title: Vesicle-associated membrane protein 2 plays a specific role in the insulin-dependent trafficking of the facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes.
abstract: Vesicle-associated membrane protein 2 (VAMP2) has been implicated in the insulin-regulated trafficking of GLUT4 in adipocytes. It has been proposed that VAMP2 co-localizes with GLUT4 in a postendocytic storage compartment (Martin, S., Tellam, J., Livingstone, C., Slot, J. W., Gould, G. W., and James, D. E. (1996) J. Cell Biol. 134, 625-635), suggesting that it may play a role distinct from endosomal v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) such as cellubrevin that are also expressed in adipocytes. The present study examines the effects of recombinant glutathione S-transferase (GST) fusion proteins encompassing the entire cytoplasmic tails of VAMP1, VAMP2, and cellubrevin on insulin-stimulated GLUT4 translocation in streptolysin O permeabilized 3T3-L1 adipocytes. GST-VAMP2 inhibited insulin-stimulated GLUT4 translocation by approximately 35%, whereas GST-VAMP1 and GST-cellubrevin were without effect. A synthetic peptide corresponding to the unique N terminus of VAMP2 also inhibited insulin-stimulated GLUT4 translocation in a dose-dependent manner. This peptide had no effect on either guanosine 5'-3-O-(thio)triphosphate-stimulated GLUT4 translocation or on insulin-stimulated GLUT1 translocation. These results imply that GLUT4 and GLUT1 may undergo insulin-stimulated translocation to the cell surface from separate intracellular compartments. To confirm this, adipocytes were incubated with a transferrin-horseradish peroxidase conjugate to fill the itinerant endocytic system after which cells were incubated with H2O2 and diaminobenzidine. This treatment completely blocked insulin-stimulated movement of GLUT1, whereas in the case of GLUT4, movement to the surface was delayed but still reached similar levels to that observed in insulin-stimulated control cells after 30 min. These results suggest that the N terminus of VAMP2 plays a unique role in the insulin-dependent recruitment of GLUT4 from its intracellular storage compartment to the cell surface.
authors.affiliation: Centre for Molecular and Cellular Biology, University of Queensland, St. Lucia, Australia.
authors.forename: L B
authors.initials: LB
authors.lastname: Martin
authors.forename: A
authors.initials: A
authors.lastname: Shewan
authors.forename: C A
authors.initials: CA
authors.lastname: Millar
authors.forename: G W
authors.initials: GW
authors.lastname: Gould
authors.forename: D E
authors.initials: DE
authors.lastname: James
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia; D002451:Cell Compartmentation; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D050683:R-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051040:Vesicle-Associated Membrane Protein 3
chemical_list: D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C495678:Slc2a1 protein, mouse; C495679:Slc2a1 protein, rat; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D051040:Vesicle-Associated Membrane Protein 3