Expression of a dominant interfering dynamin mutant in 3T3L1 adipocytes inhibits GLUT4 endocytosis without affecting insulin signaling.
To examine the role of clathrin-coated vesicle endocytosis in insulin receptor signaling and GLUT4 trafficking, we used recombinant adenovirus to express a dominant interfering mutant of dynamin (K44A/dynamin) in 3T3L1 adipocytes. Functional expression of K44A/dynamin, as measured by inhibition of transferrin receptor internalization, did not affect insulin-stimulated insulin receptor autophosphorylation, Shc tyrosine phosphorylation, or mitogen-activated protein kinase activation. Although the tyrosine phosphorylation of insulin receptor substrate-1 was slightly reduced, correlating with a 25% decrease in insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, insulin-stimulated Akt kinase activation was unaffected. In contrast, expression of K44A/dynamin resulted in the cell-surface accumulation of GLUT4 under basal conditions and an inhibition of GLUT4 endocytosis without affecting insulin-stimulated GLUT4 exocytosis. These data demonstrate that disruption of clathrin-mediated endocytosis does not significantly perturb insulin receptor signal transduction pathways. Furthermore, K44A/dynamin expression causes an accumulation of GLUT4 at the cell surface, suggesting that GLUT4 vesicles exist in at least two distinct intracellular compartments, one that undergoes continuous recycling and a second that is responsive to insulin.
Kao
A W
AW
Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242-1109, USA.
Ceresa
B P
BP
Santeler
S R
SR
Pessin
J E
JE
D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D034281:Dynamins; D004705:Endocytosis; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors; D011990:Receptors, Transferrin / Q000378:metabolism*; D015398:Signal Transduction
D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011990:Receptors, Transferrin; C495693:Slc2a4 protein, mouse; D011972:Receptor, Insulin; D020558:GTP Phosphohydrolases; D034281:Dynamins