Allosteric Regulation of E-Cadherin Adhesion

Abstract

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation.

Keywords: allosteric regulation; cadherin-1 (CDH1) (epithelial cadherin) (E-cadherin); catenin; cell adhesion; kinetics.

FIGURE 1.

Activating antibody 19A11 enhances the binding kinetics Of E-cadherin on Colo 205 cells. A, micropipette configuration used in adhesion frequency measurements. A test cell (e.g. Colo 205) expressing full-length cadherin is aspirated into the left micropipette, and an RBC surface-modified with E-cadherin-Fc is aspirated into the right micropipette. The cells are repetitively brought into contact for a defined period (and contact area) and retracted with piezoelectric manipulators. Adhesion events are quantified from visible RBC deformations and recoil upon bond failure. B, schematic of cadherin configurations on the test cell and on the RBC. C, binding probability versus contact time between RBCs displaying oriented canine E-Cad-Fc (23 or 28 cadherins/μm²) and Colo 205 cells (40 cadherins/μm²) with (black squares) or without (gray squares) treatment with whole 19A11 monoclonal antibody. The initial plateau, P1, lag, and steady state plateau P2 are shown for the kinetic time course measured with 19A11 antibody-treated Colo 205 cells. Controls (white squares) were done with RBCs without immobilized E-Cad-Fc. Each data point represents replicate measurements of 50 adhesion tests performed with at least three different cell pairs. The solid and dashed lines are the non-linear least squares fits of data for the first kinetic step to Equation 1. The best-fit parameters are summarized in Table 1. D, binding probability versus the contact time between RBCs modified with oriented E-Cad-Fc (28 and 12 cadherins/μm²) and Colo 205 cells (40 cadherins/μm²) treated with 19A11 whole mAb (black squares) and Fab fragments (gray squares). The solid and dashed lines are the non-linear least squares fits of data for the first kinetic step to Equation 1. The best-fit parameters are summarized in Table 1. E, binding probability versus the contact time between RBCs modified with oriented E-Cad-Fc (12 cadherins/μm²) and Colo 205 cells (40 cadherins/μm²) treated with Fab fragments of either the activating 19A11 (black squares) or neutral 76D5 antibody (gray squares). Controls were done with RBCs without bound cadherin extracellular domains (white squares). The solid and dashed lines are the non-linear least squares fits of data for the first kinetic step to Equation 1. The best-fit parameters are summarized in Table 1. Error bars, S.E.

FIGURE 2.

Activating 19A11 antibody does not alter homophilic binding kinetics of adhesion-competent E-cadherin. A, binding probability versus contact time between RBCs modified with adhesion-competent E-Cad-Fc (19 cadherins/μm²) treated with Fab fragments of either 19A11 (black squares) or neutral 76D5 (gray squares). Controls were with RBCs without immobilized E-Cad-Fc (white squares) or with RBCs with immobilized E-Cad-Fc without antibody (white circle). The solid, dashed, and dotted lines are nonlinear least squares fits of data corresponding to the first kinetic step to Equation 1, with best fit parameters summarized in Table 1. B, binding probability versus the contact time between RBCs modified with canine E-Cad-Fc (35 cadherins/μm²) and adhesion-competent E-cadherin on MCF7 cells (10 cadherins/μm²) with (black squares) or without (gray squares) 19A11 Fab. White squares, measurements with inhibitory, anti-E-cadherin DECMA-1 antibody (white circles). The lines indicate nonlinear least squares fits of the initial trans-binding step to Equation 1, with 19A11 Fab (solid line), without antibody (light dashed line), or with DECMA-1 inhibitory antibody (dark broken line). The best-fit parameters are summarized in Table 1. Error bars, S.E.

FIGURE 3.

Inhibiting p120 phosphorylation increases E-cadherin affinity. A, Colo 205 cells after a 2-h incubation with LiCl, relative to NaCl control. B, Western blot using the phosphospecific anti-p120^ctn antibody targeting pT310 after Colo 205 treatment with LiCl, relative to NaCl control. Tubulin was used as a loading control. E-cadherin surface expression was unaffected. C, binding probability versus contact time between RBCs modified with immobilized, oriented E-Cad-Fc (23 or 14 cadherins/μm² for staurosporine and LiCl, respectively) and Colo 205 cells (40 cadherins/μm²) treated with staurosporine (black squares) or LiCl (gray diamonds), relative to NaCl-treated control cells (white squares). Controls (white circles) used RBCs without immobilized E-Cad-Fc. The lines through the data are weighted, nonlinear least squares fits to Equation 1, with best-fit parameters summarized in Table 1. Error bars, S.E.

