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The Journal of Immunology, 2005, 175: 1014-1021.
Copyright © 2005 by The American Association of Immunologists

Critical Role of the Fifth Domain of E-Cadherin for Heterophilic Adhesion with {alpha}E{beta}7, But Not for Homophilic Adhesion

Kiyono Shiraishi1,*,{dagger}, Kensei Tsuzaka*,{dagger}, Keiko Yoshimoto{dagger}, Chika Kumazawa*,{dagger}, Kyoko Nozaki*,{dagger}, Tohru Abe{dagger}, Kazuo Tsubota{ddagger} and Tsutomu Takeuchi*,{dagger}

* Project Research Laboratory, Research Center for Genomic Medicine and{dagger} Second Department of Internal Medicine, Saitama Medical Center, Saitama Medical School, Saitama, Japan; and{ddagger} Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The integrin {alpha}E{beta}7 is expressed on intestinal intraepithelial T lymphocytes and CD8+ T lymphocytes in inflammatory lesions near epithelial cells. Adhesion between {alpha}E{beta}7+ T and epithelial cells is mediated by the adhesive interaction of {alpha}E{beta}7 and E-cadherin; this interaction plays a key role in the damage of target epithelia. To explore the structure-function relationship of the heterophilic adhesive interaction between E-cadherin and {alpha}E{beta}7, we performed cell aggregation assays using L cells transfected with an extracellular domain-deletion mutant of E-cadherin. In homophilic adhesion assays, L cells transfected with wild-type or a domain 5-deficient mutant formed aggregates, whereas transfectants with domain 1-, 2-, 3-, or 4-deficient mutants did not. These results indicate that not only domain 1, but domains 2, 3, and 4 are involved in homophilic adhesion. When {alpha}E{beta}7+ K562 cells were incubated with L cells expressing the wild type, 23% of the resulting cell aggregates consisted of {alpha}E{beta}7+ K562 cells. In contrast, the binding of {alpha}E{beta}7+ K562 cells to L cells expressing a domain 5-deficient mutant was significantly decreased, with {alpha}E{beta}7+ K562 cells accounting for only 4% of the cell aggregates, while homophilic adhesion was completely preserved. These results suggest that domain 5 is involved in heterophilic adhesion with {alpha}E{beta}7, but not in homophilic adhesion, leading to the hypothesis that the fifth domain of E-cadherin may play a critical role in the regulation of heterophilic adhesion to {alpha}E{beta}7 and may be a potential target for treatments altering the adhesion of {alpha}E{beta}7+ T cells to epithelial cells in inflammatory epithelial diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
E-cadherin, a classic member of the cadherin superfamily, is expressed on epithelial cells and mediates Ca2+-dependent homophilic cell-cell adhesion (1, 2, 3). Classic cadherins contain five extracellular domains (ECs)2 of ~110 aa each, a transmembrane domain, and a cytoplasmic domain. Structure-function analyses of the homophilic interactions of E-cadherin have largely focused on the NH2-terminal EC domain (EC1), which contains a highly conserved His-Ala-Val motif (4, 5, 6, 7). Indeed, protein fragments or peptides containing the His-Ala-Val sequence exerted limited effects on cell-cell adhesion (8, 9). Recently, crystallographic analysis has clearly demonstrated that conserved Trp in the EC1 domains of classical cadherins is critical for trans-interactions between E-cadherin molecules on different cells, serving as a strand dimer (10). However, several studies have suggested that ECs may be involved in cell adhesion in ways other than the role mediated by EC1. In human cancers, for example, E-cadherin gene mutations frequently occur in exons 7, 8, and/or 9 (corresponding to EC2 and EC3); these mutations are thought to result in the loss of the ability to undergo cell-cell adhesion (11, 12, 13, 14). A study on the binding properties of the soluble C-cadherin ectodomain suggested that EC1 was not sufficient for complete homophilic binding (15). Furthermore, Corada et al. (16, 17) demonstrated that mAbs directed against EC3-EC4 affected VE-cadherin adhesion in endothelial cells.

