THE SMALL GUANOSINE TRIPHOSPHATE-BINDING PROTEIN RAB4 IS INVOLVED IN INSULIN-INDUCED GLUT4 TRANSLOCATION AND ACTIN FILAMENT REARRANGEMENT IN 3T3-L1 CELLS

Insulin's stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter GLUT4 to the plasma mem brane. Small GTP-binding proteins have been implicated in the regulati on of vesicular traffic. We studied the effects of microinjection of w ild-type Rab4 glutathione S-transferase fusion protein (WT Rab4), a GT P-binding defective mutant (Rab4 N121I), a guanosine triphosphatase-de fective mutant (Rab4 Q67L), and a Rab4 antibody on insulin-induced GLU T4 translocation in 3T3-L1 adipocytes. Microinjection of Rab4 N121I an d Rab4 antibodies had no effect on basal GLUT4 staining, but inhibited insulin-induced GLUT4 translocation by 50% compared with that in cont rol IgG-injected cells. WT Rab4 and Rab4 Q67L microinjection had no ef fect on either basal or insulin-induced GLUT4 translocation. Premixing and coinjection of the Rab4 antibody with WT Rab4 almost completely a bolished its inhibitory effect on insulin-induced GLUT4 translocation. In contrast, microinjection of an antibody directed against the highl y conserved region of Rab3 proteins had no effect on insulin-induced G LUT4. These results point to a direct role of Rab4 in insulin-induced GLUT4 translocation, and that this effect is dependent on nucleotide b inding to the protein. We also studied the effect of microinjection of the same proteins on insulin-induced actin filament rearrangement (me mbrane ruffling) in the same cell line. Microinjection of Rab4 N121I a nd Rab4 antibodies inhibited insulin-induced membrane ruffling by 40%, whereas WT Rab4 or a Rab3 antibody injection had no effect on cytoske letal rearrangement. In summary, 1) Rab4 is a necessary component of t he insulin/GLUT4 translocation signaling pathway; 2) the function of R ab4 in this pathway requires GTP binding; 3) Rab4 also participates in the process of insulin-induced membrane ruffling; and 4) Rab3 protein s do not seem to be involved in these processes.