Cadherin conformations associated with dimerization and adhesion.
To investigate conformations of C-cadherin associated with functional activity and physiological regulation, we generated monoclonal antibodies (mAbs) that bind differentially to monomeric or dimeric forms. These mAbs recognize conformational epitopes at multiple sites along the C-cadherin ectodomain aside from the well known Trp-2-mediated dimer interface in the N-terminal EC1 domain. Group 1 mAbs, which bind monomer better than dimer and the Trp-2-mutated protein (W2A) better than wild type, recognize epitopes in EC4 or EC5. Dimerization of the W2A mutant protein via a C-terminal immunoglobulin Fc domain restored the dimeric mAb-binding properties to EC4-5 and partial homophilic binding activity but did not restore full cell adhesion activity. Group 2 and Group 3 mAbs, which bind dimer better than monomer and wild type better than W2A, recognize epitopes in EC1 and the interface between EC1 and EC2, respectively. None of the mAbs could distinguish between different physiological states of C-cadherin at the cell surface of either Xenopus embryonic cells or Colo 205 cultured cells, demonstrating that changes in dimerization do not underlie regulation of adhesion activity. On the cell surface the EC3-EC5 domains are much less accessible to mAb binding than EC1-EC2, suggesting that they are masked by the state of cadherin organization or by other molecules. Thus, the EC2-EC5 domains either reflect, or are involved in, cadherin dimerization and organization at the cell surface.
Tsuiji
Hitomi
H
Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia 22903, USA.
Xu
Liang
L
Schwartz
Kathleen
K
Gumbiner
Barry M
BM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D016466:CHO Cells; D015820:Cadherins / Q000737:chemistry* / Q000276:immunology; D002448:Cell Adhesion / Q000235:genetics / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D009154:Mutation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D014981:Xenopus
D000911:Antibodies, Monoclonal; D015820:Cadherins; D000939:Epitopes