Ligand preference of EphB2 receptor is selectively regulated by N-glycosylation

The Eph receptors and their ephrin ligands play important roles in cell communication and neuron development. Eph interacts with ephrin in a complex manner. Here, we found ephrin-B2 instead of well-recorded ephrin-A5 specifically recognize and activate EphB2 receptor in primary cortical neurons. Domain-swapping and N/Q mutagenesis results show that the ectodomain of EphB2 and its N-glycosylation sites are critical for the ephrin binding selectivity. The N265, N336, N428, and N482Q mutant EphB2 cannot distinguish ephrin-B2 from ephrin-A5. Furthermore, the N-glycosylation sites in EphB2 are evolutionarily conserved and the N-glycan-directed binding strategy is commonly used in other Eph family members. A gain-of-function EphB6 mutant restores its ephrin-B2 binding ability. Finally, EphB2 is robustly glycosylated in the mouse brain and N-glycosylation is required for EphB2 signaling-induced cell rounding and dendritic spine formation. Collectively, our findings provide a molecular basis to understand the exquisite Eph/ephrin interaction preferences.