Expression of a Dominant Interfering Dynamin Mutant in 3T3L1 Adipocytes Inhibits GLUT4 Endocytosis without Affecting Insulin Signaling*
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.
Footnotes
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↵* This work was supported in part by Research Grants DK33823 and DK25295 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Recipient of a postdoctoral fellowship award from the Juvenile Diabetes Foundation.
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↵§ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242-1109. Tel.: 319-335-7823; Fax: 319-335-7330; E-mail:jeffrey-pessin{at}uiowa.edu.
- Abbreviations:
- GLUT
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glucose transporter
- DMEM
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Dulbecco’s essential modified medium
- ERK
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extracellular-regulated kinase
- IRβ
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insulin receptor β subunit, IRS1, insulin receptor substrate-1
- MAP kinase
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mitogen-activated protein kinase
- PBS
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phosphate-buffered saline
- PI 3-kinase
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phosphatidylinositol 3-kinase
- HEK
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human embryo kidney
- PAGE
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polyacrylamide gel electrophoresis
- Shc
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Src homology 2/α-collagen-related protein.
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- Received April 27, 1998.
- Revision received July 6, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