The heterophilic interaction of E-cadherin and integrin {alpha}E{beta}7 has been previously documented (18, 19, 20, 21, 22, 23, 24). Integrin {alpha}E{beta}7 is expressed selectively on intestinal intraepithelial T lymphocytes under physiological conditions (25). Accumulating evidence indicates that {alpha}E{beta}7+ is induced on T lymphocytes in the epithelia of skin, lung, salivary, and lacrimal glands and synovial membranes during inflammation (26, 27, 28, 29, 30, 31), suggesting that this heterophilic interaction has a pathologic role. Since {alpha}E{beta}7 may have an important role in the selective localization or retention of a unique population of T cells in a specific tissue, the adhesion between {alpha}E{beta}7 and E-cadherin could be a potential target of therapeutic interventions for epithelial inflammation. Substitutions of a highly exposed and charged amino acid on mouse E-cadherin transfected into L cells demonstrated that Glu31 in EC1 is critical for binding with {alpha}E{beta}7 (32). Taraszka et al. (33) also elucidated that the substitution of the corresponding Glu in EC1 of human E-cadherin-Fc fusion proteins abrogated binding with {alpha}E{beta}7, confirming the previous observations. These studies clearly show that the specific heterophilic adhesion attributed to the exposed Glu31 in EC1 differs from homophilic adhesion. With the exception of EC1, however, the structures involved in heterophilic adhesion remain uncertain. To clarify the involvement of the EC domains in both heterophilic and homophilic adhesion, we performed cell aggregation assays using L cells transfected with specific domain-deleted E-cadherin mutations. The present report speculates on the characteristics of the E-cadherin domains involved in homophilic interactions and heterophilic interactions with {alpha}E{beta}7.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells

Mouse L-K (TK–) fibroblasts were cultured in DMEM containing 10% FBS. K562 cells were cultured in RPMI 1640 containing 10% FBS. K562 cells double-transfected with {alpha}E and {beta}7 were a gift from Dr. D. Erle (University of California, San Francisco, CA) and cultured in RPMI 1640 supplemented with 10% FBS, 500 µg/ml hygromycin B, and 500 µg/ml G418 (34).

Construction of E-cadherin deletion mutants

The expression vector containing human E-cadherin cDNA was a gift from Dr. Y. Shimoyama (National Okura Hospital, Tokyo, Japan). To examine the contribution of the different ECs to adhesion, the EC1, EC2, EC3, EC4, or EC5 domains or all of the EC domains were deleted using inverse PCR. Fig. 1 shows the domain deletion mutants ({Delta}) and the primers designed for the PCR experiments. The following primers were used: for {Delta}1, 5'-TCTCTTCTGTCTTCTGAGGCCAGGAGAGG-3' (SIG-R) and 5'-ACCCAGGAGGTCTTTAAGGGGTCTG-3' (2-F); for {Delta}2, 5'-GAATTCGGGCTTGTTGTCATTCTG-3' (1-R) and 5'-AATCCCACCACGTACAGGGTCAG-3' (3-F); for {Delta}3, 5'-GAAGATCGGAGGATTATCGTGGT-3' (2-R) and 5'-GTGCCTCCTGAAAAGAGAGTGGAA-3' (4-F); for {Delta}4, 5'-AGGCACAAAGATGGGGGCTTCATTCAC –3' (3-R) and 5'-GAACCTCGAACTATTCTTCTGT-3' (5-F); for {Delta}5, 5'-TGGTATGGGGGCGTTGTCATTCAC-3' (4-R) and 5'-ATTCCTGCCATTCTGGGGATTCTTGGAG-3' (TM-F); and for d0, SIG-R, and TM-F. Amplicons were ligated to form circular plasmids. The structures of the deletion mutants were confirmed by sequencing. Expression vectors bearing wild-type or mutated E-cadherin cDNAs were transfected into L cells using LipofectAMINE reagent (Invitrogen Life Technologies). The transfected cells were selected in DMEM supplemented with 10% FBS and 700 µg/ml G418, and colonies expressing high levels of E-cadherin were screened by Western blot analysis using the mAb 4A2C7 (Zymed).



