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Vangl2 regulates E-cadherin in epithelial cells.

Nagaoka T, Inutsuka A, Begum K, Bin hafiz Km, Kishi M - Sci Rep (2014)

Bottom Line: Vangl2 enhances the internalization of E-cadherin when overexpressed.Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis.Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuroimaging, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan.

ABSTRACT
E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2(Lpt/+) mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions.

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Related in: MedlinePlus

Regulation of cell surface levels of E-cadherin by Vangl2.(a) WB analysis of the indicated proteins included in the total cell lysates or exposed to the cell surface. The indicated expression constructs were transfected into HEK293T cells, and the cell lysates were analysed. Biotinylated surface proteins were pulled down via avidin affinity. (b–d) Quantification of the surface levels of E-cadherin (b), TfR (c) and Vangl2 (d) treated with the indicated reagents and expression constructs. The bar graphs show the ratio of the protein levels precipitated using the avidin beads to those included in the total cell lysates. (e) Co-localization of the E-cadherin (green)–Vangl2 (red) vesicles with Rab5 (blue). Expression constructs of Vangl2 and E-cadherin were transfected into HEK293T cells. E-cadherin/Vangl2 double-positive and E-cadherin/Vangl2/Rab5 triple-positive dots are indicated by arrowheads and arrows, respectively. (f) IF analysis of colocalization of transfected E-cadherin with Vangl2. The presented images were captured without EGTA treatment (0 min.), or 5 and 30 minutes after the EGTA treatment. (g) Line graph showing the transition of the ratio of the E-cadherin vesicles colocalized with Vangl2 IF. The images captured without EGTA treatment (0 min.), or 5, 15, 30, and 60 minutes after the EGTA treatment were analysed. WB images were captured using the same experimental condition for each series of experiments and for each kind of antibodies. The full-length images of the WB analyses presented in Figure 3 are included in Supplementary Figure S5. Higher magnification of the delimited region is shown in the respective lower panels. Data are presented as mean + SEM (b, c, d) or ± SEM (g). Significant differences (p < 0.05) versus control groups calculated using Student's t test are marked with *. a.u.: arbitrary unit. Scale bars: 20 μm and 5 μm for upper and lower panels, respectively.
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f3: Regulation of cell surface levels of E-cadherin by Vangl2.(a) WB analysis of the indicated proteins included in the total cell lysates or exposed to the cell surface. The indicated expression constructs were transfected into HEK293T cells, and the cell lysates were analysed. Biotinylated surface proteins were pulled down via avidin affinity. (b–d) Quantification of the surface levels of E-cadherin (b), TfR (c) and Vangl2 (d) treated with the indicated reagents and expression constructs. The bar graphs show the ratio of the protein levels precipitated using the avidin beads to those included in the total cell lysates. (e) Co-localization of the E-cadherin (green)–Vangl2 (red) vesicles with Rab5 (blue). Expression constructs of Vangl2 and E-cadherin were transfected into HEK293T cells. E-cadherin/Vangl2 double-positive and E-cadherin/Vangl2/Rab5 triple-positive dots are indicated by arrowheads and arrows, respectively. (f) IF analysis of colocalization of transfected E-cadherin with Vangl2. The presented images were captured without EGTA treatment (0 min.), or 5 and 30 minutes after the EGTA treatment. (g) Line graph showing the transition of the ratio of the E-cadherin vesicles colocalized with Vangl2 IF. The images captured without EGTA treatment (0 min.), or 5, 15, 30, and 60 minutes after the EGTA treatment were analysed. WB images were captured using the same experimental condition for each series of experiments and for each kind of antibodies. The full-length images of the WB analyses presented in Figure 3 are included in Supplementary Figure S5. Higher magnification of the delimited region is shown in the respective lower panels. Data are presented as mean + SEM (b, c, d) or ± SEM (g). Significant differences (p < 0.05) versus control groups calculated using Student's t test are marked with *. a.u.: arbitrary unit. Scale bars: 20 μm and 5 μm for upper and lower panels, respectively.

Mentions: Cell surface proteins expressed on HEK293T cells transfected with the indicated expression vectors (Figure 3a) were biotinylated and precipitated using avidin-coated beads29. The protein samples included in the precipitates as well as in the total cell lysates were analysed by WB. Treatment of the cells with 4 mM EGTA, which chelates the extracellular calcium ions, induced a reduction in cell surface levels of E-cadherin30. Similarly, cotransfection of Vangl2 decreased the E-cadherin levels exposed to the cell surface (Figure 3a and b). In these experiments, the EGTA treatment, but not Vangl2 transfection, induced the internalization of transferrin receptors (TfR; Figure 3a and c), indicating the functional specificity of Vangl2.


