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Published 9 July 2001. doi:10.1083/jcb.200103143
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© The Rockefeller University Press, 0021-9525/2001/7/231 $5.00
The Journal of Cell Biology, Volume 154, Number 1, July 9, 2001 231-243

Article

 Multiple cadherin extracellular repeats mediate homophilic binding and adhesion

Sophie Chappuis-Flament1, Ellen Wong1, Les D. Hicks2, Cyril M. Kay2 and Barry M. Gumbiner1

1 Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021
2 Protein Engineering Network of Centres of Excellence, Deptartment of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Address correspondence to Barry M. Gumbiner, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 564, New York, NY 10021. Tel.: (212) 639-6146. Fax: (212) 717-3047. E-mail: b-gumbiner{at}ski.mskcc.org

The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1–EC5). Studies on cadherin specificity have implicated the NH2-terminal EC1 domain in the homophilic binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding.

Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer–dimer equilibrium with an affinity constant of ~64 µM. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity.

The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment–based adhesion assay. A protein with only the first two NH2-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W. Brieher, N. Lavrik, B. Gumbiner, and D. Leckband. 1999. Proc. Natl. Acad. Sci. USA. 96:11820–11824; Sivasankar, S., B. Gumbiner, and D. Leckband. 2001. Biophys J. 80:1758–68). We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.

Key Words: C-cadherin; dimerization; homophilic binding; adhesion; structure


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