Vangl2 regulates E-cadherin in epithelial cells

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.

Figure 1. Association of E-cadherin with Vangl2.

(a, b, d, f) HEK293T cells were transfected with the indicated expression constructs, and the cell lysates as well as IPs were analysed by WB using the indicated Abs. IP was performed using (a) α-E-cadherin, (b) α-GFP, or (d, f) α-FLAG Abs. The protein band of E-cadherin with higher molecular weight indicates the immature form with propeptide. (c, e) Schematic representations of the mutated constructs of Vangl2 (c) and E-cadherin (e) used in the co-IP experiments shown in (d) and (f), respectively. (g) In vitro binding between recombinant E-cadherin and Vangl2. GST-Ecad incubated with either MBP-NT, nonfused MBP or MBP-CT, was immunoprecipitated using α-MBP Abs and protein G sepharose. The Inputs (0.5%) as well as IPs (approximately 30%) were separated with SDS-PAGE and analysed by WB using the indicated Abs. Significant degradation of MBP-CT occured during the purification for some unknown reason. The right lane indicates the no Ab control. Note the specific precipitation of GST-Ecad by MBP-CT. 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 1 are included in Supplementary Figure S3. IP (immunoprecipitates), IB (immunoblot), WT (wild-type Vangl2), ΔNT (Vangl2 lacking N-terminal intracellular domain), ΔCT (Vangl2 lacking the C-terminal intracellular domain), ΔPkBD (Vangl2 lacking the Prickle-binding domain), ΔETSV (Vangl2 lacking the PDZ-binding motif at the C-terminal end), S/A (E-cadherin with all serine residues within the β-catenin-binding region substituted by alanine), ΔBD (E-cadherin lacking the catenin-binding region).

Figure 2. Inhibition of complex formation between E-cadherin and Vangl2 by either β-catenin or Prickle2.

(a, c) HEK293T cells were transfected with the indicated expression plasmids, and the cell lysates as well as IPs were analysed by WB. IP was performed using α-FLAG Abs. Total DNA amount was adjusted to 3.0 μg for each well using control GFP expression vector. (b, d) Line graphs showing the ratio of the E-cadherin levels included in the IPs to those in the total cell lysates. The ratios were calculated according to the immunosignals quantified using ImageJ. The line graphs (b) and (d) were prepared according to the WB analyses (a) and (c), respectively. For quantification of E-cadherin levels, only signal strength of the protein band corresponding to the mature form (lower molecular weight) was used. Note the dose-dependent reduction of the precipitated levels of E-cadherin by increased DNA amount of β-catenin (a, b) or Prickle2 (c, d). 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 2 are included in Supplementary Figure S4. Data are presented as mean ± SEM. Significant differences (p < 0.05) versus a one stage earlier group calculated using Student's t test are marked with *. a.u.: arbitrary unit.

Figure 3. 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.

Figure 4. E-cadherin–Vangl2 association in vivo.

(a) E19 rat kidney extracts were subjected to IP using normal goat IgG or α-Vangl2 Abs, and then to WB analysis. Note the specific precipitation of E-cadherin by α-Vangl2. β-catenin or Plakoglobin were not detected in the IPs even when the blots were overexposed. (b, c) IF localization of Vangl2 (red) and E-cadherin (green) shown together with phalloidin staining (blue) on a cryosection of a P0 mouse kidney. E-cadherin is robustly expressed in the epithelial cells of the urinary tubule, which is horizontally cut on this section. Higher magnification of the delimited region (b) is shown in (c). Apical and basal limits of the epithelia are shown using white broken lines in the images of phalloidin staining (blue) and are displayed at the upper and lower side of the images of (c), respectively. The white characters in the phalloidin image of (b) denote the following: M, mesenchyme; E, epithelia; and L, lumen. (d, e) Plot profile analysis of the IF intensity along the delimited regions (c). The origin of the plot is shown as “0”. The signal intensities are plotted along one of the lateral borders between the epithelial cells (d; delimited with white dots) or transverse to these borders (e; delimited with yellow dots). The X-axis indicates the distance from the origin of the plot. The Y-axis delineates fluorescent signal intensity. For each fluorophore and each plot, the most intense signal peak was set as one arbitrary unit of the Y-axis. 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 4 are included in Supplementary Figure S6. Scale bars: 20 μm and 5 μm for (b) and (c), respectively.

Figure 5. Increased cell surface expression of E-cadherin on the epithelial cells derived from the Vangl2^Lpt/+ mutant kidneys.

(a) WB analysis of both biotinylated cell surface proteins and proteins included in the total cell lysates of the cultured kidney cells derived from the wild-type or Vangl2^Lpt/+ mutant mice. (b) Bar graphs showing the cell surface ratios of E-cadherin levels, which were quantified according to the immunosignal intensities shown in (a). (c–e) Quantitative analysis of Vangl2 levels in the kidney cells derived from wild-type or Vangl2^Lpt/+ mutant mice. The WB signal levels of total Vangl2 (c), those exposed to the cell surface (d) and their ratios (e) were compared between these two genotypes. (f, h, j) Image analysis of cultured kidney epithelial cells derived from wild-type and Vangl2^Lpt/+ mutant mice using the α-ECD and α-ICD Abs. α-ECD Abs were applied before (f) or after (j) membrane permeabilization. In (h), wild-type cells were stained either with or without permeabilization for both Abs. The images were captured using the same exposure time and gain for each fluorophore and for each series of experiments. (g, i, k) Bar graphs showing the relative average ratios of the signal intensities of α-ECD and α-ICD. The geometric mean of ratio of the wild-type (g, k) and that of the permeabilized culture was set as one arbitrary unit for each experimental condition (f), (j) and (h). 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 5 are included in Supplementary Figure S7. Data are presented as geometric mean ± 95% CI (b, e, g, i, k) or mean + SEM (c, d). Significant differences (p < 0.05) versus control groups calculated using Mann-Whitney's U test (b, e, g, i, k) or Student's t test (c, d) are marked with *. a.u.: arbitrary unit. Scale bars: 50 μm.