The functional trafficking steps used by soluble NSF attachment protein receptor (SNARE) proteins have been difficult to establish because of substantial overlap in subcellular localization and because in vitro SNARE-dependent binding and fusion reactions can be promiscuous. Therefore, to functionally identify the site of action of the vesicle-associated membrane protein (VAMP) family of R-SNAREs, we have taken advantage of the temporal requirements of adipocyte biosynthetic sorting of a dual-tagged GLUT4 reporter (myc-GLUT4-GFP) coupled with small interfering RNA gene silencing. Using this approach, we confirm the requirement of VAMP2 and VAMP7 for insulin and osmotic shock trafficking from the vesicle storage sites, respectively, and fusion with the plasma membrane. Moreover, we identify a requirement for VAMP4 for the initial biosynthetic entry of GLUT4 from the Golgi apparatus into the insulin-responsive vesicle compartment, VAMP8, for plasma membrane endocytosis and VAMP2 for sorting to the specialized insulin-responsive compartment after plasma membrane endocytosis.

Insulin stimulation of adipocytes resulted in the recruitment of atypical PKC (PKCζ/λ) to plasma membrane lipid raft microdomains. This redistribution of PKCζ/λ was prevented by Clostridium difficile toxin B and by cholesterol depletion, but was unaffected by inhibition of phosphatidylinositol (PI) 3-kinase activity. Expression of the constitutively active GTP-bound form of TC10 (TC10Q/75L), but not the inactive GDP-bound mutant (TC10/T31N), targeted PKCζ/λ to the plasma membrane through an indirect association with the Par6–Par3 protein complex. In parallel, insulin stimulation as well as TC10/Q75L resulted in the activation loop phosphorylation of PKCζ. Although PI 3-kinase activation also resulted in PKCζ/λ phosphorylation, it was not recruited to the plasma membrane. Furthermore, insulin-induced GSK-3β phosphorylation was mediated by both PI 3-kinase–PKB and the TC10–Par6–atypical PKC signaling pathways. Together, these data demonstrate that PKCζ/λ can serve as a convergent downstream target for both the PI 3-kinase and TC10 signaling pathways, but only the TC10 pathway induces a spatially restricted targeting to the plasma membrane.

Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor–mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wild-type TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the nonlipid raft domains. Similarly, only the lipid raft–localized TC10/ H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.