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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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Sedimentation equilibrium analysis of the soluble C-cadherin ectodomain (CEC1-5). (A) Sedimentation equilibrium data. (Bottom) Show global fit of data collected at six loading concentrations ranging from 2–25 μM and rotor speeds of 10,000 rpm (○) and 14,000 rpm (□) to a monomer–dimer self association model. Symbols represent measured data points, and solid lines represent theoretical fits to the model. (Top) Illustrates the deviations of the measured points from the theoretical fit lines. (B) Plots illustrating expected fractions of monomer (solid line) and dimer (dashed line) at different CEC1-5 concentrations, calculated using the Kd(1-2) of 64 μM determined by the global fit of the sedimentation equilibrium data to a monomer–dimer association model.
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fig1: Sedimentation equilibrium analysis of the soluble C-cadherin ectodomain (CEC1-5). (A) Sedimentation equilibrium data. (Bottom) Show global fit of data collected at six loading concentrations ranging from 2–25 μM and rotor speeds of 10,000 rpm (○) and 14,000 rpm (□) to a monomer–dimer self association model. Symbols represent measured data points, and solid lines represent theoretical fits to the model. (Top) Illustrates the deviations of the measured points from the theoretical fit lines. (B) Plots illustrating expected fractions of monomer (solid line) and dimer (dashed line) at different CEC1-5 concentrations, calculated using the Kd(1-2) of 64 μM determined by the global fit of the sedimentation equilibrium data to a monomer–dimer association model.

Mentions: Calculated apparent weight average molecular weights from individual sedimentation equilibrium data sets collected over a loading concentration range of 2–25 μM varied from 78,570 to 102,660, whereas an apparent weight average molecular weight of 88,850 was determined from a global fit of all the data sets to a single species model. The best global fit was obtained for a monomer–dimer self-association model, using an assumed value of 75,000 for the monomer molecular weight, and allowing the Ka for each data set to float (Fig. 1 A). There was no obvious concentration-dependent trend in the determined Kas for the various data sets, which would have indicated possible heterogeneity or non-specific aggregation. Averaging all the individual raw Ka values resulted in a calculated Molar Kd(1-2) of 64 μM. Using this value, the CEC1-5 appears to consist of ∼5–30% dimer in the concentration range at which the measurements were performed (Fig. 1 B).


Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

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

Sedimentation equilibrium analysis of the soluble C-cadherin ectodomain (CEC1-5). (A) Sedimentation equilibrium data. (Bottom) Show global fit of data collected at six loading concentrations ranging from 2–25 μM and rotor speeds of 10,000 rpm (○) and 14,000 rpm (□) to a monomer–dimer self association model. Symbols represent measured data points, and solid lines represent theoretical fits to the model. (Top) Illustrates the deviations of the measured points from the theoretical fit lines. (B) Plots illustrating expected fractions of monomer (solid line) and dimer (dashed line) at different CEC1-5 concentrations, calculated using the Kd(1-2) of 64 μM determined by the global fit of the sedimentation equilibrium data to a monomer–dimer association model.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Sedimentation equilibrium analysis of the soluble C-cadherin ectodomain (CEC1-5). (A) Sedimentation equilibrium data. (Bottom) Show global fit of data collected at six loading concentrations ranging from 2–25 μM and rotor speeds of 10,000 rpm (○) and 14,000 rpm (□) to a monomer–dimer self association model. Symbols represent measured data points, and solid lines represent theoretical fits to the model. (Top) Illustrates the deviations of the measured points from the theoretical fit lines. (B) Plots illustrating expected fractions of monomer (solid line) and dimer (dashed line) at different CEC1-5 concentrations, calculated using the Kd(1-2) of 64 μM determined by the global fit of the sedimentation equilibrium data to a monomer–dimer association model.
Mentions: Calculated apparent weight average molecular weights from individual sedimentation equilibrium data sets collected over a loading concentration range of 2–25 μM varied from 78,570 to 102,660, whereas an apparent weight average molecular weight of 88,850 was determined from a global fit of all the data sets to a single species model. The best global fit was obtained for a monomer–dimer self-association model, using an assumed value of 75,000 for the monomer molecular weight, and allowing the Ka for each data set to float (Fig. 1 A). There was no obvious concentration-dependent trend in the determined Kas for the various data sets, which would have indicated possible heterogeneity or non-specific aggregation. Averaging all the individual raw Ka values resulted in a calculated Molar Kd(1-2) of 64 μM. Using this value, the CEC1-5 appears to consist of ∼5–30% dimer in the concentration range at which the measurements were performed (Fig. 1 B).

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