Characterization of Munc-18c and syntaxin-4 in 3T3-L1 adipocytes - Putative role in insulin-dependent movement of GLUT-4

We have previously identified three mammalian Seel/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-8, 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 S. Using a three-way binding assay, it was shown that Munc-18c inhibits the binding of syntaxin-4 to VAMP-S, 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.