ASSOCIATION OF CYTOSOLIC RAB4 WITH GDI ISOFORMS IN INSULIN-SENSITIVE 3T3-L1 ADIPOCYTES

Translocation of an intracellular pool of GLUT4 glucose transporters t o the fat and muscle cell surface is thought to involve small GTP-bind ing proteins such as the Rab4 protein. The cycling of Rab proteins bet ween cytosol and intracellular membranes necessary for their function appears to be regulated by GDP-dissociation inhibitors (GDI), three of which have been cloned thus far. Previous data suggest that Rab4 bind s two of these isoforms of GDI (1 and 2) similarly when purified prote ins are employed [Shisheva, A., et al. (1994) Mel. Cell. Biol. 14, 345 9-3468]. In the present study, we have analyzed the cytosolic Rab4 in complexes with GDI-1 or GDI-2 in intact cells using a coprecipitation technique. We show here that in insulin-sensitive 3T3-L1 adipocytes an d other cultured cells, Rab4 simultaneously forms stable cytosolic com plexes with both GDI-1 and GDI-2; Acute insulin treatment of the cultu red adipocytes significantly increases cytosolic levels of Rab4 which can be quantitatively immunoprecipitated with anti-Rab4 antibodies. Su rprisingly, the increased cytosolic Rab4 due to insulin action is pred ominantly associated with cytosolic GDI-1. The levels of cytosolic Rab 4-GDI-2 complexes were virtually unaltered by insulin. Insulin-depende nt alterations of Rab4 and GDI-1 phosphorylation were not detected in P-32-labeled 3T3-L1 adipocytes, suggesting another mechanism accounts for the specificity of Rab4 binding to GDI-1. Taken together, these da ta suggest there is selective formation of Rab4-GDI-1 complexes in res ponse to insulin which plays a role in the action of insulin on membra ne trafficking.