Structurally encoded intraclass differences in EphA clusters drive distinct cell responses

The functional outcomes of ephrin binding to Eph receptors (Ephs) range from cell repulsion to adhesion. Here we used cell collapse and stripe assays to show contrasting effects of human ephrinA5 binding to EphA2 and EphA4. Despite equivalent ligand-binding affinities EphA4 triggered greater cell collapse, while EphA2-expressing cells adhered better to ephrinA5-coated surfaces. Chimeric receptors showed the ectodomain is a major determinant of cell response. We report crystal structures of EphA4 ectodomain alone and in complexes with ephrinB3 and ephrinA5. These revealed closed clusters with a dimeric or circular arrangement in the crystal lattice, contrasting with extended arrays previously observed for EphA2 ectodomain. Localization microscopy-based analyses showed ligand-stimulated EphA4 induces smaller clusters than EphA2. Mutant Ephs link these characteristics to interactions observed in the crystal lattices, suggesting a mechanism by which distinctive ectodomain surfaces determine clustering, and thereby signalling, properties.