HEMOPHILIC ADHESION OF E-CADHERIN OCCURS BY A COOPERATIVE 2-STEP INTERACTION OF N-TERMINAL DOMAINS

Cluster formation of E-cadherin on the cell surface is believed to be of major importance for cell-cell adhesion. To mimic this process the extracellular part of mouse E-cadherin (ECAD) was recombinantly fused to the assembly domain of rat cartilage oligomeric matrix protein (COM P), resulting in the chimeric protein ECAD-COMP. The COMP domain forme d a five-stranded alpha-helical coiled-coil, This enabled the formatio n of a pentameric ECAD with bundled C-termini and free N-termini. The pentameric protein construct ECAD-COMP and the monomeric ECAD were exp ressed in human embryonal kidney 293 cells. Electron microscopy, analy tical ultracentrifugation, solid phase binding and cell attachment ass ays revealed that pentamers showed strong self-association and cell at tachment, whereas monomers exhibited no activity. At the high internal concentration in the pentamer the N-terminal EC1 domains of two E-cad herin arms interact to form a ring-like structure. Then the paired dom ains interact with a corresponding pair from another pentamer. None of the four other extracellular domains of E-cadherin is involved in thi s interaction. Based on these results, an in vivo mechanism is propose d whereby two N-terminal domains of neighbouring E-cadherins at the ce ll surface first form a pair, which binds with high affinity to a simi lar complex on another cell. The strong dependence of hemophilic inter actions on C-terminal clustering points towards a regulation of E-cadh erin mediated cell-cell adhesion via lateral association.