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  		FIGURE 1. Schematic representation of the E-cadherin domain-deletion mutants and the PCR primers. Wild-type E-cadherin has five extracellular cadherin repeats, numbered 1–5 from the NH2-terminal. The numbers above the columns indicate the amino acid number, counting from the start of the coding region. The arrows represent the primers used for inverse PCR. S, Signal peptide; P, propeptide; TM, transmembrane domain; C, cytoplasmic domain.

 
RT-PCR

One microgram of total RNA from the L transfectants was used to generate single-stranded cDNA using AMV Reverse Transcriptase XL (Takara Bio). The cDNAs were amplified by PCR using Ex Taq polymerase (Takara Bio) and the following primers: for EC1, 5'-GACTGGGTTATTCCTCCCATCAGC-3' (1-F) and 1-R; for EC2, 2-F, and 2-R; for EC3, 3-F, and 3-R; for EC4, 4-F, and 4-R; and for EC5, 5-F, and 5'-TTGCAATCCTGCTTCGACAGGCTGTGC-3' (5-R).

Western blot

Cultured cells were rinsed in PBS and harvested in mammalian protein extraction reagent (Pierce). The total protein concentration was determined using a Micro BCA protein assay reagent kit (Pierce). GST fusion proteins containing the EC domains were produced in TOP10F' cells carrying pGEX-4T-2 vectors and purified using GST purification modules (Amersham Biosciences). Cell lysates and the purified fusion proteins were subjected to SDS-PAGE and transferred to an Immobilon-P membrane (Millipore).

The membrane was blocked with 5% skim milk and incubated with the primary Abs. Goat anti-GST Ab was purchased from Amersham Pharmacia. Four different mouse mAbs against human E-cadherin were used. The binding epitopes recognized by SHE78-7 and HECD-1 (Takara Bio) were unknown. 4A2C7 was generated against a recombinant protein corresponding to the cytoplasmic domain, whereas G-10 (Santa Cruz Biotechnology) was raised against a recombinant protein corresponding to aa 600–707. The membrane was then incubated with the respective HRP-conjugated secondary Abs followed by an ECL system (Amersham Biosciences) and analyzed using LAS-1000 (Fuji film).

Flow cytometry

The cell surface expression of E-cadherin in the transfectants was examined using flow cytometry. Adherent L transfectants were treated with 0.05% trypsin and 0.02% EDTA at 37°C for 10 min and then washed with DMEM containing 10% FBS. After washing with PBS, the cell suspension was incubated with anti-E-cadherin Ab or an isotype control on ice for 30 min, washed with PBS, and subjected to incubation with FITC-conjugated secondary Ab on ice for 30 min. Ten thousand stained cells were then analyzed using FACSCalibur and the CellQuest software (BD Biosciences).

Cell aggregation assays

The cell aggregation assays were performed according to a method described by Shimoyama et al. (35). Briefly, after the treatment of the adherent cells with 0.05% trypsin and 0.02% EDTA at 37°C for 10 min, the cells were washed and resuspended with DMEM containing 1.8 mM Ca2+ and 0.8 mM Mg2+ plus 10% FBS. The suspended cells were collected by centrifugation, washed with PBS, and resuspended with the above-mentioned medium.

To examine the homophilic interactions of E-cadherin, 105 L transfectants were added to each well of a 24-well plastic plate and incubated with rocking at 37°C overnight in a humidified atmosphere comprised of 7% CO2 and 93% air. The resulting cell aggregates were examined using a phase-contrast microscope (Olympus).

To examine the heterophilic interactions between E-cadherin and {alpha}E{beta}7, L transfectants and K562 transfectants were used in a coaggregation assay. The former transfectants were labeled with 3,3'-dioctadecyl-5, 5'-di(4-sulfophenyl)oxacarbocyanine sodium salt (DiO; Molecular Probes), and the latter were labeled with CellTracker CM-DiI (Molecular Probes). The transfectants were then mixed (5 x 104 cells each), incubated as described above, and observed using a confocal laser-scanning microscope (TCS SP2; Leica). Mg2+ (1 mM) was added to the medium when indicated. In some experiments, the proportion of {alpha}E{beta}7+ K562 cells in each aggregate was calculated. Micrographs of eight aggregates were taken in each assay. The numbers of K562 and L cells were counted, and the results were expressed as the percentage of K562 cells out of the total number of cells in the aggregate.