Vangl2 regulates E-cadherin in epithelial cells.

Nagaoka T, Inutsuka A, Begum K, Bin hafiz Km, Kishi M - Sci Rep (2014)

Regulation of cell surface levels of E-cadherin by Vangl2.(a) WB analysis of the indicated proteins included in the total cell lysates or exposed to the cell surface. The indicated expression constructs were transfected into HEK293T cells, and the cell lysates were analysed. Biotinylated surface proteins were pulled down via avidin affinity. (b–d) Quantification of the surface levels of E-cadherin (b), TfR (c) and Vangl2 (d) treated with the indicated reagents and expression constructs. The bar graphs show the ratio of the protein levels precipitated using the avidin beads to those included in the total cell lysates. (e) Co-localization of the E-cadherin (green)–Vangl2 (red) vesicles with Rab5 (blue). Expression constructs of Vangl2 and E-cadherin were transfected into HEK293T cells. E-cadherin/Vangl2 double-positive and E-cadherin/Vangl2/Rab5 triple-positive dots are indicated by arrowheads and arrows, respectively. (f) IF analysis of colocalization of transfected E-cadherin with Vangl2. The presented images were captured without EGTA treatment (0 min.), or 5 and 30 minutes after the EGTA treatment. (g) Line graph showing the transition of the ratio of the E-cadherin vesicles colocalized with Vangl2 IF. The images captured without EGTA treatment (0 min.), or 5, 15, 30, and 60 minutes after the EGTA treatment were analysed. WB images were captured using the same experimental condition for each series of experiments and for each kind of antibodies. The full-length images of the WB analyses presented in Figure 3 are included in Supplementary Figure S5. Higher magnification of the delimited region is shown in the respective lower panels. Data are presented as mean + SEM (b, c, d) or ± SEM (g). Significant differences (p < 0.05) versus control groups calculated using Student's t test are marked with *. a.u.: arbitrary unit. Scale bars: 20 μm and 5 μm for upper and lower panels, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f3: Regulation of cell surface levels of E-cadherin by Vangl2.(a) WB analysis of the indicated proteins included in the total cell lysates or exposed to the cell surface. The indicated expression constructs were transfected into HEK293T cells, and the cell lysates were analysed. Biotinylated surface proteins were pulled down via avidin affinity. (b–d) Quantification of the surface levels of E-cadherin (b), TfR (c) and Vangl2 (d) treated with the indicated reagents and expression constructs. The bar graphs show the ratio of the protein levels precipitated using the avidin beads to those included in the total cell lysates. (e) Co-localization of the E-cadherin (green)–Vangl2 (red) vesicles with Rab5 (blue). Expression constructs of Vangl2 and E-cadherin were transfected into HEK293T cells. E-cadherin/Vangl2 double-positive and E-cadherin/Vangl2/Rab5 triple-positive dots are indicated by arrowheads and arrows, respectively. (f) IF analysis of colocalization of transfected E-cadherin with Vangl2. The presented images were captured without EGTA treatment (0 min.), or 5 and 30 minutes after the EGTA treatment. (g) Line graph showing the transition of the ratio of the E-cadherin vesicles colocalized with Vangl2 IF. The images captured without EGTA treatment (0 min.), or 5, 15, 30, and 60 minutes after the EGTA treatment were analysed. WB images were captured using the same experimental condition for each series of experiments and for each kind of antibodies. The full-length images of the WB analyses presented in Figure 3 are included in Supplementary Figure S5. Higher magnification of the delimited region is shown in the respective lower panels. Data are presented as mean + SEM (b, c, d) or ± SEM (g). Significant differences (p < 0.05) versus control groups calculated using Student's t test are marked with *. a.u.: arbitrary unit. Scale bars: 20 μm and 5 μm for upper and lower panels, respectively.
Mentions: Cell surface proteins expressed on HEK293T cells transfected with the indicated expression vectors (Figure 3a) were biotinylated and precipitated using avidin-coated beads29. The protein samples included in the precipitates as well as in the total cell lysates were analysed by WB. Treatment of the cells with 4 mM EGTA, which chelates the extracellular calcium ions, induced a reduction in cell surface levels of E-cadherin30. Similarly, cotransfection of Vangl2 decreased the E-cadherin levels exposed to the cell surface (Figure 3a and b). In these experiments, the EGTA treatment, but not Vangl2 transfection, induced the internalization of transferrin receptors (TfR; Figure 3a and c), indicating the functional specificity of Vangl2.

Bottom Line: Vangl2 enhances the internalization of E-cadherin when overexpressed.Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis.Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuroimaging, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan.

ABSTRACT
E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2(Lpt/+) mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions.

Show MeSH
Related in: MedlinePlus