Epidermal growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions

Abstract

Ep-CAM is a new type of cell adhesion molecule (CAM) which does not structurally resemble the members of the four major families (cadherins, integrins, selectins, and CAMs of the immunoglobulin superfamily) and mediates Ca(2+)-independent, homophilic adhesions. The extracellular domain of Ep-CAM consists of a cysteine-rich region, containing two type II epidermal growth factor (EGF)-like repeats, followed by a cysteine-poor region. We generated mutated Ep-CAM forms with various deletions in the extracellular domain. These deletion mutants, together with monoclonal antibodies recognizing different epitopes in the extracellular domain, were used to investigate the role of the EGF-like repeats in the formation of intercellular contacts mediated by Ep-CAM molecules. We established that both EGF-like repeats are required for the formation of Ep-CAM-mediated homophilic adhesions, including the accumulation of Ep-CAM molecules at the cell-cell boundaries, and the anchorage of the Ep-CAM adhesion complex to F-actin via alpha-actinin. Deletion of either EGF-like repeat was sufficient to inhibit the adhesion properties of the molecule. The first EGF-like repeat of Ep-CAM is required for reciprocal interactions between Ep-CAM molecules on adjacent cells, as was demonstrated with blocking antibodies. The second EGF-like repeat was mainly required for lateral interactions between Ep-CAM molecules. Lateral interactions between Ep-CAM molecules result in the formation of tetramers, which might be the first and necessary step in the formation of Ep-CAM-mediated intercellular contacts.

FIG. 1

Heterogeneity of Ep-CAM as detected with specific MAbs. (A) Inhibition by MAb 323/A3 of the binding of various MAbs to solid-phase Ep-CAM. (B) Western blots of nonreduced (N) and β-mercaptoethanol-reduced (R) RC-6 lysates stained with MAbs 323/A3, 2G8, and 311-1K1. (C) Immunofluorescent staining for Ep-CAM in human colon carcinoma CaCo-2 cells. MAbs 2G8 and 311-1K1 detect only cytoplasmic Ep-CAM, not Ep-CAM at the cell-cell boundaries as seen with MAb 323/A3. (D) Detection of Ep-CAM with various MAbs at the cell surface of human KATO-III, U2, and RC-6 cells by flow cytometry. Although all three MAbs against Ep-CAM were used at saturating conditions, they showed differences in recognizing the cell surface Ep-CAM. (E) Cell surface and total (plus intracellular) Ep-CAM, as detected by MAbs 323/A3, 2G8, and 311-1K1. Flow cytometry was performed on intact and methanol-permeabilized RC-6 cells. MAbs 2G8 and 311-1K1 show increased levels of Ep-CAM detection compared to nonpermeabilized cells, indicating that these MAbs mainly recognize intracellular Ep-CAM. The ratio of MAb reactivity with permeabilized cells to that with nonpermeabilized cells is presented above every pair of bars. F, fluorescence.

FIG. 2

Secreted and membrane-anchored deletion mutants of Ep-CAM. (A) Secreted extracellular domain deletion mutants (lanes 10 to 16), the DNA for each mutant, the motility of each mutant in polyacrylamide gel electrophoresis, and the reactivities of various MAbs in Western blotting with mutant forms are shown. (B) Transmembrane extracellular domain mutants. Expression of the different Ep-CAM forms was verified using Western blotting. Immunoreactivities of the Ep-CAM-directed MAbs 323/A3, KS1/4, GA733, 2G8, MM104, and 311-1K1 are summarized at the bottom. (C) Detection of Ep-CAM extracellular domain mutant forms by flow cytometry. All extracellular mutant forms were expressed at the cell surface as detected with MAb MM104. (D) Amino acid sequences of the joints between various domains in the Wt and mutant Ep-CAM molecules. As can be seen, all sequences of domains and joints were unchanged in mutants. (E) Color codes for various domains of Ep-CAM as used in all schemes.

FIG. 3

An intact extracellular domain is required for the distribution of Ep-CAM to intercellular boundaries and for α-actinin binding. (A) Distribution of the different mutant Ep-CAM forms in L-cell transfectants. L cells transfected with mutant or Wt Ep-CAM were fixed and stained for mutated or Wt Ep-CAM using MAbs 323/A3 (left), 2G8 (middle), and 311-1K1 (right). Only Wt Ep-CAM as detected with the MAb 323/A3 was distributed to the intercellular boundaries. (B) Localization of Ep-CAM and α-actinin in L/Wt cells double stained for α-actinin (red) and Ep-CAM (green). Ep-CAM was detected with either MAb 323/A3 or MAb 2G8. Note that colocalization with α-actinin at the intercellular boundaries is observed for Ep-CAM recognized by MAb 323/A3 but not MAb 2G8. (C) Only Wt Ep-CAM interacts with α-actinin. Lysates and α-actinin immunoprecipitates of HCA cell transfectants expressing mutant or Wt Ep-CAM were used for Western blotting. Blots were stained for Ep-CAM (MAb 311-1K1) and α-actinin (MAb CB-11). Although all Ep-CAM forms were highly expressed by the HCA transfectants, only the Wt Ep-CAM molecules were coprecipitated with α-actinin.

