Limits...
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.

Show MeSH

Related in: MedlinePlus

Lack of adhesion activity of a construct lacking EC domains 1 and 2 (CEC3-4-5Fc) by laminar flow assay. Attachment of C-CHO cells to high concentrations of CEC3-4-5Fc (100 μg/μl) compared with CEC1-5Fc. The experiment was performed in triplicate and the mean ± SEM is shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196848&req=5

fig7: Lack of adhesion activity of a construct lacking EC domains 1 and 2 (CEC3-4-5Fc) by laminar flow assay. Attachment of C-CHO cells to high concentrations of CEC3-4-5Fc (100 μg/μl) compared with CEC1-5Fc. The experiment was performed in triplicate and the mean ± SEM is shown.

Mentions: Although EC1 and EC2 are not sufficient for effective binding activity, we wanted to test whether they are required. Therefore, we analyzed whether a construct lacking domains 1 and 2, CEC3-4-5Fc, retains bead aggregation and cell adhesion activity. Most preparations of CEC3-4-5Fc (70%) failed to induce detectable bead aggregation (not shown). In ∼30% of the preparations, there was some evidence of aggregation, but it was highly variable and irreproducible from day to day. Furthermore, CEC3-4-5Fc never exhibited detectable cell adhesion activity in the flow assay (Fig. 7) , irrespective of the preparation or day of experiment. The lack of activity in the cell adhesion assay probably cannot be attributed to the variability in the NH2-terminal propeptide cleavage that we observed because there was no detectable adhesion even at very high concentrations of the protein (100 μg/μl), which is 20 times more than needed for strong adhesion to CEC1-3Fc or for low but detectable adhesion to CEC1-2Fc. Thus, even if domains EC3, EC4, and EC5 possess some binding activity, domains 1 and 2 appear to be required for effective homophilic binding and cell adhesion. The requirement for EC1 and EC2 could be due either to the presence of a critical binding site in one or both of these two domains or to the requirement for the EC1 domain in the formation of normal cadherin cis-dimers (Shan et al., 2000). Although CEC3-4-5Fc dimerize through the COOH-terminal Fc domain in the absence of EC1, it may be ineffective in creating the proper protein conformation and/or dimeric binding interface.


Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

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

Lack of adhesion activity of a construct lacking EC domains 1 and 2 (CEC3-4-5Fc) by laminar flow assay. Attachment of C-CHO cells to high concentrations of CEC3-4-5Fc (100 μg/μl) compared with CEC1-5Fc. The experiment was performed in triplicate and the mean ± SEM is shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Lack of adhesion activity of a construct lacking EC domains 1 and 2 (CEC3-4-5Fc) by laminar flow assay. Attachment of C-CHO cells to high concentrations of CEC3-4-5Fc (100 μg/μl) compared with CEC1-5Fc. The experiment was performed in triplicate and the mean ± SEM is shown.
Mentions: Although EC1 and EC2 are not sufficient for effective binding activity, we wanted to test whether they are required. Therefore, we analyzed whether a construct lacking domains 1 and 2, CEC3-4-5Fc, retains bead aggregation and cell adhesion activity. Most preparations of CEC3-4-5Fc (70%) failed to induce detectable bead aggregation (not shown). In ∼30% of the preparations, there was some evidence of aggregation, but it was highly variable and irreproducible from day to day. Furthermore, CEC3-4-5Fc never exhibited detectable cell adhesion activity in the flow assay (Fig. 7) , irrespective of the preparation or day of experiment. The lack of activity in the cell adhesion assay probably cannot be attributed to the variability in the NH2-terminal propeptide cleavage that we observed because there was no detectable adhesion even at very high concentrations of the protein (100 μg/μl), which is 20 times more than needed for strong adhesion to CEC1-3Fc or for low but detectable adhesion to CEC1-2Fc. Thus, even if domains EC3, EC4, and EC5 possess some binding activity, domains 1 and 2 appear to be required for effective homophilic binding and cell adhesion. The requirement for EC1 and EC2 could be due either to the presence of a critical binding site in one or both of these two domains or to the requirement for the EC1 domain in the formation of normal cadherin cis-dimers (Shan et al., 2000). Although CEC3-4-5Fc dimerize through the COOH-terminal Fc domain in the absence of EC1, it may be ineffective in creating the proper protein conformation and/or dimeric binding interface.

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