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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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Two hypothetical models for role of EC1 domain in determining cadherin binding specificity. (A) Cis-dimerization specificity could influence adhesive binding specificity. Calcium binding causes the ectodomain to behave as a single structural unit. Therefore, alterations in the orientation of the EC1 domain dimerization interface are propagated to the binding sites throughout the rest of the EC domains. (B) An initial cadherin specific interaction between EC1 domains could precede the formation of the final homophilic bonds between the other EC domains. A repulsive barrier between cells would be postulated to prevent interactions between EC2-5 from occurring directly, and an initial weak binding between EC1 domains would lower the energy barrier leading to the final binding state.
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fig10: Two hypothetical models for role of EC1 domain in determining cadherin binding specificity. (A) Cis-dimerization specificity could influence adhesive binding specificity. Calcium binding causes the ectodomain to behave as a single structural unit. Therefore, alterations in the orientation of the EC1 domain dimerization interface are propagated to the binding sites throughout the rest of the EC domains. (B) An initial cadherin specific interaction between EC1 domains could precede the formation of the final homophilic bonds between the other EC domains. A repulsive barrier between cells would be postulated to prevent interactions between EC2-5 from occurring directly, and an initial weak binding between EC1 domains would lower the energy barrier leading to the final binding state.

Mentions: One theoretical model for which cis-dimerization specificity could lead to adhesive binding specificity is shown in Fig. 10 A. The model also depends on another documented structural feature of cadherins, the linking of successive EC domains together via calcium binding sites to form a rigid rod-like protein. Because of this property, we postulate that the entire ectodomain behaves as a single structural unit, and any alterations in the orientations of the EC1 domain dimer interface will be propagated throughout the rest of the EC domains. Thus small differences in the relative orientations of the EC1 dimerization interfaces for different cadherins would alter the orientations of other putative adhesive binding sites in the other EC domains (shown as large changes for emphasis), resulting in less compatible binding and/or in a reduced ability to form an extended two-dimensional lattice. Other models to explain how EC1 could determine adhesion specificity when other EC domains contribute to homophilic binding are also possible. For example, in the model shown in Fig. 10 B, an initial cadherin-specific interaction between EC1 domains could precede the formation of the final homophilic bonds between the other EC domains. For this to make sense physically, there would have to be some sort of repulsive barrier between cells to prevent interactions between EC2-5 from occurring directly, and an initial weak binding between EC1 domains would lower the energy barrier leading to the final binding state. For either of these models, there would be no cadherin-type specificity in the homophilic binding interactions between EC domains 2–5, which is consistent with the low adhesion specificity observed for many pairs of different cadherins. Irrespective of whether either of these two theoretical models is correct, this theoretical exercise demonstrates that determination of cadherin adhesion specificity by EC1 can be compatible with the participation of EC domains 2–5 in the homophilic binding interactions.


Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

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

Two hypothetical models for role of EC1 domain in determining cadherin binding specificity. (A) Cis-dimerization specificity could influence adhesive binding specificity. Calcium binding causes the ectodomain to behave as a single structural unit. Therefore, alterations in the orientation of the EC1 domain dimerization interface are propagated to the binding sites throughout the rest of the EC domains. (B) An initial cadherin specific interaction between EC1 domains could precede the formation of the final homophilic bonds between the other EC domains. A repulsive barrier between cells would be postulated to prevent interactions between EC2-5 from occurring directly, and an initial weak binding between EC1 domains would lower the energy barrier leading to the final binding state.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: Two hypothetical models for role of EC1 domain in determining cadherin binding specificity. (A) Cis-dimerization specificity could influence adhesive binding specificity. Calcium binding causes the ectodomain to behave as a single structural unit. Therefore, alterations in the orientation of the EC1 domain dimerization interface are propagated to the binding sites throughout the rest of the EC domains. (B) An initial cadherin specific interaction between EC1 domains could precede the formation of the final homophilic bonds between the other EC domains. A repulsive barrier between cells would be postulated to prevent interactions between EC2-5 from occurring directly, and an initial weak binding between EC1 domains would lower the energy barrier leading to the final binding state.
Mentions: One theoretical model for which cis-dimerization specificity could lead to adhesive binding specificity is shown in Fig. 10 A. The model also depends on another documented structural feature of cadherins, the linking of successive EC domains together via calcium binding sites to form a rigid rod-like protein. Because of this property, we postulate that the entire ectodomain behaves as a single structural unit, and any alterations in the orientations of the EC1 domain dimer interface will be propagated throughout the rest of the EC domains. Thus small differences in the relative orientations of the EC1 dimerization interfaces for different cadherins would alter the orientations of other putative adhesive binding sites in the other EC domains (shown as large changes for emphasis), resulting in less compatible binding and/or in a reduced ability to form an extended two-dimensional lattice. Other models to explain how EC1 could determine adhesion specificity when other EC domains contribute to homophilic binding are also possible. For example, in the model shown in Fig. 10 B, an initial cadherin-specific interaction between EC1 domains could precede the formation of the final homophilic bonds between the other EC domains. For this to make sense physically, there would have to be some sort of repulsive barrier between cells to prevent interactions between EC2-5 from occurring directly, and an initial weak binding between EC1 domains would lower the energy barrier leading to the final binding state. For either of these models, there would be no cadherin-type specificity in the homophilic binding interactions between EC domains 2–5, which is consistent with the low adhesion specificity observed for many pairs of different cadherins. Irrespective of whether either of these two theoretical models is correct, this theoretical exercise demonstrates that determination of cadherin adhesion specificity by EC1 can be compatible with the participation of EC domains 2–5 in the homophilic binding interactions.

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