LATERAL DIMERIZATION IS REQUIRED FOR THE HEMOPHILIC BINDING-ACTIVITY OF C-CADHERIN

Regulation of cadherin-mediated adhesion can occur rapidly at the cell surface. To understand the mechanism underlying cadherin regulation, it is essential to elucidate the hemophilic binding mechanism that und erlies all cadherin-mediated functions. Therefore, we have investigate d the structural and functional properties of the extracellular segmen t of Xenopus C-cadherin using a purified, recombinant protein (CEC 1-5 ). CEC 1-5 supported adhesion of CHO cells expressing C-cadherin. The extracellular segment was also capable of mediating aggregation of mic rospheres. Chemical cross-linking and gel filtration revealed that CEC 1-5 formed dimers in the presence as well as absence of calcium. Anal ysis of the functional activity of purified dimers and monomers demons trated that dimers retained substantially greater hemophilic binding a ctivity than monomers. These results demonstrate that lateral dimeriza tion is necessary for hemophilic binding between cadherin extracellula r segments and suggest multiple potential mechanisms for the regulatio n of cadherin activity. Since the extracellular segment alone possesse d significant hemophilic binding activity, the adhesive activity of th e extracellular segment in a cellular context was analyzed. The adhesi on of CHO cells expressing a truncated version of C-cadherin lacking t he cytoplasmic tail was compared to cells expressing the wild-type C-c adherin using a laminar flow assay on substrates coated with CEC 1-5. CHO cells expressing the truncated C-cadherin were able to attach to C EC 1-5 and to resist detachment by low shear forces, demonstrating tha t tailless C-cadherin can mediate basic, weak adhesion of CHO cells. H owever, cells expressing the truncated C-cadherin did not exhibit the complete adhesive activity of cells expressing wild-type C-cadherin. C ells expressing wild-type C-cadherin remained attached to CEC 1-5 at h igh shear forces, while cells expressing the tailless C-cadherin did n ot adhere well at high shear forces. These results suggest that there may be two states of cadherin-mediated adhesion. The first, relatively weak state can be mediated through interactions between the extracell ular segments alone. The second strong adhesive state is critically de pendent on the cytoplasmic tail.