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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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Schematic representation of Xenopus C-cadherin and the chimeric Fc fusion proteins. Full-length C-cadherin molecule consists of the ectodomain (five EC domains), transmembrane region (TM) and the cytoplasmic tail (CP). CEC1-5Fc consists of the ectodomain fused to the Fc part of the human IgG (Fc). Domains were expressed as Fc chimaeras to force dimerization, since dimerization of C-cadherin was shown to be crucial for adhesive function. CEC1-4Fc, CEC1-3Fc, and CEC1-2Fc consist of successively fewer number of cadherin repeats fused to Fc at the COOH terminus. CEC3-4-5Fc consists of the ectodomain deleted from the NH2-terminal region fused to Fc at the COOH terminus. CEC1-2FNFc consists of the first two domains fused to the two fibronectin type III repeats of the chicken N-CAM (FN III), still having Fc as the COOH-terminal region. CEC1-2-4Fc consists of successively domains 1-2 and 4 fused to Fc at the COOH terminus and on the same scheme CEC1-2-4-5Fc of domains 1-2 and 4-5.
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fig2: Schematic representation of Xenopus C-cadherin and the chimeric Fc fusion proteins. Full-length C-cadherin molecule consists of the ectodomain (five EC domains), transmembrane region (TM) and the cytoplasmic tail (CP). CEC1-5Fc consists of the ectodomain fused to the Fc part of the human IgG (Fc). Domains were expressed as Fc chimaeras to force dimerization, since dimerization of C-cadherin was shown to be crucial for adhesive function. CEC1-4Fc, CEC1-3Fc, and CEC1-2Fc consist of successively fewer number of cadherin repeats fused to Fc at the COOH terminus. CEC3-4-5Fc consists of the ectodomain deleted from the NH2-terminal region fused to Fc at the COOH terminus. CEC1-2FNFc consists of the first two domains fused to the two fibronectin type III repeats of the chicken N-CAM (FN III), still having Fc as the COOH-terminal region. CEC1-2-4Fc consists of successively domains 1-2 and 4 fused to Fc at the COOH terminus and on the same scheme CEC1-2-4-5Fc of domains 1-2 and 4-5.

Mentions: To examine the contribution of the different extracellular (EC) cadherin domains of C-cadherin, a series of C-cadherin mutants were designed (Fig. 2) . First, we sequentially deleted the EC domains from the COOH terminus according to the described sequence of C-cadherin (Lee and Gumbiner, 1995) and the structures of the cadherin repeats observed by x-ray crystallography (Shapiro et al., 1995; Nagar et al., 1996; Tamura et al., 1998). After analyzing the first constructs, we decided to make additional deletion constructs also shown in Fig. 2. Previous studies on the soluble C-cadherin ectodomain showed that dimerization was necessary for adhesive function (Brieher et al., 1996). During initial attempts to express C-cadherin with EC domain deletions, it was difficult to obtain active dimeric forms (not shown); therefore, chimeras having the IgG Fc domain (Fc) fused to the COOH terminus were constructed to force dimerization (the IgFc domain forms parallel stable disulfide-linked dimers). A similar approach has been used to produce functional soluble dimers of N-cadherin and VE–cadherin (Baumgartner et al., 2000; Lambert et al., 2000) and human E-cadherin (unpublished data). We also made a construct in which a linker was inserted between the COOH terminus of EC1-2 and the IgFc domains, CEC1-2FNFc (Fig. 2). This linker consists of the two fibronectin-like domains founded in the extracellular domain of the chicken N-CAM, each of which is similar in size and in shape to the EC domains and have not been found to have any kind of adhesive activity (Cunningham et al., 1987; Ranheim et al., 1996). Additionally, constructs were also made with deletions of the either the first two NH2-terminal domains (CEC3-4-5Fc) or a deletion of domain 3 (CEC1-2-4Fc and CEC1-2-4-5Fc). We also tried to make a construct having only the EC1 domain fused to Fc (CEC1Fc), but it was poorly expressed and could not be recovered in reasonable quantities.


Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

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

Schematic representation of Xenopus C-cadherin and the chimeric Fc fusion proteins. Full-length C-cadherin molecule consists of the ectodomain (five EC domains), transmembrane region (TM) and the cytoplasmic tail (CP). CEC1-5Fc consists of the ectodomain fused to the Fc part of the human IgG (Fc). Domains were expressed as Fc chimaeras to force dimerization, since dimerization of C-cadherin was shown to be crucial for adhesive function. CEC1-4Fc, CEC1-3Fc, and CEC1-2Fc consist of successively fewer number of cadherin repeats fused to Fc at the COOH terminus. CEC3-4-5Fc consists of the ectodomain deleted from the NH2-terminal region fused to Fc at the COOH terminus. CEC1-2FNFc consists of the first two domains fused to the two fibronectin type III repeats of the chicken N-CAM (FN III), still having Fc as the COOH-terminal region. CEC1-2-4Fc consists of successively domains 1-2 and 4 fused to Fc at the COOH terminus and on the same scheme CEC1-2-4-5Fc of domains 1-2 and 4-5.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Schematic representation of Xenopus C-cadherin and the chimeric Fc fusion proteins. Full-length C-cadherin molecule consists of the ectodomain (five EC domains), transmembrane region (TM) and the cytoplasmic tail (CP). CEC1-5Fc consists of the ectodomain fused to the Fc part of the human IgG (Fc). Domains were expressed as Fc chimaeras to force dimerization, since dimerization of C-cadherin was shown to be crucial for adhesive function. CEC1-4Fc, CEC1-3Fc, and CEC1-2Fc consist of successively fewer number of cadherin repeats fused to Fc at the COOH terminus. CEC3-4-5Fc consists of the ectodomain deleted from the NH2-terminal region fused to Fc at the COOH terminus. CEC1-2FNFc consists of the first two domains fused to the two fibronectin type III repeats of the chicken N-CAM (FN III), still having Fc as the COOH-terminal region. CEC1-2-4Fc consists of successively domains 1-2 and 4 fused to Fc at the COOH terminus and on the same scheme CEC1-2-4-5Fc of domains 1-2 and 4-5.
Mentions: To examine the contribution of the different extracellular (EC) cadherin domains of C-cadherin, a series of C-cadherin mutants were designed (Fig. 2) . First, we sequentially deleted the EC domains from the COOH terminus according to the described sequence of C-cadherin (Lee and Gumbiner, 1995) and the structures of the cadherin repeats observed by x-ray crystallography (Shapiro et al., 1995; Nagar et al., 1996; Tamura et al., 1998). After analyzing the first constructs, we decided to make additional deletion constructs also shown in Fig. 2. Previous studies on the soluble C-cadherin ectodomain showed that dimerization was necessary for adhesive function (Brieher et al., 1996). During initial attempts to express C-cadherin with EC domain deletions, it was difficult to obtain active dimeric forms (not shown); therefore, chimeras having the IgG Fc domain (Fc) fused to the COOH terminus were constructed to force dimerization (the IgFc domain forms parallel stable disulfide-linked dimers). A similar approach has been used to produce functional soluble dimers of N-cadherin and VE–cadherin (Baumgartner et al., 2000; Lambert et al., 2000) and human E-cadherin (unpublished data). We also made a construct in which a linker was inserted between the COOH terminus of EC1-2 and the IgFc domains, CEC1-2FNFc (Fig. 2). This linker consists of the two fibronectin-like domains founded in the extracellular domain of the chicken N-CAM, each of which is similar in size and in shape to the EC domains and have not been found to have any kind of adhesive activity (Cunningham et al., 1987; Ranheim et al., 1996). Additionally, constructs were also made with deletions of the either the first two NH2-terminal domains (CEC3-4-5Fc) or a deletion of domain 3 (CEC1-2-4Fc and CEC1-2-4-5Fc). We also tried to make a construct having only the EC1 domain fused to Fc (CEC1Fc), but it was poorly expressed and could not be recovered in reasonable quantities.

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