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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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Individual plasma membrane caveolae are clustered into higher-order structures in adipocytes. (A) Plasma membrane sheets were prepared from differentiated 3T3L1 adipocytes, incubated with a monoclonal caveolin 1 antibody, and subjected to confocal fluorescent microscopy at low magnification (a) or at high magnification (b) as indicated by the inset bar. (B) Plasma membrane sheets were prepared, fixed, and incubated with a monoclonal caveolin 1 antibody and a 10-nm gold-conjugated rabbit anti–mouse antibody as described in Materials and methods. The samples where then sectioned and visualized at low magnification (a) or high magnification (b) as indicated by the inset bar. Arrowheads indicate caveolae clustered into ring-shaped structures and arrows indicate individual caveolae.
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fig5: Individual plasma membrane caveolae are clustered into higher-order structures in adipocytes. (A) Plasma membrane sheets were prepared from differentiated 3T3L1 adipocytes, incubated with a monoclonal caveolin 1 antibody, and subjected to confocal fluorescent microscopy at low magnification (a) or at high magnification (b) as indicated by the inset bar. (B) Plasma membrane sheets were prepared, fixed, and incubated with a monoclonal caveolin 1 antibody and a 10-nm gold-conjugated rabbit anti–mouse antibody as described in Materials and methods. The samples where then sectioned and visualized at low magnification (a) or high magnification (b) as indicated by the inset bar. Arrowheads indicate caveolae clustered into ring-shaped structures and arrows indicate individual caveolae.

Mentions: The organization of the large, caveolin-positive structures was examined at higher magnification (Fig. 5 A, a and b). These torus-shaped configurations were found to range in size from 0.5 to 1.5 μm. The assembly of individual caveolae into these ringed structures was confirmed by caveolin-immunogold electron microscopy (Fig. 5 B, a and b). At low magnification, both individual and multiple aggregates of immunopositive caveolin–labeled structures were present in the adipocyte plasma membrane (Fig. 5 B, a). At higher magnification, these aggregates of caveolin-positive structures were clearly visualized as clusters of individual immunopositive caveolae organized into ring-like shapes. Furthermore, the size distribution of these aggregates were in excellent agreement with the dimensions determined by confocal fluorescent microscopy.


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)

Individual plasma membrane caveolae are clustered into higher-order structures in adipocytes. (A) Plasma membrane sheets were prepared from differentiated 3T3L1 adipocytes, incubated with a monoclonal caveolin 1 antibody, and subjected to confocal fluorescent microscopy at low magnification (a) or at high magnification (b) as indicated by the inset bar. (B) Plasma membrane sheets were prepared, fixed, and incubated with a monoclonal caveolin 1 antibody and a 10-nm gold-conjugated rabbit anti–mouse antibody as described in Materials and methods. The samples where then sectioned and visualized at low magnification (a) or high magnification (b) as indicated by the inset bar. Arrowheads indicate caveolae clustered into ring-shaped structures and arrows indicate individual caveolae.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Individual plasma membrane caveolae are clustered into higher-order structures in adipocytes. (A) Plasma membrane sheets were prepared from differentiated 3T3L1 adipocytes, incubated with a monoclonal caveolin 1 antibody, and subjected to confocal fluorescent microscopy at low magnification (a) or at high magnification (b) as indicated by the inset bar. (B) Plasma membrane sheets were prepared, fixed, and incubated with a monoclonal caveolin 1 antibody and a 10-nm gold-conjugated rabbit anti–mouse antibody as described in Materials and methods. The samples where then sectioned and visualized at low magnification (a) or high magnification (b) as indicated by the inset bar. Arrowheads indicate caveolae clustered into ring-shaped structures and arrows indicate individual caveolae.
Mentions: The organization of the large, caveolin-positive structures was examined at higher magnification (Fig. 5 A, a and b). These torus-shaped configurations were found to range in size from 0.5 to 1.5 μm. The assembly of individual caveolae into these ringed structures was confirmed by caveolin-immunogold electron microscopy (Fig. 5 B, a and b). At low magnification, both individual and multiple aggregates of immunopositive caveolin–labeled structures were present in the adipocyte plasma membrane (Fig. 5 B, a). At higher magnification, these aggregates of caveolin-positive structures were clearly visualized as clusters of individual immunopositive caveolae organized into ring-like shapes. Furthermore, the size distribution of these aggregates were in excellent agreement with the dimensions determined by confocal fluorescent microscopy.

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