title: Characterization of Munc-18c and syntaxin-4 in 3T3-L1 adipocytes. Putative role in insulin-dependent movement of GLUT-4.
abstract: We have previously identified three mammalian Sec1/Munc-18 homologues in adipocytes (Tellam, J. T., McIntosh, S., and James, D. E. (1995) J. Biol. Chem. 270, 5857-5863). These proteins are thought to modulate the interaction between vesicle membrane and target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and thus regulate intracellular vesicular transport. This study aimed to further characterize these Munc-18 isoforms and to define their potential role in the trafficking of GLUT-4 in adipocytes, a process reported to involve the vesicle membrane SNARE, VAMP-2. Using an in vitro binding assay with recombinant fusion proteins, we show that Munc-18a and Munc-18b bind to syntaxin-1A, -2, and -3, while Munc-18c binds only to syntaxin-2 and -4. The specific interaction between Munc-18c and syntaxin-4 is of interest because aside from syntaxin-1A, which is not expressed in adipocytes, syntaxin-4 is the only syntaxin that binds to VAMP-2. Using a three-way binding assay, it was shown that Munc-18c inhibits the binding of syntaxin-4 to VAMP-2. The subcellular distribution of syntaxin-4 and Munc-18c was almost identical, both being enriched in the plasma membrane, and both exhibiting an insulin-dependent movement out of an intracellular membrane fraction similar to that observed for GLUT-4. Munc-18b had a similar distribution to Munc-18c and so may also be involved in vesicle transport to the cell surface, whereas Munc-18a was undetectable by immunoblotting in adipocytes. Microinjection of a syntaxin-4 antibody into 3T3-L1 adipocytes blocked the insulin-dependent recruitment of GLUT-4 to the cell surface. These data suggest that syntaxin-4/Munc-18c/VAMP-2 may play a role in the docking/fusion of intracellular GLUT-4-containing vesicles with the cell surface in adipocytes.
authors.affiliation: Centre for Molecular and Cellular Biology and Department of Physiology and Pharmacology, University of Queensland, St. Lucia 4072, Queensland, Australia.
authors.forename: J T
authors.initials: JT
authors.lastname: Tellam
authors.forename: S L
authors.initials: SL
authors.lastname: Macaulay
authors.forename: S
authors.initials: S
authors.lastname: McIntosh
authors.forename: D R
authors.initials: DR
authors.lastname: Hewish
authors.forename: C W
authors.initials: CW
authors.lastname: Ward
authors.forename: D E
authors.initials: DE
authors.lastname: James
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000502:physiology; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000502:physiology*; D051938:Munc18 Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins / Q000502:physiology*; D009842:Oligopeptides / Q000276:immunology; D015388:Organelles / Q000378:metabolism; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
chemical_list: D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D051938:Munc18 Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C495693:Slc2a4 protein, mouse; C494214:Stx1a protein, mouse; C497938:Stxbp1 protein, mouse; C497941:Stxbp2 protein, mouse; C497943:Stxbp3 protein, mouse; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins