Ligand-independent GLUT4 translocation induced by guanosine 5'-O-(3-thiotriphosphate) involves tyrosine phosphorylation.
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) (GTPgammaS), into 3T3-L1 adipocytes. Thirty minutes after the injection of 5 mM GTPgammaS, 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 GTPgammaS-mediated GLUT4 translocation. On the other hand, coinjection of antiphosphotyrosine antibodies (PY20) blocked GTPgammaS-induced GLUT4 translocation by 65%. Furthermore, microinjection of GTPgammaS 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 GTPgammaS stimulated 2-deoxy-D-glucose transport to 70% of the extent of insulin stimulation. In addition, immunoblotting with phosphotyrosine antibodies after electroporation of GTPgammaS revealed increased tyrosine phosphorylation of several proteins, including 70- to 80-kDa and 120- to 130-kDa species. These results suggest that GTPgammaS 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).
Haruta
T
T
Department of Medicine, University of California-San Diego, La Jolla 92093-0673, USA.
Morris
A J
AJ
Vollenweider
P
P
Nelson
J G
JG
Rose
D W
DW
Mueckler
M
M
Olefsky
J M
JM
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D018274:Electroporation; D051275:Glucose Transporter Type 4; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D051379:Mice; D008845:Microinjections; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010766:Phosphorylation; D014443:Tyrosine / Q000378:metabolism*
D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495693:Slc2a4 protein, mouse; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases