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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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Related in: MedlinePlus

Expression and purification of the mutant cadherin proteins secreted from transfected CHO cells. (A) Western blotting of purified proteins with an anti–human Fc; (B) Coomassie staining of purified proteins on a non reducing gel; (C) Coomassie staining of purified proteins separated by an 8% reducing gel. *Indicate processed mature full-length protein as confirmed by NH2-terminal sequencing.
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fig3: Expression and purification of the mutant cadherin proteins secreted from transfected CHO cells. (A) Western blotting of purified proteins with an anti–human Fc; (B) Coomassie staining of purified proteins on a non reducing gel; (C) Coomassie staining of purified proteins separated by an 8% reducing gel. *Indicate processed mature full-length protein as confirmed by NH2-terminal sequencing.

Mentions: Proteins were stably expressed in CHO cells and were purified from conditioned media on Protein A column. These polypeptides are recognized by an anti–human Fc antibody, demonstrating that the Fc part of the IgG is present (Fig. 3 A), and by anti–C-cadherin antibody (not shown). Assuming these proteins are modified by glycosylation or other posttranslational modifications (Lee and Gumbiner, 1995), the molecular weights are the expected sizes for the mature secreted proteins. We also made sure that those proteins were dimeric by running them on a nonreducing gel (Fig. 3 B). Between 1 and 2 mg of each purified protein were obtained from two liters of conditioned media as determined by Coomassie staining (Fig. 3 C). Minor bands of higher and lower molecular weight than mature full-length protein probably correspond to precursor forms and breakdown products, respectively, since they are recognized by anti–human Fc and by anti–C-cadherin antibodies.


Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

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

Expression and purification of the mutant cadherin proteins secreted from transfected CHO cells. (A) Western blotting of purified proteins with an anti–human Fc; (B) Coomassie staining of purified proteins on a non reducing gel; (C) Coomassie staining of purified proteins separated by an 8% reducing gel. *Indicate processed mature full-length protein as confirmed by NH2-terminal sequencing.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Expression and purification of the mutant cadherin proteins secreted from transfected CHO cells. (A) Western blotting of purified proteins with an anti–human Fc; (B) Coomassie staining of purified proteins on a non reducing gel; (C) Coomassie staining of purified proteins separated by an 8% reducing gel. *Indicate processed mature full-length protein as confirmed by NH2-terminal sequencing.
Mentions: Proteins were stably expressed in CHO cells and were purified from conditioned media on Protein A column. These polypeptides are recognized by an anti–human Fc antibody, demonstrating that the Fc part of the IgG is present (Fig. 3 A), and by anti–C-cadherin antibody (not shown). Assuming these proteins are modified by glycosylation or other posttranslational modifications (Lee and Gumbiner, 1995), the molecular weights are the expected sizes for the mature secreted proteins. We also made sure that those proteins were dimeric by running them on a nonreducing gel (Fig. 3 B). Between 1 and 2 mg of each purified protein were obtained from two liters of conditioned media as determined by Coomassie staining (Fig. 3 C). Minor bands of higher and lower molecular weight than mature full-length protein probably correspond to precursor forms and breakdown products, respectively, since they are recognized by anti–human Fc and by anti–C-cadherin antibodies.

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