Ligand-independent GLUT4 translocation induced by guanosine 5′-O-(3-thiotriphosphate) involves tyrosine phosphorylation

被引:23
作者
Haruta, T
Morris, AJ
Vollenweider, P
Nelson, JG
Rose, DW
Mueckler, M
Olefsky, JM
机构
[1] Univ Calif San Diego, Dept Med 0673, La Jolla, CA 92093 USA
[2] Washington Univ, Sch Med, Dept Cell Biol & Physiol, St Louis, MO 63110 USA
[3] Vet Adm Res Serv, San Diego, CA 92161 USA
关键词
D O I
10.1210/en.139.1.358
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
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) (GTP gamma S), into 3T3-L1 adipocytes. Thirty minutes after the injection of 5 mM GTP gamma S, 40% of injected cells displayed surface GLUT4 staining indicative of GLUM 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 GTP gamma S-mediated GLUT4 translocation. On the other hand, coinjection of antiphosphotyrosine antibodies (PY20) blocked GTP gamma S-induced GLUT4 translocation by 65%. Furthermore, microinjection of GTP gamma S 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 GTP gamma S stimulated 2-deoxy-D-glucose transport to 70% of the extent of insulin stimulation. In addition, immunoblotting with phosphotyrosine antibodies after electroporation of GTP gamma S revealed increased tyrosine phosphorylation of several proteins, including 70- to 80-kDa and 120- to 130-kDa species. These results suggest that GTP gamma S 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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页码:358 / 364
页数:7
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