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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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Expression of a dominant- interfering caveolin 3 mutant disrupts the plasma membrane subdomain compartmentalization of TC10. Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of HA-TC10 plus 200 μg of the empty vector (a–c), wild-type caveolin 3 (Cav3/WT; d–f), or the dominant-interfering caveolin 3 mutant (Cav3/DGV; g–i). 36 h later, the cells were fixed, plasma membrane sheets were prepared and subjected to confocal fluorescent microscopy with a polyclonal caveolin 1 antibody (a, d, and g), and a monoclonal HA antibody (b, e, and h). The merged images are shown in panels c, f, and i. These are representative field of cells from two independent determinations. Bar, 10 μM.
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fig8: Expression of a dominant- interfering caveolin 3 mutant disrupts the plasma membrane subdomain compartmentalization of TC10. Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of HA-TC10 plus 200 μg of the empty vector (a–c), wild-type caveolin 3 (Cav3/WT; d–f), or the dominant-interfering caveolin 3 mutant (Cav3/DGV; g–i). 36 h later, the cells were fixed, plasma membrane sheets were prepared and subjected to confocal fluorescent microscopy with a polyclonal caveolin 1 antibody (a, d, and g), and a monoclonal HA antibody (b, e, and h). The merged images are shown in panels c, f, and i. These are representative field of cells from two independent determinations. Bar, 10 μM.

Mentions: A dominant-interfering caveolin 3 mutant (Cav3/DGV) has been found to sequester cholesterol away from the endogenous lipid raft microdomains (Roy et al., 1999; Pol et al., 2001). Furthermore, expression of the Cav3/DGV mutant partially misdirected H-Ras out of membrane microdomains and functionally inhibited H-Ras, but not K-Ras, signaling responses (Roy et al., 1999). Since TC10 localizes to caveolar structures and shows a trafficking pattern indistinguishable from H-Ras, we determined whether the Cav3/DGV mutant would also effect the plasma membrane microdomain distribution of TC10. Similar to the endogenous TC10 protein, the expressed EGFP-TC10/WT fusion protein was colocalized with endogenous plasma membrane caveolin in the torus-shaped structures (Fig. 8 , a–c). Similarly, expression of Cav3/WT had no significant effect on the plasma membrane microdomain localization of either the coexpressed EGFP-TC10 protein or on endogenous caveolin (Fig. 8, d–f). In contrast, expression of Cav3/DGV resulted in a partial dispersion of caveolin concomitant with a marked reduction in the number of torus-shaped caveolin-positive structures (Fig. 8 g). More importantly, there was a near complete scattering of TC10 throughout the plasma membrane (Fig. 8, h and i). The greater effect of Cav3/DGV on TC10 compared with caveolin parallels the effect of Cav3/DGV on the plasma membrane microdomain distribution of caveolin and H-Ras reported previously for fibroblasts (Roy et al., 1999). In any case, the ability of Cav3/DGV to disrupt the plasma membrane organization of TC10 is consistent with the effect of β-CD and further supports the specific plasma membrane microdomain compartmentalization of TC10.


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)

Expression of a dominant- interfering caveolin 3 mutant disrupts the plasma membrane subdomain compartmentalization of TC10. Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of HA-TC10 plus 200 μg of the empty vector (a–c), wild-type caveolin 3 (Cav3/WT; d–f), or the dominant-interfering caveolin 3 mutant (Cav3/DGV; g–i). 36 h later, the cells were fixed, plasma membrane sheets were prepared and subjected to confocal fluorescent microscopy with a polyclonal caveolin 1 antibody (a, d, and g), and a monoclonal HA antibody (b, e, and h). The merged images are shown in panels c, f, and i. These are representative field of cells from two independent determinations. Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Expression of a dominant- interfering caveolin 3 mutant disrupts the plasma membrane subdomain compartmentalization of TC10. Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of HA-TC10 plus 200 μg of the empty vector (a–c), wild-type caveolin 3 (Cav3/WT; d–f), or the dominant-interfering caveolin 3 mutant (Cav3/DGV; g–i). 36 h later, the cells were fixed, plasma membrane sheets were prepared and subjected to confocal fluorescent microscopy with a polyclonal caveolin 1 antibody (a, d, and g), and a monoclonal HA antibody (b, e, and h). The merged images are shown in panels c, f, and i. These are representative field of cells from two independent determinations. Bar, 10 μM.
Mentions: A dominant-interfering caveolin 3 mutant (Cav3/DGV) has been found to sequester cholesterol away from the endogenous lipid raft microdomains (Roy et al., 1999; Pol et al., 2001). Furthermore, expression of the Cav3/DGV mutant partially misdirected H-Ras out of membrane microdomains and functionally inhibited H-Ras, but not K-Ras, signaling responses (Roy et al., 1999). Since TC10 localizes to caveolar structures and shows a trafficking pattern indistinguishable from H-Ras, we determined whether the Cav3/DGV mutant would also effect the plasma membrane microdomain distribution of TC10. Similar to the endogenous TC10 protein, the expressed EGFP-TC10/WT fusion protein was colocalized with endogenous plasma membrane caveolin in the torus-shaped structures (Fig. 8 , a–c). Similarly, expression of Cav3/WT had no significant effect on the plasma membrane microdomain localization of either the coexpressed EGFP-TC10 protein or on endogenous caveolin (Fig. 8, d–f). In contrast, expression of Cav3/DGV resulted in a partial dispersion of caveolin concomitant with a marked reduction in the number of torus-shaped caveolin-positive structures (Fig. 8 g). More importantly, there was a near complete scattering of TC10 throughout the plasma membrane (Fig. 8, h and i). The greater effect of Cav3/DGV on TC10 compared with caveolin parallels the effect of Cav3/DGV on the plasma membrane microdomain distribution of caveolin and H-Ras reported previously for fibroblasts (Roy et al., 1999). In any case, the ability of Cav3/DGV to disrupt the plasma membrane organization of TC10 is consistent with the effect of β-CD and further supports the specific plasma membrane microdomain compartmentalization of TC10.

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