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Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.

Watson RT, Shigematsu S, Chiang SH, Mora S, Kanzaki M, Macara IG, Saltiel AR, Pessin JE - J. Cell Biol. (2001)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
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.

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The expressed TC10/WT and TC10/H-Ras chimeras specifically colocalize with caveolin. Differentiated 3T3L1 adipocytes were electroporated with 50 μg of HA epitope–tagged TC10 (a–c), TC10/H-Ras chimera (d–f), and TC10/K-Ras chimera (g–i) cDNAs, and plasma membrane sheets were prepared 18 h later. The plasma membrane sheets were then colabeled with a polyclonal caveolin 1 antibody (a, d, and g) and a monoclonal HA antibody (b, e, and h). Merged images are presented in panels c, f, and i. These are representative plasma membrane sheets from three independent determinations.
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fig7: The expressed TC10/WT and TC10/H-Ras chimeras specifically colocalize with caveolin. Differentiated 3T3L1 adipocytes were electroporated with 50 μg of HA epitope–tagged TC10 (a–c), TC10/H-Ras chimera (d–f), and TC10/K-Ras chimera (g–i) cDNAs, and plasma membrane sheets were prepared 18 h later. The plasma membrane sheets were then colabeled with a polyclonal caveolin 1 antibody (a, d, and g) and a monoclonal HA antibody (b, e, and h). Merged images are presented in panels c, f, and i. These are representative plasma membrane sheets from three independent determinations.

Mentions: To confirm the plasma membrane compartmentalization of the expressed TC10 and TC10/Ras chimera proteins, we next compared their colocalization with caveolin in isolated plasma membrane sheets. As reported previously (Li et al., 1996; Mineo et al., 1996; Song et al., 1996; Roy et al., 1999), H-Ras was found to colocalize with the caveolin-positive torus-shaped structures, whereas K-Ras was uniformly distributed across the plasma membrane sheets and did not specifically colocalize with caveolin (data not shown). As observed for the endogenous TC10 protein, the expressed TC10/WT protein was strongly colocalized with caveolin in the torus-shaped structures (Fig. 7 , a–c). A similar plasma membrane distribution was detected for the expressed TC10/H-Ras chimera (Fig. 7, d–f). In contrast, the TC10/K-Ras chimera was found to be uniformly scattered throughout the plasma membrane (Fig. 7, g–i).


Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation.

Watson RT, Shigematsu S, Chiang SH, Mora S, Kanzaki M, Macara IG, Saltiel AR, Pessin JE - J. Cell Biol. (2001)

The expressed TC10/WT and TC10/H-Ras chimeras specifically colocalize with caveolin. Differentiated 3T3L1 adipocytes were electroporated with 50 μg of HA epitope–tagged TC10 (a–c), TC10/H-Ras chimera (d–f), and TC10/K-Ras chimera (g–i) cDNAs, and plasma membrane sheets were prepared 18 h later. The plasma membrane sheets were then colabeled with a polyclonal caveolin 1 antibody (a, d, and g) and a monoclonal HA antibody (b, e, and h). Merged images are presented in panels c, f, and i. These are representative plasma membrane sheets from three independent determinations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196453&req=5

fig7: The expressed TC10/WT and TC10/H-Ras chimeras specifically colocalize with caveolin. Differentiated 3T3L1 adipocytes were electroporated with 50 μg of HA epitope–tagged TC10 (a–c), TC10/H-Ras chimera (d–f), and TC10/K-Ras chimera (g–i) cDNAs, and plasma membrane sheets were prepared 18 h later. The plasma membrane sheets were then colabeled with a polyclonal caveolin 1 antibody (a, d, and g) and a monoclonal HA antibody (b, e, and h). Merged images are presented in panels c, f, and i. These are representative plasma membrane sheets from three independent determinations.
Mentions: To confirm the plasma membrane compartmentalization of the expressed TC10 and TC10/Ras chimera proteins, we next compared their colocalization with caveolin in isolated plasma membrane sheets. As reported previously (Li et al., 1996; Mineo et al., 1996; Song et al., 1996; Roy et al., 1999), H-Ras was found to colocalize with the caveolin-positive torus-shaped structures, whereas K-Ras was uniformly distributed across the plasma membrane sheets and did not specifically colocalize with caveolin (data not shown). As observed for the endogenous TC10 protein, the expressed TC10/WT protein was strongly colocalized with caveolin in the torus-shaped structures (Fig. 7 , a–c). A similar plasma membrane distribution was detected for the expressed TC10/H-Ras chimera (Fig. 7, d–f). In contrast, the TC10/K-Ras chimera was found to be uniformly scattered throughout the plasma membrane (Fig. 7, g–i).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus