Abstract
Insulin stimulates glucose transporter (GLUT) 4 vesicle translocation from intracellular storage sites to the plasma membrane in 3T3L1 adipocytes through a VAMP2- and syntaxin 4-dependent mechanism. We have observed that Munc18c, a mammalian homolog of the yeast syntaxin-binding protein n-Sec1p, competed for the binding of VAMP2 to syntaxin 4. Consistent with an inhibitory function for Munc18c, expression of Munc18c, but not the related Munc18b isoform, prevented the insulin stimulation of GLUT4 and IRAP/vp165 translocation to the plasma membrane without any significant effect on GLUT1 trafficking. As expected, overexpressed Munc18c was found to co-immunoprecipitate with syntaxin 4 in the basal state. However, these complexes were found to dissociate upon insulin stimulation. Furthermore, endogenous Munc18c was predominantly localized to the plasma membrane and its distribution was not altered by insulin stimulation. Although expression of enhanced green fluorescent protein-Munc18c primarily resulted in a dispersed cytosolic distribution, co-expression with syntaxin 4 resulted in increased localization to the plasma membrane. Together, these data suggest that Munc18c inhibits the docking/fusion of GLUT4-containing vesicles by blocking the binding of VAMP2 to syntaxin 4. Insulin relieves this inhibition by inducing the dissociation of Munc18c from syntaxin 4 and by sequestering Munc18c to an alternative plasma membrane binding site.
Highlights
The binding of insulin to its heterotetrameric integral-membrane receptor activates its intracellular tyrosine kinase domain and thereby triggers a signaling cascade resulting in the translocation and fusion of intracellular GLUT41 isoform-containing vesicles to the plasma membrane [1,2,3]
Subcellular Distribution of v- and t-SNARE Proteins in 3T3L1 Adipocytes—To examine the intracellular localization of Munc18c in comparison to GLUT4 and several SNARE proteins involved in insulin-stimulated GLUT4 translocation, we used differential centrifugation to isolate subcellular fractions from 3T3L1 adipocytes (Fig. 1)
Immunoblot analysis demonstrated the presence of the GLUT4 protein in the whole cell extract, which was unaffected by insulin treatment (Fig. 1, lanes 1 and 2)
Summary
The binding of insulin to its heterotetrameric integral-membrane receptor activates its intracellular tyrosine kinase domain and thereby triggers a signaling cascade resulting in the translocation and fusion of intracellular GLUT41 isoform-containing vesicles to the plasma membrane [1,2,3]. Expression of Munc18c Inhibits Insulin-stimulated GLUT4 and IRAP/vp165 Translocation—Previous studies have indicated that the interaction of syntaxin 4 with VAMP2 is required for insulin-stimulated GLUT4 translocation to the plasma membrane (10 –12, 28). Co-expression of Munc18c did not alter the localization of GLUT4-eGFP in the absence of insulin, there was a marked reduction of insulin-stimulated plasma membrane fluorescence (Fig. 3, panels e and f).
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