FIG. 4

Effects of truncations in the extracellular domain on the cell adhesion properties of Ep-CAM. (A) Degree of aggregation in suspension of L cells transfected with mutant or Wt Ep-CAM. Cells were allowed to aggregate in either the absence or presence of CCD, which inhibits Ep-CAM-mediated cell aggregation. (B) Micrographs of aggregates formed by L-cell transfectants in the presence of antibodies to Ep-CAM. L/C and L/Wt cells were allowed to aggregate for 16 h in the absence or presence of MAb 323/A3 or 2G8. (C) The 16-h aggregation of L/C and L/Wt cells in the absence or presence of MAbs 323/A3 or 2G8, presented as the degree of aggregation.

FIG. 5

Multimerization of Ep-CAM. (A) Western blot with freshly prepared lysates of H/Wt or H/M1 cells, stained with MAb 323/A3. The presence of dimers (×2), tetramers (×4), and monomers (×1) in the lysates is indicated. (B) Western blot with lysates of H/Wt cells incubated in the absence (−CCD) or presence (+CCD) of CCD for 2 h. (C) Western blots of 323/A3 immunoprecipitates after cell surface biotinylation. H/C and H/Wt cells were labeled, lysed, and used for immunoprecipitation of Ep-CAM using the MAb 323/A3. Blots were stained with streptavidin to detect biotinylated Ep-CAM or with MAb 323/A3 to detect all Ep-CAM present in the immunoprecipitates. Note the relative enrichment of the surface fraction for Ep-CAM by dimers and tetramers. Asterisks mark the nonspecific band detected in both a sample and the control probes. (D) Western blot presenting multimerization of M1 (lacking the cytoplasmic domain) and Wt Ep-CAM with or without chemical cross-linking with DSP. HCA cells transfected with blank vector (C), M1, or Wt Ep-CAM were incubated in the presence (+DSP) or absence (−DSP) of a chemical cross-linker. (E) Western blot presenting cytoskeleton-anchored (P) and soluble (S) Ep-CAM forms. H/Wt cells were extracted with 50 mM CHAPS buffer, and lysates of the pellet and of the detergent-soluble fractions were prepared. Blots were stained with MAb 323/A3. Ep-CAM dimers and tetramers are relatively increased in the cytoskeleton-anchored fraction. (F) Western blot showing lower reactivity of MAbs 2G8 and 311-1K1 than of MAb 323/A3 with multimeric forms of the Wt Ep-CAM.

FIG. 6

Role for EGF-like repeats in the formation of multimers. (A) Western blot for the secreted mutants of Ep-CAM. Note effective tetramerization or M10, M11, and M14, some dimerization of M12 and M15, and no dimers formed by M13 and M16. (B) Dimeric and multimeric forms of the deletion mutants of Ep-CAM with intact transmembrane and cytoplasmic domains. (C) Dimerization and multimerization of Ep-CAM forms cotransfected into HCA cells (the mutants were expressed in the same cells). Note the absence of mixed multimers-dimers formed by M6 either on its own or in combination with any other forms tested. The MAbs used for immunoblotting are restricted in their reactivity with mutants: C220 reacts with all forms except M5 and M7; 2G8 does not react with M6 and M7. (D) M5 forms dimers with M1 upon cotransfection of the constructs into the same cells. M1, with an intact extracellular domain, tagged with a FLAG sequence at the COOH terminus, was immunoprecipitated from the cotransfected cells using anti-FLAG antibody. M5 but not M6 was coprecipitated, as well as the control Wt molecules. (E) Cells expressing individual mutants were mixed. No dimers of mixed type were found between M5, M6, and Wt Ep-CAM. For immunoprecipitation we used MAbs that do not recognize either M5 or M6 but do recognize the Wt Ep-CAM. ×1, ×2, and ×4 indicate positions of the monomer, dimer, and tetramer Ep-CAM bands. Note that the position is only indicative, since the mobility of the respective n-mers for mutants is usually higher.

FIG. 7

Ep-CAM molecules and their adhesions. (A) Domain structure map of the Ep-CAM molecule with the regions recognized by three groups of MAbs indicated. (B) Hypothetical structure of the Ep-CAM molecule as suggested by Schön et al. (36) (left). Our data demonstrate that the EGF domains of Ep-CAM are folded independently (right). (C) Hypothetical model of Ep-CAM-mediated adhesions with lateral interactions of the molecules mediated by EGF II (empty arrow) and reciprocal interactions mediated by EGF I (solid arrow). (D) Immunolocalization of Ep-CAM in human colon cells (upper panel with inserts) and L-cell transfectants (lower panel). The immunogold beads are present as pairs at the sites of adhesions but are single outside these areas. The distribution of gold particles suggests a symmetrical and closed, non-zipper-like model for the Ep-CAM mediated adhesions. SP, signal peptide; CPR, cysteine-poor region; Cyt, cytoplasmic domain.