title: The small guanosine triphosphate-binding protein Rab4 is involved in insulin-induced GLUT4 translocation and actin filament rearrangement in 3T3-L1 cells.
abstract: Insulin's stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter GLUT4 to the plasma membrane. Small GTP-binding proteins have been implicated in the regulation of vesicular traffic. We studied the effects of microinjection of wild-type Rab4 glutathione S-transferase fusion protein (WT Rab4), a GTP-binding defective mutant (Rab4 N121I), a guanosine triphosphatase-defective mutant (Rab4 Q67L), and a Rab4 antibody on insulin-induced GLUT4 translocation in 3T3-L1 adipocytes. Microinjection of Rab4 N121I and Rab4 antibodies had no effect on basal GLUT4 staining, but inhibited insulin-induced GLUT4 translocation by 50% compared with that in control IgG-injected cells. WT Rab4 and Rab4 Q67L microinjection had no effect on either basal or insulin-induced GLUT4 translocation. Premixing and coinjection of the Rab4 antibody with WT Rab4 almost completely abolished its inhibitory effect on insulin-induced GLUT4 translocation. In contrast, microinjection of an antibody directed against the highly conserved region of Rab3 proteins had no effect on insulin-induced GLUT4. These results point to a direct role of Rab4 in insulin-induced GLUT4 translocation, and that this effect is dependent on nucleotide binding to the protein. We also studied the effect of microinjection of the same proteins on insulin-induced actin filament rearrangement (membrane ruffling) in the same cell line. Microinjection of Rab4 N121I and Rab4 antibodies inhibited insulin-induced membrane ruffling by 40%, whereas WT Rab4 or a Rab3 antibody injection had no effect on cytoskeletal rearrangement. In summary, 1) Rab4 is a necessary component of the insulin/GLUT4 translocation signaling pathway; 2) the function of Rab4 in this pathway requires GTP binding; 3) Rab4 also participates in the process of insulin-induced membrane ruffling; and 4) Rab3 proteins do not seem to be involved in these processes.
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla 92093, USA.
authors.forename: P
authors.initials: P
authors.lastname: Vollenweider
authors.forename: S S
authors.initials: SS
authors.lastname: Martin
authors.forename: T
authors.initials: T
authors.lastname: Haruta
authors.forename: A J
authors.initials: AJ
authors.lastname: Morris
authors.forename: J G
authors.initials: JG
authors.lastname: Nelson
authors.forename: M
authors.initials: M
authors.lastname: Cormont
authors.forename: Y
authors.initials: Y
authors.lastname: Le Marchand-Brustel
authors.forename: D W
authors.initials: DW
authors.lastname: Rose
authors.forename: J M
authors.initials: JM
authors.lastname: Olefsky
mesh_terms: D016475:3T3 Cells; D000199:Actins / Q000502:physiology*; D017667:Adipocytes / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D051275:Glucose Transporter Type 4; D005982:Glutathione Transferase / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008845:Microinjections; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009154:Mutation; D020692:rab3 GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins
chemical_list: D000199:Actins; D000906:Antibodies; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495693:Slc2a4 protein, mouse; D005982:Glutathione Transferase; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins