Analysis of Heterophilic and Homophilic Interactions of Cadherins Using the c-Jun/c-Fos Dimerization Domains

Thomas Ahrens,Therese Schulthess,Olivier Pertz,Jürgen Engel,Charlotte Fauser,Daniel Häussinger

doi:10.1074/jbc.m200606200

Abstract

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 Ca(2+)-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 Ca(2+) and double-ringed trans contacts above 2 mm Ca(2+). The Ca(2+)-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.

Highlights

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
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
Pentamerization increased the intrinsic concentration of ectodomains to estimated levels (ϳ0.1–2 mM) at which cis and trans interactions occur. cis dimerization was detected in electron micrographs as a ringlike interaction at the N terminus of two ectodomains and trans contacts by subsequent association of two rings

Summary

EXPERIMENTAL PROCEDURES

DNA Constructions—Sequences encoding the leucine zippers of human c-Jun [29] and human c-Fos [30], respectively, were amplified by PCR. PCADFos was 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, 5Ј-GAAGTCGGTACCTGCGGCCGCCACGGG-3Ј and COMP-Strep-Taglow, 5Ј-GACTCACCGCGGTCTAGACTATTTTTCGAACTGTGGGTGACTCCATCCACTTCCCACGCTCAGACC-3Ј. A transfection mix was prepared containing 5 ␮l of LipofectAMINE, 200 ␮l of DMEM F12 without FBS and 1 ␮g of plasmid DNA This mix was incubated for 45 min at room temperature. When confluency of transfected cells was reached on 20-cm plates (Falcon), 23 ml of expression medium (DMEM F12, 1% glutamine, 10 ␮g/ml penicillin/streptomycin) was added for 48 h. Electron Microscopy—Purified cadherin proteins were used at a protein concentration of 1–2 ␮M in 20 mM Tris-HCl, pH 7.4, containing EDTA or CaCl2 as indicated. Five fields were evaluated, resulting in a total of ϳ150 molecules

RESULTS

DISCUSSION