Spatiotemporal Proteomics Deciphers Functional Selectivity of EGFR ligands

The epidermal growth factor receptor (EGFR) induces different signaling outputs depending on ligand identity and biological context. This phenomenon is known as functional selectivity, but the underlying molecular mechanisms remain elusive. Here, we investigated this on a global scale and time-resolved by high-throughput multilayered proteomics integrating dynamic changes in the EGFR interaction network by proximity biotinylation using EGFR- TurboID, phosphoproteome, and proteome in response to stimulation with the six highest-affinity EGFR ligands. We obtained comprehensive temporal profiles of protein recruitment and phosphosite changes pinpointing signaling proteins differentially regulated by the six ligands with key impact on EGFR endocytic fate, e.g. degradation or recycling. Specifically, the Epsin family protein, Clint1 was identified to control the endocytic trafficking of EGFR towards degradation. Moreover, we characterized the protein interaction selectivity of EGFR C-terminally phosphorylated tyrosine residues using a panel of tyrosine mutated constructs showing STAT5 specificity for EGFR Y1173. These data provide a comprehensive resource deciphering functional selectivity of EGFR signaling to support discovery of novel drug targets.