Endocytosis of the Glucose Transporter GLUT4 Is Mediated by the GTPase Dynamin*
- From the Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
Abstract
To study the role of the GTPase dynamin in GLUT4 intracellular recycling, we have overexpressed dynamin−1 wild type and a GTPase-negative mutant (K44A) in primary rat adipose cells. Transfection was accomplished by electroporation using an hemagglutinin (HA)-tagged GLUT4 as a reporter protein. In cells expressing HA-GLUT4 alone, insulin results in an ≈7-fold increase in cell surface anti-HA antibody binding. Studies with wortmannin indicate that the kinetics of HA-GLUT4-trafficking parallel those of the native GLUT4 and in addition, that newly synthesized HA-GLUT4 goes to the plasma membrane before being sorted into the insulin-responsive compartments. Short term (4 h) coexpression of dynamin-K44A and HA-GLUT4 increases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. Under conditions of maximal expression of dynamin-K44A (24 h), most or all of the intracellular HA-GLUT4 appears to be present on the cell surface in the basal state, and insulin has no further effect. Measurements of the kinetics of HA-GLUT4 endocytosis show that dynamin-K44A blocks internalization of the glucose transporters. In contrast, expression of dynamin wild type decreases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. These data demonstrate that the endocytosis of GLUT4 is largely mediated by processes which require dynamin.
Footnotes
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↵* This work was supported in part by a fellowship from the Deutsche Forschungsgemeinschaft (to H. A.).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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↵‡ To whom correspondence should be addressed: EDMNS, DB, NIDDK, National Institutes of Health, Bldg. 10, Rm. 5N102, 10 Center Dr. MSC 1420, Bethesda, MD 20892-1420. Tel.: 301-496-5953; Fax: 301-402-0432; E-mail: hadi{at}helix.nih.gov.
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↵1 The abbreviations used are: HA, hemagglutinin; TGN, trans-Golgi network.
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- Received January 27, 1998.
- Revision received April 23, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
