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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 inhibits insulin-stimulated GLUT4 translocation. (A) Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of GLUT4-EGFP plus 200 μg of the empty vector (a and b), Myc epitope–tagged wild-type caveolin 3 (Cav3/WT; c and d), or Myc epitope tagged dominant-interfering caveolin 3 mutant (Cav3/DGV; e and f). 36 h later, the cells were then incubated for 30 min in the absence (a, c, and e) or presence (b, d, and f) of 100 nM insulin. The cells were then fixed and the subcellular localization of GLUT4-EGFP was determined by confocal fluorescent microscopy. These are a representative field of cells from four independent determinations. (B) The subcellular distribution of expressed Cav3/WT (a and b) or the Cav3/DGV mutant (c and d) was determined by confocal fluorescent microscopy. These are a representative field of cells from four independent determinations. Bar, 10 μM.
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fig9: Expression of a dominant-interfering caveolin 3 mutant inhibits insulin-stimulated GLUT4 translocation. (A) Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of GLUT4-EGFP plus 200 μg of the empty vector (a and b), Myc epitope–tagged wild-type caveolin 3 (Cav3/WT; c and d), or Myc epitope tagged dominant-interfering caveolin 3 mutant (Cav3/DGV; e and f). 36 h later, the cells were then incubated for 30 min in the absence (a, c, and e) or presence (b, d, and f) of 100 nM insulin. The cells were then fixed and the subcellular localization of GLUT4-EGFP was determined by confocal fluorescent microscopy. These are a representative field of cells from four independent determinations. (B) The subcellular distribution of expressed Cav3/WT (a and b) or the Cav3/DGV mutant (c and d) was determined by confocal fluorescent microscopy. These are a representative field of cells from four independent determinations. Bar, 10 μM.

Mentions: Based on these results, we next examined the effect of Cav3/DGV on insulin-stimulated GLUT4 translocation (Fig. 9) . Expression of the wild-type caveolin 3 protein (Cav3/WT) had no effect on GLUT4-EGFP translocation, which was essentially identical to the control transfected cells (Fig. 9 A, a–d). In contrast, the Cav3/DGV mutant inhibited insulin-stimulated GLUT4-EGFP translocation (Fig. 9 A, e and f). The expression patterns of the Cav3/WT and Cav3/DGV constructs were also determined (Fig. 9 B). As reported previously, Cav3/WT was predominantly localized to the plasma membrane, with a small amount also present in the perinuclear region (Fig. 9 B, a and b). In contrast, Cav3/DGV showed strong labeling in the cytoplasm, where it formed distinct vesicle structures (Fig. 9 B, c and d) similar to the cholesterol-enriched vesicles previously reported in fibroblasts (Roy et al., 1999; Pol et al., 2001). Insulin stimulation had no significant effect on the localization of either Cav3/WT or Cav3/DGV (Fig. 9 B, a–d).


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 inhibits insulin-stimulated GLUT4 translocation. (A) Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of GLUT4-EGFP plus 200 μg of the empty vector (a and b), Myc epitope–tagged wild-type caveolin 3 (Cav3/WT; c and d), or Myc epitope tagged dominant-interfering caveolin 3 mutant (Cav3/DGV; e and f). 36 h later, the cells were then incubated for 30 min in the absence (a, c, and e) or presence (b, d, and f) of 100 nM insulin. The cells were then fixed and the subcellular localization of GLUT4-EGFP was determined by confocal fluorescent microscopy. These are a representative field of cells from four independent determinations. (B) The subcellular distribution of expressed Cav3/WT (a and b) or the Cav3/DGV mutant (c and d) was determined by confocal fluorescent microscopy. These are a representative field of cells from four independent determinations. Bar, 10 μM.
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Related In: Results  -  Collection

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

fig9: Expression of a dominant-interfering caveolin 3 mutant inhibits insulin-stimulated GLUT4 translocation. (A) Differentiated 3T3L1 adipocytes were coelectroporated with 50 μg of GLUT4-EGFP plus 200 μg of the empty vector (a and b), Myc epitope–tagged wild-type caveolin 3 (Cav3/WT; c and d), or Myc epitope tagged dominant-interfering caveolin 3 mutant (Cav3/DGV; e and f). 36 h later, the cells were then incubated for 30 min in the absence (a, c, and e) or presence (b, d, and f) of 100 nM insulin. The cells were then fixed and the subcellular localization of GLUT4-EGFP was determined by confocal fluorescent microscopy. These are a representative field of cells from four independent determinations. (B) The subcellular distribution of expressed Cav3/WT (a and b) or the Cav3/DGV mutant (c and d) was determined by confocal fluorescent microscopy. These are a representative field of cells from four independent determinations. Bar, 10 μM.
Mentions: Based on these results, we next examined the effect of Cav3/DGV on insulin-stimulated GLUT4 translocation (Fig. 9) . Expression of the wild-type caveolin 3 protein (Cav3/WT) had no effect on GLUT4-EGFP translocation, which was essentially identical to the control transfected cells (Fig. 9 A, a–d). In contrast, the Cav3/DGV mutant inhibited insulin-stimulated GLUT4-EGFP translocation (Fig. 9 A, e and f). The expression patterns of the Cav3/WT and Cav3/DGV constructs were also determined (Fig. 9 B). As reported previously, Cav3/WT was predominantly localized to the plasma membrane, with a small amount also present in the perinuclear region (Fig. 9 B, a and b). In contrast, Cav3/DGV showed strong labeling in the cytoplasm, where it formed distinct vesicle structures (Fig. 9 B, c and d) similar to the cholesterol-enriched vesicles previously reported in fibroblasts (Roy et al., 1999; Pol et al., 2001). Insulin stimulation had no significant effect on the localization of either Cav3/WT or Cav3/DGV (Fig. 9 B, a–d).

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