EphA2 and Ephrin-A1 Utilize the Same Interface for Both and Interactions That Differentially Regulate Cell Signaling and Function.

The 14 members of Eph receptor tyrosine kinases (RTK) bind to membrane-tethered ligand called ephrins and mediate cell contact signaling where the receptors and ligands engage on adjacent cells. Previous studies reveal that some Eph and ephrin pairs are coexpressed on the same cells, including EphA3-ephrin-A3 and EphA4/ephrin-A5, can also interact with each other . However, significant discrepancies persist as to the molecular basis and functional significance of the interactions, owning to the difficulties to directly interrogate the interactions. Here, we utilize time-resolved live cell fluorescence spectroscopy to demonstrate direct interactions between EphA2 and ephrin-A1. Structure-guided mutagenesis mapped interactions to two salt bridges between the ligand- and receptor-binding domains of EphA2 and ephrin-A1. Interestingly, the same interface is shared with interaction. Consequently, EphA2-ephrin-A1 interaction competes with their interaction , which leads to attenuation of EphA2 canonical signaling and inhibition of cell rounding when ligand is presented . EphA2 and ephrin-A1 are widely coexpressed in many epithelial tissues, and dysregulation of their expression is known to contribute to tumor initiation and progression. The detailed molecular characterization of the mutually exclusive and interactions uncovers a new mechanism underpinning their unique roles in oncogenesis.