In the Ab-blocking experiments, the cells were incubated in the presence of 0.8 µg/ml of appropriate Ab.

Immunocytochemistry

Following the cell aggregation assays, the cell aggregates were collected and washed with PBS. For immunofluorescence staining, the aggregates were incubated with anti-E-cadherin Ab (HECD-1). Subsequently, the samples were incubated with Alexa Fluor 568-conjugated goat anti-mouse IgG Ab (Molecular Probes), followed by incubation with FITC-conjugated anti-{alpha}E{beta}7 Ab (Beckman Coulter). The specimens were observed using a confocal laser-scanning microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
To confirm whether the transfectants expressed the desired region of E-cadherin, RT-PCR was performed for EC1, EC2, EC3, EC4, and EC5. Fig. 2 shows that the parent L cells did not exhibit any products amplified by human E-cadherin and that all five ECs were expressed in wild-type transfectants. All clones transfected with domain-deletion mutations of E-cadherin exhibited the expected expression patterns: {Delta}1 lacked EC1, {Delta}2 lacked EC2, {Delta}3 lacked EC3, {Delta}4 lacked EC4, and {Delta}5 lacked EC5.



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  		FIGURE 2. Expression of EC mRNA in L cells transfected with E-cadherin domain-deletion mutations. The primers were designed to amplify the various ECs. The mRNAs from parent L cells (L) and L transfectants with wild-type (w), mock (m), and mutated E-cadherin were analyzed using RT-PCR.

 
The protein expression of mutated E-cadherin was examined by Western blot analysis using the appropriate Abs (Fig. 3a). 4A2C7, raised against a recombinant protein corresponding to the cytoplasmic domain, detected all of the deletion mutants. G-10, an alternative E-cadherin Ab that cross-reacts with mouse form (manufacturer’s data), could not detect endogenous mouse E-cadherin in mock transfectants (data not shown). To clarify the binding epitopes recognized by the SHE78-7, HECD-1, and G-10 Abs, GST fusion proteins containing the EC domains were analyzed (Fig. 3b). SHE78-7 reacted with EC1, whereas HECD-1 reacted with EC2. G-10, raised against a recombinant protein corresponding to EC5, correctly detected EC5.



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  		FIGURE 3. a, Immunoblot analysis of mutated E-cadherin expression in transfectants. Protein extracts of L cells transfected with mock (m), wild-type (w), and mutated E-cadherin were separated on a 7.5% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was then incubated with anti-E-cadherin Ab 4A2C7, which visualized the 120-kDa band of wild-type E-cadherin. b, Identification of the binding epitopes recognized by the mAbs. Purified GST fusion proteins containing the ECs were separated on a 7.5% polyacrylamide gel and transferred to polyvinylidene difluoride membranes. The membranes were incubated with Abs against GST and E-cadherin (SHE78-7, HECD-1, and G-10).

 
The expression of the mutated E-cadherin proteins on the cell surfaces of L transfectants was analyzed using flow cytometry (Fig. 4). Approximately 90% of the wild-type and {Delta}1, {Delta}3, {Delta}4, and {Delta}5 transfectants stained positive when incubated with HECD-1, but the {Delta}2 transfectants did not. In contrast, the {Delta}2 transfectants stained positive when incubated with SHE78-7 (Fig. 4); the wild-type, {Delta}3, {Delta}4, and {Delta}5 transfectants also stained positive when incubated with SHE78-7 (data not shown). These results show that the mutated E-cadherin proteins were expressed on the outer cell surface, indicating that protein transport to the plasma membrane was not impaired by the mutations.



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  		FIGURE 4. Flow cytometry analysis of E-cadherin expression on the cell surface of L transfectants. Mock (a), wild-type (b), {Delta}1 (c), {Delta}3 (e), {Delta}4 (f), and {Delta}5 (g) transfectants were stained with HECD-1, and {Delta}2 (d) transfectants were stained with SHE78-7. Open histograms indicate background labeling obtained with isotype controls, whereas filled histograms indicate labeling with the Ag-specific Abs.

 
To examine the homophilic interactions of the mutated E-cadherin proteins, L transfectants were subjected to a cell aggregation assay (Fig. 5). Mock transfectants were dispersed, whereas L cells expressing wild-type E-cadherin formed closely packed aggregates. Aggregation was strongly inhibited by the addition of SHE78-7 to the assay medium. HECD-1 also inhibited cell aggregation, but its inhibitory effect was not as strong as that of SHE78-7; G-10 did not have any effect on the aggregation. These results indicate that the adhesion of the L cells was mediated through E-cadherin, especially through the EC1 and EC2 domains, but not through the EC5 domain. Higher concentrations of Abs did not influence the results (data not shown).



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  		FIGURE 5. Homophilic interaction of wild-type E-cadherin. L cells transfected with mock (a) and wild-type (b) E-cadherin were used in cell aggregation assays. Wild-type transfectants were incubated in the presence of the anti-E cadherin mAbs SHE78-7 (c), HECD-1 (d), G-10 (e), or an isotype control (f). Bars, 300 µm.

 
Next, we examined the interaction of L cells transfected with domain-deletion mutants (Fig. 6). Cell aggregation was completely inhibited not only in the {Delta}1 transfectants, but also in the {Delta}2, {Delta}3, and {Delta}4 transfectants. In contrast, EC5-deficient transfectants formed cell aggregates. SHE78-7 and HECD-1 Abs also inhibited {Delta}5 aggregation (Fig. 6, f and g), indicating that {Delta}5 aggregates were similar to those produced by wild-type transfectants.



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  		FIGURE 6. Homophilic interactions of domain-deletion mutants. L cells transfected with {Delta}1 (a), {Delta}2 (b), {Delta}3 (c), {Delta}4 (d), and {Delta}5 (e) were used in a cell aggregation assay. {Delta}5 transfectants were incubated with SHE78-7 (f), HECD-1 (g), or an isotype control (h). Bars, 300 µm.

 
To analyze heterophilic interactions, {alpha}E{beta}7+ K562 cells and wild-type E-cadherin+ L cells were mixed. Initially, the {alpha}E{beta}7+ K562 cells did not adhere with the E-cadherin+ L cells; in other words, only the E-cadherin+ L cells adhered and formed cell aggregates (Fig. 7a). Thus, we modified the concentration of divalent cations in the assay medium, which originally contained Ca2+ and Mg2+. Increasing the Ca2+ or Mg2+ concentrations had no effect on the aggregate constituents (data not shown). In contrast, Mn2+ supplementation led to a number of {alpha}E{beta}7+ K562 cells being included in the cell aggregates (Fig. 7a). To confirm this observation, the numbers of the two cell types in each aggregate were counted. As shown in Fig. 8, the percentage of {alpha}E{beta}7+ K562 cells in the cell aggregates significantly increased with Mn2+ supplementation (from 5.0 ± 0.8% without Mn2+ supplementation to 22.8 ± 0.9% with Mn2+ supplementation). Immunofluorescence staining of the cell aggregates clearly showed that E-cadherin and {alpha}E{beta}7 were localized on the cell membranes and in close contact (Fig. 7b). In subsequent coaggregation assays, Mn2+ was added to the assay medium.



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  		FIGURE 7. a, Heterophilic interaction of wild-type E-cadherin with {alpha}E{beta}7. Following the labeling of E-cadherin+ L cells with DiO (green) and {alpha}E{beta}7+ K562 cells with DiI (red), the cells were allowed to aggregate in assay medium with (lower) or without (upper) 1 mM Mn2+. The dye localization into cytoplasmic vesicle made DiI-labeled K562 cells look smaller than they really are, while the plasma membrane of L cells was successfully labeled by DiO. Bars, 100 µm. b, Immunofluorescence detection of heterophilic aggregates. Cell aggregates were stained with anti-E-cadherin Ab, followed by an Alexa Fluor 568-conjugated secondary Ab, and with FITC-conjugated anti-{alpha}E{beta}7 Ab. E-cadherin (red) and {alpha}E{beta}7 (green) were localized on the cell membrane and in close contact with each other (arrowheads). Nomarski differential interference micrographs are shown to the left of each fluorescent photograph. Bars, 8 µm.

 


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  		FIGURE 8. Effects of manganese, mAbs, and domain-deletions on heterophilic interactions. The numbers of K562 and L cells were counted, and the results were expressed as the percentage of K562 cells out of the total number of cells in the cell aggregate. Results are shown as the mean ± SEM (n = 8). Student’s t test was used for the statistical analysis. The asterisks indicate a significant difference of p < 0.001, compared with wild-type transfectants cultured in the presence of Mn2+.

 
The adhesion of {alpha}E{beta}7+ K562 cells to E-cadherin+ L cells was clearly inhibited by the presence of mAb against {alpha}E{beta}7, with the percentage of {alpha}E{beta}7+ K562 cells in the cell aggregates decreasing to 2.9 ± 1.1% (Figs. 8 and 9a). This result shows that the {alpha}E{beta}7+ K562 cells adhere to the E-cadherin+ L cells via {alpha}E{beta}7 during cell aggregation. SHE78-7 inhibited aggregation in the heterophilic assay (Fig. 9b), whereas HECD-1 allowed the formation of some aggregates in which {alpha}E{beta}7+ K562 cells were adhered to E-cadherin+ L (23.0 ± 3.1%, Figs. 8 and 9c). G-10 did not inhibit the adhesion of the {alpha}E{beta}7+ K562 cells to E-cadherin+ L (25.9 ± 1.2%, Figs. 8 and 9d).



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  		FIGURE 9. Heterophilic interaction of E-cadherin with {alpha}E{beta}7. Following the labeling of E-cadherin+ L cells with DiO (green) and {alpha}E{beta}7+ K562 cells with DiI (red), the cells were allowed to aggregate in assay medium containing 1 mM Mn2+ in the presence of anti-{alpha}E{beta}7 Ab (a) or one of the anti-E-cadherin Abs SHE78-7 (b), HECD-1 (c), or G-10 (d) or an isotype control (e). Nomarski differential interference micrographs are shown to the left of each fluorescent photograph. Bars, 100 µm.

 
Next, the interactions of {alpha}E{beta}7 and the mutated E-cadherin proteins were examined. When {alpha}E{beta}7+ K562 cells were mixed with the mock transfectants, no cell aggregates were formed (Fig. 10a). Similarly, the {Delta}1, {Delta}2, {Delta}3, or {Delta}4 transfectants and {alpha}E{beta}7+ K562 cells formed no cell aggregates (data not shown). The mixture of {alpha}E{beta}7+ K562 cells and {Delta}5 transfectants produced cell aggregates, but the percentage of {alpha}E{beta}7+ K562 cells in the aggregates was significantly lower than that in aggregates formed from the mixture of {alpha}E{beta}7+ K562 cells and wild-type transfectants (4.0 ± 0.9% vs 22.8 ± 0.9%; Figs. 8 and 10b).



