PI(3,4,5)P3 to intact cells significantly increased lawn-associated myc signal without C-terminus gain. Conversely, PI3P increased the detection of C-terminus epitope without any myc gain. We propose that insulin regulates GLUT4 membrane arrival, fusion and C-terminus unmasking, through distinct phosphoinositides. PI(3,4,5)P3 causes arrival, fusion without unmasking, whereas PI3P causes arrival and unmasking without fusion.
J. Biol. Chem, 10.1074/jbc.M500501200
Submitted on January 14, 2005
Revised on June 6, 2005
Accepted on June 13, 2005
Insulin regulates the membrane arrival, fusion and C-terminal unmasking of Glut4 via distinct phosphoinositides
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