EphA2 and Ephrin-A1 Use the Same Interface for Both in cis and in trans Interactions That Regulate Distinct 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 in trans 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 in cis. However, significant discrepancies persist as to the molecular basis and functional significance of the cis interactions, owning to the difficulties to directly interrogate the interactions. Here, we utilize time-resolved live cell fluorescence spectroscopy to demonstrate direct in cis 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 in trans interaction. Consequently, EphA2-ephrin-A1 interaction in cis competes with their interaction in trans, which leads to attenuation of EphA2 canonical signaling and inhibition of cell rounding when ligand is presented in trans. 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 cis and trans interactions uncovers a new mechanism underpinning their unique roles in oncogenesis. Significance StatementEphA2 exerts dual functions in tumorigenesis, depending on the binding status of its membrane-tethered ephrin-A ligands. Ligands presented in trans on adjacent cells activate EphA2 canonical signaling associated with tumor suppression, whereas loss of ligand expression promotes oncogenic noncanonical signaling of EphA2 via serine 897 phosphorylation. Combining time-resolved spectroscopy in live cells, structure-guided mutagenesis, we show strong in cis interactions between EphA2 and ephrin-A1, which shares the same interface as interaction in trans. Moreover, the cis interaction interferes with ligand binding in trans, attenuates EphA2 canonical signaling. Our results uncover a new mechanism of EphA2 regulation by its co-expressed ligand ephrin-A1 with important implications in its known roles in oncogenesis as well as other disease processes including development of cataract.