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Originally published In Press as doi:10.1074/jbc.M500501200 on June 13, 2005
J. Biol. Chem., Vol. 280, Issue 31, 28792-28802, August 5, 2005
Insulin Regulates the Membrane Arrival, Fusion, and C-terminal Unmasking of Glucose Transporter-4 via Distinct Phosphoinositides*
Manabu Ishiki  ,
Varinder K. Randhawa¶||,
Vincent Poon¶,
Lellean JeBailey¶**, and
Amira Klip¶
From the
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8 and the ¶Department of Biochemistry, the University of Toronto, Toronto, Ontario M5S 1A8, Canada
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 the following: (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 phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) to intact cells significantly increased lawn-associated myc signal without C terminus gain. Conversely, phosphatidylinositol 3-phosphate (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 and fusion without unmasking, whereas PI3P causes arrival and unmasking without fusion.
Received for publication, January 14, 2005
, and in revised form, June 6, 2005.
This report is dedicated to the memory of Dr. Tetsuro Kono.
* This work was supported by the Canadian Institutes of Health Research (CIHR) (Grant MT7307 to A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
 Supported by a fellowship from the Canadian Diabetes Association.
|| Supported by a CIHR Doctoral Award Studentship.
** Supported by a studentship from the Ontario Student Opportunity Trust Fund-Hospital for Sick Children Foundation Student Scholarship Program.
To whom correspondence should be addressed: Programme in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-6392; Fax: 416-813-5028; E-mail: amira{at}sickkids.ca.
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Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
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