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

• Published in: Endocrinology (Philadelphia) 1998-01, Vol.139 (1), p.358-364
• Main Authors: Haruta, Tetsuro, Morris, Aaron J., Vollenweider, Peter, Nelson, James G., Rose, David W., Mueckler, Michael, Olefsky, Jerrold M.
• Format: Article
• Language: English
• Subjects: 1-Phosphatidylinositol 3-kinase; 3T3 Cells; Adipocytes; Adipocytes - metabolism; Animals; Antibodies; Biological Transport - drug effects; Coinjection; Electroporation; Fusion protein; Glucose; Glucose transport; Glucose Transporter Type 4; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Guanosines; Immunoblotting; Immunoglobulin G; Insulin; Kinases; Mice; Microinjection; Microinjections; Molecular Weight; Monosaccharide Transport Proteins - metabolism; Muscle Proteins; Phosphatidylinositol 3-Kinases - physiology; Phosphorylation; Phosphotyrosine; Protein transport; Proteins; Signal transduction; Staining; Translocation; Tyrosine; Tyrosine - metabolism; Wortmannin
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/endo.139.1.5698

Abstract 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) (GTPγS), into 3T3-L1 adipocytes. Thirty minutes after the injection of 5 mm GTPγS, 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 GTPγS-mediated GLUT4 translocation. On the other hand, coinjection of antiphosphotyrosine antibodies (PY20) blocked GTPγS-induced GLUT4 translocation by 65%. Furthermore, microinjection of GTPγS 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 GTPγS stimulated 2-deoxy-d-glucose transport to 70% of the extent of insulin stimulation. In addition, immunoblotting with phosphotyrosine antibodies after electroporation of GTPγS revealed increased tyrosine phosphorylation of several proteins, including 70- to 80-kDa and 120- to 130-kDa species. These results suggest that GTPγS 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).