FIGURE 4.

p120^ctn phosphorylation status regulates E-cadherin binding. A, immunostained E-cadherin and p120 catenin in adherent Colo 205 mouse p120 catenin transfectants. Colo 205 cells were transfected with the neomycin vector (Neo Vector), with a vector encoding wild type mouse p120^ctn (WT mp120ctn), or with a vector encoding the mouse p120^ctn phosphorylation mutant (6S,T→A mp120ctn). The top row shows cells stained with anti-human E-cadherin antibody (green). The bottom row shows cells immunostained with anti-mouse p120^ctn. B, binding probability versus the contact time between RBCs modified with immobilized, oriented E-Cad-Fc (12 or 25 cadherins/μm²) and Colo 205 cells expressing WT mouse p120^ctn (gray squares; 40 cadherins/μm²), expressing the mouse p120^ctn 6S,T→A phosphomutant (black squares; 51 cadherins/μm²), or infected with the neomycin vector (white squares; 44 cadherins/μm²). Controls (white circles) used RBCs without immobilized E-Cad-Fc and untreated Colo 205 cells. In B, each data point represents replicate measurements of 50 adhesion tests performed with at least three different cell pairs. The lines through the data are weighted, nonlinear least squares fits of kinetic data to Equation 1, with best-fit parameters summarized in Table 1. Error bars, S.E.

FIGURE 5.

A proposed cis-interface mutant does not affect E-cadherin affinity. Binding probability versus contact time between RBCs surface-modified with either oriented mouse E-Cad-His₆ WT (21 cadherins/μm²; filled squares) or mouse E-Cad-His₆ L175D (22 cadherins/μm²; white squares) on both sides. The solid and dashed lines are the non-linear least squares fits of data for the initial, trans-binding step. The best-fit parameters summarized in Table 1. Error bars, S.E.

FIGURE 6.

Superresolution images of cadherin clusters on Colo 205 cells. A, structured illumination microscopy of Colo 205 cells. Superresolution SIM of live Colo 205 derivatives was performed at 37 °C, after directly labeling live cells with the Alexa-568-labeled Fab fragment of 76D5, which binds the human E-cadherin extracellular domain: i, untransfected Colo 205; ii, Colo 205 expressing mouse p120 (mp120) WT; iii, Colo 205 expressing mouse p120 6S,T→A. B, STORM of E-cadherin clusters on Colo 205 cells immunostained with control anti-rat Alexa 647 secondary antibody (i) and with anti-E-cadherin primary DECMA-1 and secondary anti-rat antibodies (ii–iv). E-cadherin clusters are concentrated at the cell periphery. Control data show negligible clusters. v, an expanded section of the cell surface in iv.

FIGURE 7.

Analysis of E-cadherin clusters on Colo 205 cells. Comparison of E-cadherin clusters on WT Colo 205 cells, Colo 205 expressing WT mouse p120, and Colo 205 cells transfected with mouse p120 6S,T→A. A, cluster diameter; B, number of points per cluster; C, total number of clusters per cell; D, percent clustering ((points per cluster × number of clusters)/total points). Apart from the control, there are no significant differences between the features of clusters on the three Colo 205 cell lines. E, histograms of the number of E-cadherin clusters on Colo 205 cells versus the cluster diameter (nm). n > 6 cells were analyzed each for untransfected Colo 205 cells and Colo 205 cells transfected with WT mouse p120^ctn or mouse p120^ctn 6S,T→A. The gray curves are Gaussian fits of the cluster distributions, obtained with each cell line. Error bars, S.E. n.s., not significant.

FIGURE 8.

Proposed model for the allosteric activation Of E-cadherin. A, the low E-cadherin affinity on untreated Colo 205 results in little bond formation and low cell-cell binding probabilities. B, activating antibody treatment or the expression of the phosphorylation mutant mouse p120^ctn 6S,T→A decreases p120^ctn phosphorylation and activates E-cadherin-mediated cell aggregation. C, the substantially higher E-cadherin following activating antibody treatment or expression of the p120^ctn phosphomutant 6S,T→A increases the frequency of homophilic E-cadherin bonds and the measured cell-cell binding probabilities.