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Expression of a Dominant Interfering Dynamin Mutant in 3T3L1 Adipocytes Inhibits GLUT4 Endocytosis without Affecting Insulin Signaling

Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242-1109, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/1998; 273(39):25450-7. DOI: 10.1074/jbc.273.39.25450
Source: PubMed

ABSTRACT 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.

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    • "Insulin-stimulated transfected adipocytes were chilled to 4 ° C and incubated with the myc monoclonal antibody for 1 h to label GLUT4 at the plasma membrane. Cells were then washed to remove insulin and excess myc antibody as described previously ( Kao et al., 1998 ). The cells were placed at 37 ° C and incubated for various times to allow the myc antibody – bound GLUT4 to internalize. "
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    • "Basal golgin-160 knockdown cells and insulin-stimulated control adipocytes were chilled and incubated with the myc monoclonal antibody for 1 h at 4°C to label the GLUT4 at the plasma membrane. Cells were then washed to remove insulin and excess myc antibody as described previously (Kao et al., 1998). The cells were placed at 37°C and incubated for various times to allow myc antibody-bound GLUT4 to internalize. "
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    • "In order to investigate whether HMIT internalization was dependent on dynamin-mediated endocytosis, we infected HMIT-expressing cells with the GTPase-deficient dominantnegative form of dynamin (dynaminK44A) (Ceresa et al, 1998; Kao et al, 1998). Under basal conditions, cell surface expression of HMIT could be clearly detected in cells expressing dynaminK44A but not in control cells. "
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