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  		FIGURE 10. Heterophilic interactions of E-cadherin-deletion mutants with {alpha}E{beta}7. L cells transfected with mock (a) and {Delta}5 (b) were labeled with DiO (green), while the {alpha}E{beta}7+ K562 cells were labeled with DiI (red). The cells were allowed to aggregate in assay medium containing 1 mM Mg2+. Nomarski differential interference micrographs are shown to the left of each fluorescent photograph. Bars, 100 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we attempted to clarify the overall binding capability of E-cadherin for both homophilic adhesion and heterophilic adhesion to the integrin {alpha}E{beta}7 through the generation of domain-deletion E-cadherin mutants and the transfection of these mutants into L cells to determine the roles of the individual domains in cell adhesion. EC1, EC2, EC3, and EC4 domain-deletion mutants lost their homophilic binding ability, suggesting that these EC domains are indispensable for homophilic adhesion. Substantial evidence suggests that the combination of cis-dimerization of two cadherin molecules on the same cell surface and trans-interactions between cadherin dimers on opposing cell surfaces maximizes homophilic adhesion. The widely accepted linear zipper model attributes the adhesive interfaces in the cis- and trans-interactions to EC1 (6, 7, 36). However, recent studies have introduced a new model for homophilic adhesion. Through the analysis of domain deletions in the Xenopus C-cadherin ectodomain using bead aggregation and cell adhesion assays, Clappuis-Flament et al. (15) demonstrated that the combination of at least three EC domains, such as EC1-EC2-EC3 or EC1-EC2-EC4, was required for trans-interaction and proposed an alternative model in which multiple ECs are required to achieve full adhesive capability. Several studies have provided data to support this model, suggesting the existence of multiple adhesive interfaces. For example, a mAb recognizing a potential epitope spanning the EC3–EC4 domains affected VE-cadherin adhesion in endothelial cells from umbilical veins (16). A mutant lacking the EC1 domain failed to exhibit homophilic adhesion, although the mutant could adhere to other mutants expressing wild-type E-cadherin (37). In contrast, a crystallographic analysis of C-cadherin supported the linear zipper model by showing that EC1 interacts with EC2 on other E-cadherin molecules on the same cell surface, leading to cis-dimerization (10). Our results showing that EC1- or EC2-deficient mutants failed to exhibit full homophilic adhesion are basically consistent with the above findings. However, mutants lacking either the EC3 or EC4 domains also failed to exhibit maximum adhesion. The fact that the present study used mammalian cadherin, whereas the previous study used Xenopus cadherin, may partially explain this discrepancy in findings. Another possible explanation may be that intracellular events were affected by the domain deletion in our aggregation assay, since the transmembrane and cytoplasmic domains were included in the domain-deletion human E-cadherin mutants. Compelling evidence suggests that the adhesive strength of cadherin is regulated by the transmembrane and cytoplasmic domains (3, 38, 39, 40, 41, 42). For example, the cytoplasmic tail of cadherin interacts with {beta}-catenin and p120, which link cadherin to the actin cytoskeleton through {alpha}-catenin, and the regulation of the cadherin-catenin complex by diverse phosphorylation reactions influences the adhesive function of cadherin (43). In addition, the interaction of a motif in domain 4 of N-cadherin with the fibroblast growth factor receptor is required for neurite outgrowth (44), suggesting that EC4 may be involved in a cell signaling pathway in which fibroblast growth factor receptor controls the gene transcription and adhesive activity of cadherin via Snail, resulting in a loss of cell-cell adhesion (43). To the best of our knowledge, little information is available about the involvement of the membrane proximal domain EC5 in homophilic adhesion. EC5 might not participate in the adhesive bond, possibly explaining the preservation of homophilic adhesion in EC5-deficient mutants. Alternatively, conformational changes in E-cadherin resulting from the EC5 deletion may cause cell aggregation via a process different from that occurring with the native molecule. However, current data that the same mAbs inhibiting homotypic aggretgates by wild-type transfectants (SHE78-7, HECD-1) also inhibited those by the {Delta}5 mutant would be evidence that the process of cell aggregation is similar.

Consistent with previous studies (21, 22, 45), we found that Mn2+ stimulated heterophilic interactions between {alpha}E{beta}7 and E-cadherin. The functional activity of integrins is regulated through an inside-out signaling mechanism that quickly switches inactive forms to active forms. Divalent cations are also required for the acquisition of the active state. In particular, manganese has been shown to promote ligand binding by inducing a conformational change in the metal ion-dependent adhesion site (46, 47, 48).

To examine homophilic and heterophilic interactions, we adopted a cell aggregation assay that has been frequently used to evaluate cell-cell adhesion activity (4, 15, 35, 37). This assay method produced reproducible and clear results in the present study. When {Delta}1, {Delta}2, {Delta}3, or {Delta}4 transfectants were mixed with {alpha}E{beta}7+ K562 cells, aggregation did not occur, indicating that all four domain-deletion mutants had lost their ability to undergo heterophilic adhesion with {alpha}E{beta}7. Alternatively, aggregation may have been prevented by a disruption in homophilic adhesion, although the ability to undergo heterophilic adhesion was retained. Heterophilic interactions have been suggested to be so weak that cell aggregation may not occur without homophilic adhesion; alternatively, {alpha}E{beta}7 adhesion may require an E-cadherin homophilic bond to be formed by trans-interactions among cells. The mAb HECD-1 against EC2 did not inhibit homophilic aggregation completely, allowing heterophilic adhesion and supporting the possibility that a homophilic scaffold may be necessary for heterophilic interactions. If so, the effect of the {Delta}1, {Delta}2, {Delta}3, and {Delta}4 mutants cannot be evaluated because these mutants prevent homophilic adhesion, and some degree of homophilic adhesion by the transfected L cells is a prerequisite to allowing a determination of the percentage of K562 cells included in the aggregates of E-cadherin-expressing cells. Therefore, the EC5 deletion mutant is the only mutant of the entire set used in this article that can be evaluated for loss of heterophilic adhesion in the K562 coaggregation assay.

