Journal
JOURNAL OF CELL BIOLOGY
Volume 154, Issue 1, Pages 231-243Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1083/jcb.200103143
Keywords
C-cadherin; dimerization; hemophilic binding; adhesion; structure
Categories
Funding
- NCI NIH HHS [P30-CA-08784] Funding Source: Medline
- NIGMS NIH HHS [R01 GM052717, GM52717] Funding Source: Medline
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The extracellular hemophilic-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 hemophilic 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 hemophilic 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 similar to 64 muM. No higher order oligomers were detected, indicating that hemophilic binding between cis-dimers is of significantly lower affinity. The hemophilic 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 hemophilic binding. CEC1-3 Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5 Fc. EC3 is not required for hemophilic 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 hemophilic 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.
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