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

Mixed bead aggregation assay to assess homophilic binding activity between different cadherin mutants. Flow cytometry was used to detect and quantify mixed aggregates formed between yellow fluorescent beads (Y) and red fluorescent beads (R) coated with different cadherin EC constructs. Mixed aggregates appear in the region to the right of and above the lines drawn on the graph. (Yellow only singlets and small aggregates appear in the lower right region, but red only singlets and small aggregates do not appear on the graph because they lie on the y axis.) (A) Analysis of aggregation between CEC1-5Fc on both sets of beads. (B) Analysis of aggregation between CEC1-2Fc on both sets of beads. (C) Analysis of aggregation between CEC3-4-5FC on both sets of beads. (D) Analysis of aggregation between CEC1-2Fc–coated yellow beads and CEC3-4-5FC–coated red beads.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196848&req=5

fig8: Mixed bead aggregation assay to assess homophilic binding activity between different cadherin mutants. Flow cytometry was used to detect and quantify mixed aggregates formed between yellow fluorescent beads (Y) and red fluorescent beads (R) coated with different cadherin EC constructs. Mixed aggregates appear in the region to the right of and above the lines drawn on the graph. (Yellow only singlets and small aggregates appear in the lower right region, but red only singlets and small aggregates do not appear on the graph because they lie on the y axis.) (A) Analysis of aggregation between CEC1-5Fc on both sets of beads. (B) Analysis of aggregation between CEC1-2Fc on both sets of beads. (C) Analysis of aggregation between CEC3-4-5FC on both sets of beads. (D) Analysis of aggregation between CEC1-2Fc–coated yellow beads and CEC3-4-5FC–coated red beads.

Mentions: Since domains 1 and 2 are required but not sufficient for homophilic binding activity, it is possible that domains 1 and 2 need to bind to EC domains 3, 4, or 5 in the full C-cadherin ectodomain. To try to test this possibility, bead mixing experiments were performed. A flow cytometry assay with different color fluorescent beads (yellow and red) was used to determine whether CEC1-2Fc–coated beads and CEC3-4-5Fc–coated beads aggregate better with each other than they do by themselves (Fig. 8 ; Table I). A positive control for the assay is shown by an analysis of mixed aggregates formed by two sets of beads coated with full-length C-cadherin (CEC1-5Fc) in Fig. 8 A. Aggregates containing both fluorescent colors (yellow and red) appear along the diagonal of the fluorescence intensity graph. The formation of mixed aggregates was quite extensive at this time in the assay, since each point on the graph is a single fluorescent event that corresponds to a single aggregate, each of which can contain a large number of beads. For CEC1-5Fc, >90% of the detected events and ∼790,000 beads are present in mixed aggregates (Table I). As expected, mixed aggregates between two sets of beads, which both contained CEC1-2Fc, were smaller and fewer (Fig. 8 B). Only 27% of the events contained mixed aggregates (i.e., 73% were either single beads or small unmixed aggregates), with only ∼28,000 beads present in mixed aggregates (Table I). The negative control (i.e., background) is shown by analysis of CEC3-4-5Fc by itself, which formed even fewer and smaller mixed aggregates (Fig. 8 C), with <10% of events in mixed aggregates (i.e., >90% single or small unmixed) and <5,000 beads present in mixed aggregates (Table I). The experimental analysis of the mixing between CEC1-2Fc beads and CEC3-4-5Fc beads is shown in Fig. 8 D (one case) and Table I (both the case in Fig. 8 D and the reciprocal mixture). These samples formed mixed aggregates no better than CEC3-4-5Fc alone, and worse than CEC1-2Fc alone. Therefore, using this assay for mixed aggregation, it was not possible to detect a direct binding interaction between EC domains 1-2 and domains 3, 4, and 5.


Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

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

Mixed bead aggregation assay to assess homophilic binding activity between different cadherin mutants. Flow cytometry was used to detect and quantify mixed aggregates formed between yellow fluorescent beads (Y) and red fluorescent beads (R) coated with different cadherin EC constructs. Mixed aggregates appear in the region to the right of and above the lines drawn on the graph. (Yellow only singlets and small aggregates appear in the lower right region, but red only singlets and small aggregates do not appear on the graph because they lie on the y axis.) (A) Analysis of aggregation between CEC1-5Fc on both sets of beads. (B) Analysis of aggregation between CEC1-2Fc on both sets of beads. (C) Analysis of aggregation between CEC3-4-5FC on both sets of beads. (D) Analysis of aggregation between CEC1-2Fc–coated yellow beads and CEC3-4-5FC–coated red beads.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Mixed bead aggregation assay to assess homophilic binding activity between different cadherin mutants. Flow cytometry was used to detect and quantify mixed aggregates formed between yellow fluorescent beads (Y) and red fluorescent beads (R) coated with different cadherin EC constructs. Mixed aggregates appear in the region to the right of and above the lines drawn on the graph. (Yellow only singlets and small aggregates appear in the lower right region, but red only singlets and small aggregates do not appear on the graph because they lie on the y axis.) (A) Analysis of aggregation between CEC1-5Fc on both sets of beads. (B) Analysis of aggregation between CEC1-2Fc on both sets of beads. (C) Analysis of aggregation between CEC3-4-5FC on both sets of beads. (D) Analysis of aggregation between CEC1-2Fc–coated yellow beads and CEC3-4-5FC–coated red beads.
Mentions: Since domains 1 and 2 are required but not sufficient for homophilic binding activity, it is possible that domains 1 and 2 need to bind to EC domains 3, 4, or 5 in the full C-cadherin ectodomain. To try to test this possibility, bead mixing experiments were performed. A flow cytometry assay with different color fluorescent beads (yellow and red) was used to determine whether CEC1-2Fc–coated beads and CEC3-4-5Fc–coated beads aggregate better with each other than they do by themselves (Fig. 8 ; Table I). A positive control for the assay is shown by an analysis of mixed aggregates formed by two sets of beads coated with full-length C-cadherin (CEC1-5Fc) in Fig. 8 A. Aggregates containing both fluorescent colors (yellow and red) appear along the diagonal of the fluorescence intensity graph. The formation of mixed aggregates was quite extensive at this time in the assay, since each point on the graph is a single fluorescent event that corresponds to a single aggregate, each of which can contain a large number of beads. For CEC1-5Fc, >90% of the detected events and ∼790,000 beads are present in mixed aggregates (Table I). As expected, mixed aggregates between two sets of beads, which both contained CEC1-2Fc, were smaller and fewer (Fig. 8 B). Only 27% of the events contained mixed aggregates (i.e., 73% were either single beads or small unmixed aggregates), with only ∼28,000 beads present in mixed aggregates (Table I). The negative control (i.e., background) is shown by analysis of CEC3-4-5Fc by itself, which formed even fewer and smaller mixed aggregates (Fig. 8 C), with <10% of events in mixed aggregates (i.e., >90% single or small unmixed) and <5,000 beads present in mixed aggregates (Table I). The experimental analysis of the mixing between CEC1-2Fc beads and CEC3-4-5Fc beads is shown in Fig. 8 D (one case) and Table I (both the case in Fig. 8 D and the reciprocal mixture). These samples formed mixed aggregates no better than CEC3-4-5Fc alone, and worse than CEC1-2Fc alone. Therefore, using this assay for mixed aggregation, it was not possible to detect a direct binding interaction between EC domains 1-2 and domains 3, 4, and 5.

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