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Ligand-Independent GLUT4 Translocation Induced by Guanosine 5'-O-(3-Thiotriphosphate) Involves Tyrosine Phosphorylation¹

Department of Medicine, University of California-San Diego, La Jolla, California 92093; the Department of Cell Biology and Physiology, Washington University School of Medicine (M.M.), St. Louis, Missouri 63110; and the Veterans Administration Research Service (J.M.O.), San Diego, California 92161

Address all correspondence and requests for reprints to: Jerrold M. Olefsky, M.D., Department of Medicine (0673), University of California-San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673.

To delineate the signaling pathway leading to glucose transport protein (GLUT4) translocation, we examined the effect of microinjection of the nonhydrolyzable GTP analog, guanosine 5'-O-(3-thiotriphosphate) (GTPS), into 3T3-L1 adipocytes. Thirty minutes after the injection of 5 mM GTPS, 40% of injected cells displayed surface GLUT4 staining indicative of GLUT4 translocation compared with 55% for insulin-treated cells and 10% in control IgG-injected cells. Treatment of the cells with the phosphatidylinositol 3-kinase inhibitor wortmannin or coinjection of GST-p85 SH2 fusion protein had no effect on GTPS-mediated GLUT4 translocation. On the other hand, coinjection of antiphosphotyrosine antibodies (PY20) blocked GTPS-induced GLUT4 translocation by 65%. Furthermore, microinjection of GTPS led to the appearance of tyrosine-phosphorylated proteins around the periphery of the plasma membrane, as observed by immunostaining with PY20. Treatment of the cells with insulin caused a similar phosphotyrosine-staining pattern. Electroporation of GTPS stimulated 2-deoxy-D-glucose transport to 70% of the extent of insulin stimulation. In addition, immunoblotting with phosphotyrosine antibodies after electroporation of GTPS revealed increased tyrosine phosphorylation of several proteins, including 70- to 80-kDa and 120- to 130-kDa species. These results suggest that GTPS acts upon a signaling pathway either downstream of or parallel to activation of phosphatidylinositol 3-kinase and that this pathway involves tyrosine-phosphorylated protein(s).

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