ATP-SENSITIVE BINDING OF A 70-KDA CYTOSOLIC PROTEIN TO THE GLUCOSE-TRANSPORTER IN RAT ADIPOCYTES

We have identified a 70-kDa cytosolic protein (GTBP70) in rat adipocyt es that binds to glutathione S-transferase fusion proteins correspondi ng to the cytoplasmic domains of the facilitative glucose transporter isoforms Glut1, Glut2, and Glut4. GTBP70 did not bind to irrelevant fu sion proteins, indicating that the binding is specific to the glucose transporter. GTBP70 binding to the glucose transporter showed little i soform specificity but was significantly subdomain-specific; it bound to the C-terminal domain and the central loop, but not to the N-termin al domain of Glut4. The GTBP70 binding to Glut4 was not affected by th e presence of 2 mM EDTA, 2.4 mM Ca2+, or 150 mm K+. The binding was in hibited by ATP in a dose-dependent manner, with 50% inhibition at 10 m M ATP. This inhibition was specific to ATP, as ADP and AMP-PCP (adenos ine 5'-(beta,gamma-methylenetriphosphate)) were without effect. GTBP70 did not react with antibodies against phosphotyrosine, phosphothreoni ne, or phosphoserine, suggesting that it is not a phosphoprotein. The binding of GTBP70 to Glut4 was not affected by the pretreatment of adi pocytes with insulin. When these experiments were repeated using rat h epatocyte cytosols, no ATP-sensitive 70-kDa protein binding to the glu cose transporter fusion proteins was evident, suggesting that either G TBP70 expression or its function is cell-specific. These findings stro ngly suggest the possibility that GTBP70 may play a key role in glucos e transporter regulation in insulin target cells such as adipocytes.