The membrane-proximal region of the E-cadherin cytoplasmic domain prevents dimerization and negatively regulates adhesion activity

Abstract

Cadherins are transmembrane glycoproteins involved in Ca2+-dependent cell-cell adhesion. Deletion of the COOH-terminal residues of the E-cadherin cytoplasmic domain has been shown to abolish its cell adhesive activity, which has been ascribed to the failure of the deletion mutants to associate with catenins. Based on our present results, this concept needs revision. As was reported previously, leukemia cells (K562) expressing E-cadherin with COOH-terminal deletion of 37 or 71 amino acid residues showed almost no aggregation. Cells expressing E-cadherin with further deletion of 144 or 151 amino acid residues, which eliminates the membrane-proximal region of the cytoplasmic domain, showed E-cadherin-dependent aggregation. Thus, deletion of the membrane-proximal region results in activation of the nonfunctional E-cadherin polypeptides. However, these cells did not show compaction. Chemical cross-linking revealed that the activated E-cadherin polypeptides can be cross-linked to a dimer on the surface of cells, whereas the inactive polypeptides, as well as the wild-type E-cadherin polypeptide containing the membrane-proximal region, can not. Therefore, the membrane-proximal region participates in regulation of the adhesive activity by preventing lateral dimerization of the extracellular domain.

Figure 1

Schematic representation of the transmembrane and cytoplasmic domains of E-cadherin, and the mutant polypeptides with deletions of the 15–amino acid cytoplasmic domain. EΔC37 and EΔC71 are mutant E-cadherin polypeptides with COOH-terminal deletions of 37 and 71 amino acid residues, respectively. EC43, EC18, EC7, and EC0 are mutant E-cadherin polypeptides with COOH-terminal deletions of 108, 133, 144, and 151 amino acid residues, respectively. Thus, EC43, EC18, and EC7 retain 43, 18, and 7 NH₂-terminal residues of the cytoplasmic domain whereas EC0 completely lacks the cytoplasmic domain.

Figure 2

Immunoblot detection of E-cadherin polypeptides. K562 cells expressing the wild-type E-cadherin (EK cells) or the mutant polypeptides with deletions of the cytoplasmic domain as shown in Fig. 1 were lysed in SDS sample buffer, and then subjected to immunoblot analysis with DECMA-1, an E-cadherin mAb.

Figure 3

Aggregation of K562 cells expressing the mutant E-cadherin polypeptides. K562 cells expressing the wild-type E-cadherin (EK) or the mutant polypeptides with deletions of the cytoplasmic domain shown in Fig. 1, or K562 cells transfected with the control vector (nK) were allowed to aggregate for 30 min in the absence (filled bars) or presence (open bars) of DECMA-1. Error bars represent SD.

Figure 4

EK cells and EC0K cells show similar aggregation properties. (a) Aggregation of EK cells (open bars) and EC0K cells (shaded bars) under different shear forces generated by rotation. Cells were allowed to aggregate at 37°C for 30 min. (b) Aggregation kinetics of EK cells and EC0K cells. Cells were allowed to aggregate for the indicated times at 37°C (circles) or 4°C (squares) using a rotational force of 70 rpm. Error bars represent SD.

Figure 4

EK cells and EC0K cells show similar aggregation properties. (a) Aggregation of EK cells (open bars) and EC0K cells (shaded bars) under different shear forces generated by rotation. Cells were allowed to aggregate at 37°C for 30 min. (b) Aggregation kinetics of EK cells and EC0K cells. Cells were allowed to aggregate for the indicated times at 37°C (circles) or 4°C (squares) using a rotational force of 70 rpm. Error bars represent SD.

Figure 5

EK cells in aggregates show compaction but EC0K cells in aggregates remain uncompacted. Phase-contrast micrographs (a and b) of small aggregates formed by EK cells (a) and EC0K cells (b). Cells were photographed without fixation. Immunofluorescence localization of E-cadherin polypeptides (c and d). Cells were stained with DECMA-1 after fixation. Bar, 50 μm.

Figure 6

Chemical cross-linking of E-cadherin polypeptides on the surface of cells. K562 cells expressing either the wild-type (EK), a nonfunctional mutant (EΔC71K), or an active tail-less E-cadherin polypeptide (EC0K) were incubated with the indicated concentrations of DTSSP, and then subjected to immunoblot analysis with DECMA-1. The arrow indicates a cross-linked product of 170 kD found in EC0K cells.

Figure 7

Two-dimensional (nonreducing/reducing) SDS-PAGE analysis of the 170-kD cross-linked product. EC0K cells labeled with [³⁵S]methionine were cross-linked with 50 μg/ml of DTSSP and then lysed with Triton X-100/NP-40. The lysates were subjected to immunoprecipitation with E-cadherin antibodies, and the immunoprecipitates were analyzed by SDS-PAGE on a first-dimension (nonreducing) gel and fluorography (a). Part of the cross-linked product in the first-dimension gel was further analyzed on a second-dimension (reducing) gel (b). The horizontal arrow indicates the direction of electrophoresis in the first-dimension gel and the vertical arrow indicates that in the second-dimension gel. The positions of EC0 monomers and dimers, (EC0)₂, are indicated at the top and on the right, respectively.

Figure 8

p120 associates with the truncated E-cadherin retaining the membrane-proximal region but not with the completely tail-less E-cadherin. (a) Lack of coprecipitation of [³⁵S]methionine-labeled polypeptides with mutant E-cadherin polypeptides. Cells labeled overnight with [³⁵S]methionine were lysed and then subjected to immunoprecipitation with E-cadherin antibodies. The immunoprecipitates were analyzed by SDS-PAGE and fluorography. K562 cells expressing E-cadherin (EK) were used as a positive control and K562 cells transfected with the control vector (nK) as a negative control. The nature of a faint 110-kD band present in the EΔC71 immunoprecipitate is unknown at present. Although it comigrates with an isoform of p120, it seems not to be the isoform, because it is not present in the E-cadherin immunoprecipitate, in which a larger amount of the isoforms was coprecipitated (see Fig. 8 b). (b) Immunoblot detection of p120 coprecipitated with E-cadherin polypeptides. Cells were lysed as described under Materials and Methods, and then E-cadherin was collected using E-cadherin antibodies. After SDS-PAGE and transfer to nitrocellulose membranes, the proteins were stained with anti-p120 antibodies. The band corresponding to ∼70 kD appears to be a degradation product of p120. K562 cells seem to have a high proteolytic activity compared with other cell types. Immunoprecipitation experiments resulted in the partial degradation of catenins even in the presence of a cocktail of protease inhibitors (Ozawa and Kemler, 1998).