Association of cytosolic Rab4 with GDI isoforms in insulin-sensitive 3T3-L1 adipocytes.
Translocation of an intracellular pool of GLUT4 glucose transporters to the fat and muscle cell surface is thought to involve small GTP-binding proteins such as the Rab4 protein. The cycling of Rab proteins between 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 binds two of these isoforms of GDI (1 and 2) similarly when purified proteins are employed [Shisheva, A., et al. (1994) Mol. Cell. Biol. 14, 3459-3468]. 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 and other cultured cells, Rab4 simultaneously forms stable cytosolic complexes with both GDI-1 and GDI-2. Acute insulin treatment of the cultured adipocytes significantly increases cytosolic levels of Rab4 which can be quantitatively immunoprecipitated with anti-Rab4 antibodies. Surprisingly, the increased cytosolic Rab4 due to insulin action is predominantly associated with cytosolic GDI-1. The levels of cytosolic Rab4-GDI-2 complexes were virtually unaltered by insulin. Insulin-dependent alterations of Rab4 and GDI-1 phosphorylation were not detected in 32P-labeled 3T3-L1 adipocytes, suggesting another mechanism accounts for the specificity of Rab4 binding to GDI-1. Taken together, these data suggest there is selective formation of Rab4-GDI-1 complexes in response to insulin which plays a role in the action of insulin on membrane trafficking.
Shisheva
A
A
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. ashishev@moose.med.wayne.edu
Czech
M P
MP
D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D000818:Animals; D003600:Cytosol / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D006801:Humans; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D020695:rab4 GTP-Binding Proteins
C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D007328:Insulin; D019204:GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins