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Originally published In Press as doi:10.1074/jbc.M200606200 on March 21, 2002

J. Biol. Chem., Vol. 277, Issue 22, 19455-19460, May 31, 2002
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Analysis of Heterophilic and Homophilic Interactions of Cadherins Using the c-Jun/c-Fos Dimerization Domains*

Thomas AhrensDagger , Olivier Pertz§, Daniel Häussinger, Charlotte FauserDagger , Therese SchulthessDagger , and Jürgen EngelDagger ||

From the Dagger  Department of Biophysical Chemistry and  Department of Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel 4056, Switzerland and the § Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, January 22, 2002, and in revised form, February 26, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cadherin-mediated cell-cell adhesion is initiated by cis dimerization of cadherin ectodomains at the cell surface followed by an antiparallel trans interaction of dimers on opposing cells. To resolve open questions concerning the molecular details and specificity of cis and trans interactions, ectodomains of E- and P-cadherin were analyzed by chemical cross-linking and by electron microscopy. At the high intrinsic concentration created by artificial oligomerization the N-terminal cadherin (CAD)-domain of P-cadherin are forming ring-like cis dimers. At 2 mM Ca2+-associated rings involving two cis dimers indicate trans contacts in electron micrographs. cis and trans interactions were further analyzed by heterodimerization of the ectodomains of E-cadherin (ECAD) and P-cadherin (PCAD) through the leucine zipper domains of c-Jun and c-Fos. ECADJun/ECADFos dimers predominantly form ring-like cis dimers at 1 mM Ca2+ and double-ringed trans contacts above 2 mM Ca2+. The Ca2+-dependent tetrameric trans contacts of ECADJun/ECADFos dimers are also detectable after chemical cross-linking. Only cis contacts but no trans interactions are observed for heterodimers of ECADFos and the Trp-2 to Ala mutant ECADW2AJun arguing for a decisive role of Trp-2 in trans but not cis interaction. Neither cis nor trans interaction was found for heterodimers of ECADJun and PCADFos suggesting that specificity for homophilic interactions already exists at the level of cis dimerization.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cadherins (Ca2+-dependent adhesion receptors)1 are transmembrane receptors that are involved in selective cell-cell recognition and adhesion in biological and pathological processes such as early embryogenesis, morphogenesis, synapse formation and tumor invasion (1). The cadherin superfamily of proteins to date contains around 100 members (2). The "classic" members of this protein family (E-, N-, P-, and C-cadherin) are characterized by five, highly similar Ig-like extracellular CAD-domains (3, 4) followed by a transmembrane region and a cytoplasmic domain, which connects the protein to the actin cytoskeleton through alpha - and beta -catenins.

Several models are currently discussed for adhesion mediated by classic cadherins (5-7). The models jointly propose that cadherin-mediated adhesion is initiated by lateral, parallel cis dimerization followed by an antiparallel adhesive trans contact of cis dimers on opposing cells. They are controversial concerning the CAD-domains involved, the contact sites, and the specificity of interactions.

Structural studies identifying the interfaces involved in the adhesive trans interaction of cadherins are limited, because of the weakness of this interaction (8). Candidate interfaces for trans interaction have been described for the N-terminal CAD1 and the CAD1-2 domain pair crystal structures of N-cadherin (9, 10). These contacts have been criticized in that they are caused by crystal packing forces rather than protein-protein interactions (2, 11).

Structural investigations on cis and trans interactions of the full-length, glycosylated ectodomain of cadherins so far are restricted to ultrastructural studies using electron microscopy and atomic force microscopy. Upon calcium binding the ectodomain of classic cadherins adopts a bent rod-like shape, which was first detected in a monomeric state in electron micrographs (12, 13). To analyze cis and trans contacts of recombinantly expressed cadherin ectodomains the intrinsic protein concentration must be increased. Therefore the coiled-coil domain of cartilage oligomeric matrix protein (14), which forms a pentameric coiled-coil structure (15), has been fused to the CAD-domains 1-5 of E-cadherin (16). Pentamerization increased the intrinsic concentration of ectodomains to estimated levels (~0.1-2 mM) at which cis and trans interactions occur. cis dimerization was detected in electron micrographs as a ring-like interaction at the N terminus of two ectodomains and trans contacts by subsequent association of two rings. The method was also used to monitor the Ca2+ dependence of different adhesion steps of E-cadherin (17). The authors observed rigidification and cis dimerization of two molecules at Ca2+ concentrations <1 mM and trans interaction above this threshold. In contrast to the importance of Trp-2 for cis dimerization of N-cadherin (10), site-directed mutagenesis of Trp-2 to alanine abolished trans but not cis interactions of E-cadherin (17).

The concept of specific interactions between the N-terminal domains 1 and 2 in cis and trans interactions was challenged by a model based on force measurements between lipid bilayers to which ectodomains of Xenopus C-cadherin were attached (7, 18). Here, a ribbon model of multiple adhesive interactions involving variable ectodomains as interaction partners was suggested.

The specificity of cadherin interactions is another important feature of cadherin function (19). The importance of cadherin specificity in tissue morphogenesis has been shown by a number of investigators (20-22). Early studies in which CAD-domains were exchanged from one cadherin to another clearly proved that cadherin specificity is determined by the N-terminal domain CAD1 (23). Although specificity normally leads to homophilic cell-cell adhesion, heterophilic trans association of cadherins has been observed, which could be important during cell migration or tissue remodeling (24-26). In addition, heterophilic cis interactions of cadherins on the same cell surface have also been reported recently (27, 28).

Based on contacts between CAD1 domains of NCAD a "zipper model" was proposed (10) that is at variance with electron microscopic data of interacting complete extracellular regions (16, 17). It may be argued that steric hindrance imposed by the pentamerization domain may prevent contacts between relevant CAD-domains and thus prevent zipper formation. To resolve this problem homophilic and heterophilic interactions of cadherins were further analyzed by using different linkers and a different oligomerization system. Cadherin ectodomains were artificially clustered using the leucine zipper domains of c-Jun and c-Fos, which are known to preferentially form heterodimers. This oligomerization system enabled the production of ectodomain heterodimers of murine P- and E-cadherin. Using electron microscopy and chemical cross-linking we demonstrate that the ring formation is independent of the type of oligomerization domain and that specificity for homophilic interactions already exists for cis dimerization.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA Constructions-- Sequences encoding the leucine zippers of human c-Jun (29) and human c-Fos (30), respectively, were amplified by PCR. The coding sequence for a His6-tag at the 3'-end of the construct was also added.

Primers used were (restriction sites are indicated as boldface letters): cJunLZ-upp, 5'-AATAAGAATGCGGCCGCAGGATCAGGTTCTGGAAGAATCGCCCGGCTG-3'; cJunLZ-low, 5'-GCGCTCTAGACTAATGATGGTGGTGGTGATGTCCACTTCCGTGGTTCATGACTTT-3'; cFosLZ-upp, 5'-AATAAGAATGCGGCCGCAGGATCAGGTTCTGGACTGACTGATACACTC-3'; cFosLZ-low, 5'-GCGCTCTAGACTAATGATGGTGG- TGGTGATGTCCACTTCCCAGGATGAACTCTAG-3'.

Polymerase chain reaction followed standard protocols with Pwo polymerase (Roche Molecular Biochemicals). The resulting fragments were NotI/XbaI-digested and cloned into NotI/XbaI-digested murine ECADCOMPpBS (17) thus exchanging the COMPcc for the c-Jun/c-Fos leucine zippers, respectively. Finally, ECADJun and ECADFos were KpnI/XbaI-digested and cloned into KpnI/NheI sites of the eucaryotic expression vector pCepPu (31).

The cDNA of the extracellular domain of murine P-cadherin (32) was amplified by PCR using following primers: PCAD-upp, 5'-GGGGTACCATGGAGCTTCTTAGTGGGCCTCAC-3'; PCAD-low, 5'-ATAAGAATGCGGCCGCGGGTTCCGAGGGTCTGGGGCAGTC-3'. The cDNA fragment of P-cadherin (amino acids 1-643) was then KpnI/NotI-digested and cloned into KpnI/NotI-digested pBSFos plasmid. PCADFos was finally cloned into the KpnI/NheI sites of pCepPu. For constructing PCADCOMP-StrepTag (33), ratCOMPcc was amplified by PCR from ECADCOMPpBS using the primers: COMP-upp, 5'-GAAGTCGGTACCTGCGGCCGCCACGGG-3' and COMP-Strep-Taglow, 5'-GACTCACCGCGGTCTAGACTATTTTTCGAACTGTGGGTGACTCCATCCACTTCCCACGCTCAGACC-3'. The PCR product was NotI/XbaI-digested and cloned into NotI/XbaI-digested pBSPCADFos and finally cloned into pCepPu using KpnI/NheI sites.

The ECADW2AJun construct was generated using the pCep4-ECADW2ACOMP plasmid described before (17). The ECADW2A cDNA was cloned into KpnI/NotI-digested pBSJun. ECADW2AJun was eventually cloned into KpnI/NheI-digested pCepPu vector. Escherichia coli DH10b was used as cloning host strain. All constructs were sequenced with a BigDye sequencing kit (PerkinElmer Life Sciences) and analyzed with an ABI Prism 310 sequencer (PerkinElmer Life Sciences).

Cell Culture, Transfection, and Expression of Recombinant Proteins-- Human embryonal kidney 293-EBNA cells (Invitrogen) were cultured in DMEM F12 supplemented with 10% fetal bovine serum, 1% glutamine, and 10 µg/ml penicillin/streptomycin. All reagents were purchased from Invitrogen. Stable transfections were carried out using LipofectAMINE (Invitrogen). Cells were plated at a concentration of 5 × 105 cells/well in 6-well plates (Falcon) and were grown over night. A transfection mix was prepared containing 5 µl of LipofectAMINE, 200 µl of DMEM F12 without FBS and 1 µg of plasmid DNA. This mix was incubated for 45 min at room temperature. The cells were washed once with DMEM F12 without FBS, and 0.8 ml of medium without FBS was added per well. The transfection mix was then added for 16 h at 37 °C. The wells were washed again, and 2 ml of FBS-containing medium/well was added. Selection of transfected cells was performed in culture medium containing 2 µg/ml puromycin. Bulk cultures of transfected cells were used for protein expression. When confluency of transfected cells was reached on 20-cm plates (Falcon), 23 ml of expression medium (DMEM F12, 1% glutamine, 10 µg/ml penicillin/streptomycin) was added for 48 h. Conditioned medium was then collected, and new expression medium was added. This procedure was repeated 8-10 times. Supernatants were centrifuged at 2500 × g for 10 min, buffered in 20 mM Hepes, pH 7.1, and stored at -20 °C.

Protein Purification-- Supernatants containing recombinant proteins were sterile-filtered, dialyzed against 50 mM Tris, pH 7.9, and concentrated by DEAE-Sepharose chromatography (Amersham Biosciences, Inc.). Purification of His6-tagged proteins followed standard procedures under native conditions (Novagen). Purification of PCADCOMP-StrepTag was performed using the StrepTactin matrix (IBA, Goettingen, Germany). Shortly thereafter, the concentrated supernatant in 100 mM Tris/HCl, pH 7.9, was applied to a column packed with 2 ml of the StrepTactin resin. Applying a single-step affinity purification under native conditions, the protein was eluted with 5 mM of the specific competitor desthiobiotin (Sigma). Finally, the fractions containing the purified protein were dialyzed against 20 mM Tris, pH 7.9, 1 mM CaCl2.

Western Blotting-- 5 µg of PCADCOMP-StrepTag was loaded onto an 8% SDS-polyacrylamide gel. The gel was blotted onto a nitrocellulose membrane (Schleicher & Schuell). Unspecific binding sites on the membrane were blocked with 25 mM Tris, pH 8.1, 125 mM NaCl, 0.5% Tween 20 (Merck), 3% bovine serum albumin (Sigma). After several washes with blocking buffer, the membrane was incubated with 0.2 µg/ml HRP-streptavidin (Pierce) for 2 h at room temperature. After two final washing steps with 25 mM Tris, pH 8.1, 125 mM NaCl, the chromogenic reaction was initiated using ECL-Plus (Amersham Biosciences, Inc.).

CD Spectroscopy-- An Aviv 62DS circular dichroism spectropolarimeter was used with thermostatted 1-mm quartz cuvettes. Far-ultraviolet spectra (195-250 nm) were recorded at 25 °C. Proteins were dialyzed against 5 mM Tris-HCl, pH 7.4, with or without 5 mM CaCl2. Each spectrum was the average of six scans and was corrected for the buffer contribution.

Chemical Cross-linking-- Chemical cross-linking of purified cadherins was performed using bis(sulfosuccinimidyl)suberate (BS3), which is a homobifunctional sulfo-N-hydroxysuccinimide ester analog with a spacer arm length of 1.14 nm (Pierce). Cadherin chimera were dialyzed against 10 mM Hepes, pH 7.1, containing EDTA or CaCl2 as indicated for each experiment. The purified proteins (4 µM) were incubated at a 10- to 50-fold molar excess of cross-linker for 120 min at 25 °C. The reaction was stopped by adding 100 mM Tris-HCl, pH 7.4, or reducing (50 mM dithiothreitol) gel sample buffer (2× Laemmli), and the oligomerization state was analyzed by SDS-PAGE (3-15% gradient gels). The oligomerization states of cadherins after cross-linking were visualized by silver staining of the gels.

Electron Microscopy-- Purified cadherin proteins were used at a protein concentration of 1-2 µM in 20 mM Tris-HCl, pH 7.4, containing EDTA or CaCl2 as indicated. For electron microscopy at different Ca2+ concentrations, samples were Ca2+-depleted with EDTA and then dialyzed against Ca2+-containing buffers. Each sample was diluted 1:1 (v/v) with 80% glycerol and sprayed onto freshly cleaved mica. Rotary shadowing was performed with platinum/carbon at an angle of 9° and carbon shadowing at an angle of 90°. Replica formation and electron microscopy were performed as described elsewhere (34). For statistical evaluations, the protein species ("rings" and "double rings") were counted on electron micrographs. Five fields were evaluated, resulting in a total of ~150 molecules.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cis and trans Interactions of P-cadherin Are Formed as Ring-like Structures and Associated Rings-- Using a well established method to analyze cis and trans association of E-cadherin (16, 17) a PCADCOMP protein chimera was designed (Fig. 1) and expressed in the eucaryotic cell line HEK 293-EBNA. The fusion protein contained the five extracellular CAD-domains of P-cadherin connected by a linker region to the coiled-coil domain of cartilage oligomeric matrix protein (COMP). A sequence eight amino acids long with high affinity for modified streptavidin was added at the C terminus of the protein to facilitate purification (35). Purification of the secreted protein was performed as described under "Experimental Procedures." A silver-stained SDS-polyacrylamide gel and a Western blot of the purified protein are shown in Fig. 2A. PCADCOMP-StrepTag was readily detected after blotting as a 90-kDa protein by incubation with HRP-conjugated streptavidin and enhanced chemiluminescence.


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  		Fig. 1.   Schematic illustration of cadherin fusion proteins used for analysis of cis and trans association of classic cadherins. E-cadherin or P-cadherin ectodomains (CAD1-5) were connected by flexible linkers (hatched bars) to the leucine zipper domains of c-Jun (gray), c-Fos (black), or the coiled-coil domain of COMP. For protein purification either an His6-tag or a streptavidin-tag was added at the C terminus of the chimeric proteins.


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  		Fig. 2.   Purification and analysis of cis and trans association of PCADCOMP. A, silver staining and Western blotting of purified PCADCOMP. PCADCOMP was purified using the StrepTag II purification system. The protein was analyzed by silver staining after 8% SDS-PAGE (molecular mass markers are indicated by arrows on the left) and blotted onto a nitrocellulose membrane. Protein detection was performed by incubation with HRP-conjugated streptavidin followed by ECL. B, electron microscopy of PCADCOMP. PCADCOMP was subjected to rotary shadowing in the presence of 2 mM Ca2+. Representative fields of the electron micrographs are shown, and individual molecules are shown in higher magnification.

PCADCOMP was subjected to rotary shadowing. In previous work cis and trans interactions of E-cadherin were detected in such electron micrographs as ring-like structures and associated rings, respectively. Similar structures for PCADCOMP, as for E-cadherin, were found, indicating cis and trans interaction (Fig. 2B).

Cloning and Expression of Cadherin Heterodimers to Study cis and trans Interactions-- Analysis of cis and trans association of cadherin heterodimers was investigated by chemical cross-linking and electron microscopy. Constructs were designed based on the preferential capacity of the c-Jun and c-Fos leucine zippers to form heterodimers (36, 37). These heterodimerizing coiled-coil domains have already been used successfully to generate bispecific F(ab')2 antibody fragments (38), a soluble T-cell receptor (39), and soluble human alpha 3beta 1 integrin (40). The extracellular domains of E-cadherin, P-cadherin, and the W2A mutant of E-cadherin were connected by an AAAGSGSG linker to either the c-Jun or the c-Fos leucine zipper domains (Fig. 1). A coding sequence for a His6-tag was added at the C-terminal end of the constructs. Silver-stained gels of the purified proteins are shown in Fig. 3A. A second protein band of ~110 kDa was observed after purification of ECADJun and ECADFos (Fig. 3A, lanes 3 and 4). These bands represent a minor proportion of E-cadherin molecules where the N-terminal propeptide domain was not properly processed. Correct folding of the heterodimers was tested by CD spectroscopy (Fig. 3B). A dominant beta -sheet like secondary structure of E-cadherin with minima of the CD signal around 215 nm is seen at 5 mM Ca2+ (Fig. 3B; triangles). Calcium depletion results in a significant change in the circular dichroism spectra of the protein indicated by a shift of the minima of the CD signal toward 200 nm (Fig. 3B; open circles). These findings are supported by similar CD changes of the monomeric extracellular domain of E-cadherin (13).


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  		Fig. 3.   Purification and CD spectroscopy of cadherin c-Jun/c-Fos leucine zipper fusion proteins. A, silver staining of purified cadherin fusion proteins. The proteins were expressed individually in HEK 293-EBNA cells. Supernatants were collected and recombinant proteins were purified by nickel-affinity chromatography and analyzed by 10% SDS-PAGE. Molecular mass markers are indicated by arrows on the left. Lane 1, PCADFos; lane 2, ECADW2AJun; lane 3, ECADJun; lane 4, ECADFos. B, dimerized proteins were analyzed for correct protein folding by CD spectroscopy. Far-UV spectra of each protein mixture ranging from 250 to 195 nm were recorded in the presence of 5 mM Ca2+ (closed triangles) or without Ca2+ (open circles).

Specificity and Ca2+ Dependence of Cadherin trans Interactions Evaluated by Chemical Cross-linking-- In homodimers of ECAD interlinked by the c-Jun and c-Fos leucine zipper domains, the two polypeptide chains were chemically cross-linked by BS3 as indicated by stable 180-kDa dimers on SDS-PAGE under reducing conditions (Fig. 4A). In addition, a complex of around 400 kDa was cross-linked in the presence of Ca2+ but not in the presence of EDTA (Fig. 4A). This 400-kDa complex most likely represents the trans association of two ECAD dimers. In contrast, no analogous indication for a trans complex was observed for heterodimers of ECADJun and PCADFos, neither in the presence nor in the absence of Ca2+ (Fig. 4B). The possibility that the observed tetramerization in Fig. 4A is caused by association of the C-terminal His6-tags can be excluded because the His6-tag is present in both dimers.


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  		Fig. 4.   Chemical cross-linking of cadherin heterodimers. A, ECADFos/ECADJun (4 µM) heterodimers were cross-linked for 2 h at 20 °C with 10-50× molar excess of BS3. The reaction was performed in the presence of either 10 mM EDTA or 10 mM Ca2+ as indicated. The cross-linked proteins were separated under reducing conditions on a 3-15% gradient SDS gel. The gel was finally silver-stained. B, cross-linking of ECADJun/PCADFos (4 µM) heterodimers with BS3 using the same conditions as in A.

cis and trans Association of Cadherin Heterodimers Analyzed by Electron Microscopy-- To get information on cis and trans dimerization on the ultrastructural level, we performed electron microscopy experiments (Figs. 5 and 6). Three different subsets of heterodimeric proteins were rotary-shadowed and analyzed. For ECADJun/ECADFos dimers, a mixture of non-associated dimers, ring-forming cis dimers, and only a few eight-shaped complexes of two rings indicating trans interactions were observed in the presence of 1 mM Ca2+ (Fig. 5A). The latter fraction was, however, the most prominent one in the presence of 5 mM Ca2+ (Fig. 5B).


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  		Fig. 5.   Electron microscopy of ECADJun/ECADFos dimers. ECADJun/ECADFos dimers in the presence of 1 mM Ca2+ (A) and 5 mM Ca2+ (B) were subjected to rotary shadowing. Representative fields are shown, and individual molecules are shown in higher magnification.


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  		Fig. 6.   Electron microscopy of ECADW2AJun/ECADFos and ECADJun/PCADFos heterodimers. ECADW2AJun/ECADFos (A) and ECADJun/PCADFos heterodimers (B) were subjected to rotary shadowing in the presence of 5 mM Ca2+. Representative fields of the electron micrographs are shown, and individual heterodimers are shown in higher magnification.

To see whether Trp-2 is involved in cis or trans interactions a ECADFos/ECADW2AJun heterodimer was investigated, and the results are depicted in Fig. 6A. Mutating Trp-2 in one of the chains of the heterodimer into an alanine totally abolished trans association of ECAD (Fig. 6A). cis interactions observed as ring-like structures involving CAD1 and 2 are still formed, confirming and extending previous studies with the ECADCOMPW2A mutant (17). A small proportion of W-shaped tetramers was detected for the ECADFos/ECADW2AJun heterodimer (Fig. 6A), perhaps indicating rare events of cis interaction of non-mutated ECAD chains of different dimers.

Finally, analyzing ECADJun/PCADFos heterodimers, neither cis nor trans interactions were detectable (Fig. 6B). Antiparallel head-to-head interactions of monomers were also not observed. These data are in agreement with the strong homophilic specificity of P- and E-cadherin proven by other assays and provide the novel insight that specificity is already determined at the level of cis dimerization.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report we extended the ECADCOMP model system of analyzing interactions of clustered cadherin ectodomains (16). Realizing the potential risk that the oligomerization domain introduced by protein engineering may influence the mode of cadherin interactions due to steric hindrance, different oligomerization domains and different linkers were used. These include the c-Jun and c-Fos leucine zippers, which have been used successfully for heterodimerized protein complexes (38, 40, 41).

For E-cadherin ectodomains dimerized by c-Jun and c-Fos leucine zippers a high proportion of ring-like structures indicating cis interactions was detected at Ca2+ concentrations of around 1 mM. At Ca2+ concentrations above 2 mM trans contacts of two cis dimers are formed confirming and extending our model of cadherin association (16). Our data strongly suggest a contribution of mainly CAD1 of E-cadherin or P-cadherin to cis and trans association. Most importantly, the rings had the same shape as the earlier observed rings in ECADCOMP. Because the dimeric Jun/Fos leucine zipper and the pentameric COMP coiled-coil are very different, an induction of binding by these domains is highly unlikely. It should also be noted that different spacers were used. On the other hand, ectodomains of classic cadherins intrinsically show a slightly bended shape after Ca2+ binding (12, 13). Glycosylation of CAD-domains 3-5 may also prevent a parallel alignment, thus leading to ring-like cis dimers.

The importance of Trp-2 in the adhesive function of classic cadherins has been proven by many researchers (9, 10, 17, 27). Alternatively, it was proposed that Trp-2 is already essential for cis dimer formation (10) or necessary for Ca2+-dependent trans association (17). Our data favor the latter hypothesis, because ECADW2AJun/ECADFos heterodimers are still able to form cis dimers but not trans dimers.

Heterophilic cis interactions of different cadherins have been observed by co-immunoprecipitations of total cell lysates for N- and R-cadherin (27) and for E- and P-cadherin (28). These co-immunoprecipitation experiments do not necessarily describe the nature of the cis contact, because there is the risk that large cadherin complexes connected by catenins and the cytoskeleton are also co-precipitated. On the ultrastructural level we were unable to detect heterophilic lateral contacts of E-cadherin and P-cadherin. The results presented here favor the strong homophilic specificity of these cadherins, which has already been observed in a pioneering publication (19). In addition, domain swapping experiments between E-cadherin and P-cadherin proved that the N-terminal CAD1-domain determined the specificity of cadherin-mediated cell-cell adhesion (23). Our data now suggest that the specificity of cadherin adhesion is already determined at the level of cis dimerization, a feature that will be further defined by site-directed mutagenesis.

In contrast to the above-mentioned finding that the specificity is located in domain 1 and that a point mutation in this domain abolishes adhesion, a ribbon model of multiple adhesive interactions involving several CAD-domains was proposed for C-cadherin (7, 18) and a contribution of the CAD-domains 3-5 has been suggested (11). These results obtained for C-cadherin are hard to reconcile with the formation of rings and associated rings we observe for E-cadherin and P-cadherin, which suggests exclusive interactions between the N-terminal domains. In addition, an instructive study analyzing the zonula adherens in chicken retinal epithelium by cryo-electron microscopic methods recently showed that rods of about ~20 nm in length were extending from the cell surface very much resembling cadherins (42). Cells were connected by head-to-head interactions of these rod-like structures with a total length of ~35-45 nm, whereas the ribbon model suggests an overlap with 22 nm (7, 18).

Several other members of the Ig family of cell adhesion molecules have been analyzed by electron microscopy studies. In one study a conformational shift of the L1 cell adhesion molecule from a compact horseshoe (detected by negative staining) to an elongated form (detected by rotary shadowing) was demonstrated (43). In contrast, for E-cadherin the same kind of associated rings are detected when rotary shadowing is compared with negative staining (16). A homodimeric ICAM1-GCN4 protein chimera has been shown to associate to ring-like structures as well as W-shaped tetramers. This has been interpreted to account for different modes of cis interactions (44). A close parallel alignment of these members of the Ig family of CAMs, which would indicate direct interactions of variable extracellular domains, was not detected by rotary shadowing. Close alignment of Ig-domains nevertheless can be observed by electron microscopy as was shown for the D1-D4 domains of the Ig family member hemolin (43).

In summary, cis and trans association of dimerized E-cadherin or pentamerized P-cadherin involves only the N-terminal domains CAD1 and 2 resulting in ring-like structures formed during cis and trans contacts. No parallel alignment of whole cadherin ectodomains was observed. A decisive role of Trp-2 was found for trans but not for cis interaction of E-cadherin. The specificity of cadherin interactions was proven to be determined at the level of cis dimerization. It will be interesting to define the contact sites in cis and trans association and the residues involved in cadherin specificity in future studies.

    ACKNOWLEDGEMENTS

The cDNAs of the human c-Jun and c-Fos leucine zippers were kindly provided by Johannes Eble (Münster, Germany). We thank Masatoshi Takeichi (Kyoto, Japan) for providing the murine P-cadherin cDNA.

    FOOTNOTES

* This work was supported by the Swiss National Science Foundation (Grant 31-49281.96 to J. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel.: 41-61-267-2250; Fax: 41-61-267-2189; E-mail: Juergen.Engel@unibas.ch.

Published, JBC Papers in Press, March 21, 2002, DOI 10.1074/jbc.M200606200

    ABBREVIATIONS

The abbreviations used are: cadherin, Ca2+-dependent adhesion receptor (CAD); BS3, bis(sulfosuccinimidyl)suberate; cc, coiled-coil; CD, circular dichroism; COMP, cartilage oligomeric matrix protein; EM, electron microscopy; Ig, immunoglobulin; HRP, horse radish peroxidase; LZ, leucine zipper; DMEM, Dulbecco's modified Eagle's medium.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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