EGFR phosphorylates and associates with EFNB1 to regulate cell adhesion to fibronectin.

Ephrin-Bs (EFNB1-3) are ligands for members of the largest subfamily of receptor tyrosine kinases (RTKs) in humans, the EPH receptors. Interestingly, ephrin-Bs are transmembrane proteins that may also act as receptors themselves upon EPH binding, activating so-called reverse signaling pathways that are critical for multiple cellular processes. Although a number of ephrin-B signaling effectors have been identified, the molecular mechanisms underlying ephrin-B-driven cellular processes remain unresolved, suggesting that multiple signaling effectors are yet to be discovered. Here, we employed proximity labeling proteomics to delineate the proximity network of EFNB1 in steady state and under active reverse signaling conditions. This allowed us to identify 90 uncharacterized EFNB1 proximity partners, from which we could distinguish three main groups: EPH receptor stimulation-dependent, stimulation-independent and negatively modulated by EPH receptor stimulation. We further investigated the functional relationship between EFNB1 and one of the candidates identified, the epidermal growth factor receptor (EGFR). We found that EFNB1 and EGFR associate in cells and showed that the formation of this complex relies on EFNB1's PDZ-binding motif (PBM). Strikingly, we demonstrate that EGFR directly phosphorylates tyrosine residues within EFNB1's PBM, which results in the disruption of the EFNB1-EGFR complex. Furthermore, we show that the EFNB1-EGFR association is required for EFNB1-dependent cell adhesion to fibronectin. Taken together, our results shed light on a functional relationship between EFNB1 and EGFR.