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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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Cholesterol depletion with methyl-β-cyclodextrin disrupts the plasma membrane subdomain compartmentalization of caveolin and TC10. Differentiated 3T3L1 adipocytes were incubated with 10 mM β-CD for the times indicated. Plasma membrane sheets were prepared and colabeled with a rabbit caveolin 1 antibody (a–e) and a chicken TC10 antibody (f–j). Merged images are presented in panels k–o. These are representative plasma membrane sheets from two independent determinations. Bar, 10 μM.
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fig6: Cholesterol depletion with methyl-β-cyclodextrin disrupts the plasma membrane subdomain compartmentalization of caveolin and TC10. Differentiated 3T3L1 adipocytes were incubated with 10 mM β-CD for the times indicated. Plasma membrane sheets were prepared and colabeled with a rabbit caveolin 1 antibody (a–e) and a chicken TC10 antibody (f–j). Merged images are presented in panels k–o. These are representative plasma membrane sheets from two independent determinations. Bar, 10 μM.

Mentions: The morphological effects of cholesterol extraction on these caveolin-positive structures were examined in Fig. 6 . Previous studies have demonstrated that treatment of cells with methyl-β-cyclodextrin (β-CD) results in cholesterol depletion and disruption of plasma membrane caveolae (Chang et al., 1992; Rothberg et al., 1992; Hailstones et al., 1998; Parpal et al., 2001). Similarly, treatment of adipocytes with β-CD resulted in a time-dependent dissolution of these torus-shaped caveolin structures, which was nearly complete by 60 min (Fig. 6, a–e). In addition, treatment with β-CD resulted in a parallel disruption of TC10 organization and colocalization with caveolin (Fig. 6, f–o). Thus, these data demonstrate that the adipocyte plasma membrane has highly organized aggregates of individual caveolin-enriched lipid rafts that form large morphologically distinct torus-shaped structures.


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)

Cholesterol depletion with methyl-β-cyclodextrin disrupts the plasma membrane subdomain compartmentalization of caveolin and TC10. Differentiated 3T3L1 adipocytes were incubated with 10 mM β-CD for the times indicated. Plasma membrane sheets were prepared and colabeled with a rabbit caveolin 1 antibody (a–e) and a chicken TC10 antibody (f–j). Merged images are presented in panels k–o. These are representative plasma membrane sheets from two independent determinations. Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Cholesterol depletion with methyl-β-cyclodextrin disrupts the plasma membrane subdomain compartmentalization of caveolin and TC10. Differentiated 3T3L1 adipocytes were incubated with 10 mM β-CD for the times indicated. Plasma membrane sheets were prepared and colabeled with a rabbit caveolin 1 antibody (a–e) and a chicken TC10 antibody (f–j). Merged images are presented in panels k–o. These are representative plasma membrane sheets from two independent determinations. Bar, 10 μM.
Mentions: The morphological effects of cholesterol extraction on these caveolin-positive structures were examined in Fig. 6 . Previous studies have demonstrated that treatment of cells with methyl-β-cyclodextrin (β-CD) results in cholesterol depletion and disruption of plasma membrane caveolae (Chang et al., 1992; Rothberg et al., 1992; Hailstones et al., 1998; Parpal et al., 2001). Similarly, treatment of adipocytes with β-CD resulted in a time-dependent dissolution of these torus-shaped caveolin structures, which was nearly complete by 60 min (Fig. 6, a–e). In addition, treatment with β-CD resulted in a parallel disruption of TC10 organization and colocalization with caveolin (Fig. 6, f–o). Thus, these data demonstrate that the adipocyte plasma membrane has highly organized aggregates of individual caveolin-enriched lipid rafts that form large morphologically distinct torus-shaped structures.

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