We demonstrated that EC5 is critical for heterophilic adhesion with {alpha}E{beta}7+ cells, but not for homophilic adhesion. This is the first evidence showing the involvement of ECs other than EC1 in heterophilic adhesion with {alpha}E{beta}7. Since the integrin {alpha}E{beta}7 plays a critical role in the selective localization of T cells in inflamed epithelia (26, 27, 28, 29, 30, 31, 49), the adhesive interaction could be a potential target for therapeutic interventions. In contrast, mutational analyses of selected residues clearly revealed that the side chain of Glu31 is important for integrin {alpha}E{beta}7 recognition, but not for homophilic adhesion, indicating that the E-cadherin residues critical for heterophilic adhesion to {alpha}E{beta}7 are distinct from those required for homophilic adhesion (32, 33). Higgins et al. (50) proposed a docking model involving the {alpha}E A domain and EC1 in which the metal ion-dependent adhesion site cleft of {alpha}E comes in contact with Glu31 of EC1 and the Phe298 projection of {alpha}E coordinates with the hydrophobic pocket of EC1. Given a previous report showing that synthetic peptides encompassing Asn27-Val34 in EC1 had very little inhibitory effect on the interaction with {alpha}E{beta}7 (32), however, full adhesive activity may require other binding sites or events, such as a conformational change in the cadherin or integrin molecules. Since domain 5 is located proximal to the membrane, it is less likely to serve as a direct binding site for the integrin, implying that the effect of the EC5 deletion on binding of the {alpha}E{beta}7 may be due to long-range effects that alter the conformation of more membrane distal domain of the molecules. However, the {Delta}5 mutants retained the ability to participate in homophilic adhesion and still bound the EC1-specific Ab SHE78-7 and the EC2-specific Ab HECD-1, indicating that a gross disruption of the conformation of the domain distal to EC5 is probably not occurring. The complexity of the EC5 conformational structure, containing asparagine residues of N-linked glycosylation and two intramolecular disulfide bonds, may favor the hypothesis that EC5 plays a role in the regulation of heterophilic adhesion via a change in conformation of the membrane proximal domain. Although the inside-out signaling mechanism that alters the conformational change in E-cadherin remains to be clarified, further functional and structural studies should provide an insight into our understanding of the role of EC5 in heterophilic adhesion. Identification of smaller deletions or point mutations in EC5 that also affected heterophilic adhesion by {alpha}E{beta}7 would be of considerable interest. The current data did not show that EC5-specific Ab G-10 block heterophilic adhesion of {alpha}E{beta}7 to E-cadherin. The adhesion was also not inhibited by the polyclonal Ab, which we newly generated in rabbits using a synthetic peptide corresponding to part of EC5 (our unpublished observations). If any anti-EC5 mAb blocks heterophilic adhesion to the same degree as anti-{alpha}E, this would also be good supporting evidence for a role of EC5 in heterophilic adhesion.


   Acknowledgments
 
We are grateful to Dr. D. Erle for providing K562-{alpha}E{beta}7 cells and Dr. Y. Shimoyama for providing the vector containing E-cadherin cDNA. We acknowledge the use of equipment belonging to the Saitama Medical School Research Center for Genomic Medicine


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence to Dr. Kiyono Shiraishi, Project Research Laboratory, Research Center for Genomic Medicine, Saitama Medical School, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan. E-mail address: kiyono{at}saitama-med.ac.jp Back

2 Abbreviations used in this paper: EC, extracellular domain; DiO, 3,3'-deoctadecyl-5, 5'-di(4-sulfophenyl)oxacarbocyanine sodium salt. Back

Received for publication January 5, 2005. Accepted for publication May 13, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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