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Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

Chappuis-Flament S, Wong E, Hicks LD, Kay CM, Gumbiner BM - J. Cell Biol. (2001)

Bottom Line: A protein with only the first two NH(2)-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.These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W.Biophys J. 80:1758-68).

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA.

ABSTRACT
The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH(2)-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 approximately 64 microm. 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 NH(2)-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.

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Model of adhesive bond formation via the extracellular cadherin repeats. (A) Binding via multiple EC domains, as demonstrated in this study. If binding sites have different orientations, cadherin dimers could form two-dimensional lattices (not shown). (B) Linear zipper model for the homophilic bond via the EC1 domains alone, as proposed in Shapiro et al. (1995).
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fig9: Model of adhesive bond formation via the extracellular cadherin repeats. (A) Binding via multiple EC domains, as demonstrated in this study. If binding sites have different orientations, cadherin dimers could form two-dimensional lattices (not shown). (B) Linear zipper model for the homophilic bond via the EC1 domains alone, as proposed in Shapiro et al. (1995).

Mentions: A thorough structure–function analysis of the homophilic binding properties of the soluble C-cadherin ectodomain reveals that multiple cadherin EC repeats contribute to a low affinity interaction between cadherin cis-dimers. Although the EC1 domain appears to be required for the formation of an effective adhesive bond, perhaps due to its role in cis-dimerization, it cannot account for the entire homophilic binding interaction as has been previously believed. A minimum of three of the EC domains is required for effective homophilic binding and adhesion, since domains EC1-2 are not sufficient. Although domains EC4 and EC5 do not seem to be absolutely required, they can contribute to the binding interaction. CEC1-4Fc and CEC1-5Fc do exhibit a somewhat better binding activity than CEC1-3Fc. Moreover, EC3 is not specifically required for binding, and EC4 is able to substitute for EC3, since CEC1-2-4-5Fc and CEC1-2-4Fc have high binding and adhesion activity. Together, these findings suggest that the homophilic bond formed between cadherins involves extensive overlap between the extracellular domains and may arise from multiple interactions or different combinations of interactions between EC domains (Fig. 9 A).


Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

Chappuis-Flament S, Wong E, Hicks LD, Kay CM, Gumbiner BM - J. Cell Biol. (2001)

Model of adhesive bond formation via the extracellular cadherin repeats. (A) Binding via multiple EC domains, as demonstrated in this study. If binding sites have different orientations, cadherin dimers could form two-dimensional lattices (not shown). (B) Linear zipper model for the homophilic bond via the EC1 domains alone, as proposed in Shapiro et al. (1995).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196848&req=5

fig9: Model of adhesive bond formation via the extracellular cadherin repeats. (A) Binding via multiple EC domains, as demonstrated in this study. If binding sites have different orientations, cadherin dimers could form two-dimensional lattices (not shown). (B) Linear zipper model for the homophilic bond via the EC1 domains alone, as proposed in Shapiro et al. (1995).
Mentions: A thorough structure–function analysis of the homophilic binding properties of the soluble C-cadherin ectodomain reveals that multiple cadherin EC repeats contribute to a low affinity interaction between cadherin cis-dimers. Although the EC1 domain appears to be required for the formation of an effective adhesive bond, perhaps due to its role in cis-dimerization, it cannot account for the entire homophilic binding interaction as has been previously believed. A minimum of three of the EC domains is required for effective homophilic binding and adhesion, since domains EC1-2 are not sufficient. Although domains EC4 and EC5 do not seem to be absolutely required, they can contribute to the binding interaction. CEC1-4Fc and CEC1-5Fc do exhibit a somewhat better binding activity than CEC1-3Fc. Moreover, EC3 is not specifically required for binding, and EC4 is able to substitute for EC3, since CEC1-2-4-5Fc and CEC1-2-4Fc have high binding and adhesion activity. Together, these findings suggest that the homophilic bond formed between cadherins involves extensive overlap between the extracellular domains and may arise from multiple interactions or different combinations of interactions between EC domains (Fig. 9 A).

Bottom Line: A protein with only the first two NH(2)-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.These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W.Biophys J. 80:1758-68).

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA.

ABSTRACT
The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH(2)-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 approximately 64 microm. 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 NH(2)-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.

Show MeSH
Related in: MedlinePlus