Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR.

Ligand binding to the EGF receptor (EGFR) triggers multiple signal-transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its cross-talk with signaling are poorly understood. Here, we combine peroxidase-catalyzed proximity labeling, isobaric peptide tagging, and quantitative mass spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identify a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We show that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, is enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes and demonstrate that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases. [Display omitted] • Proximity proteome of activated EGFR tracked by time-resolved APEX-mediated labeling • Multiple signaling proteins remain proximal to EGFR during its endocytosis • Trk-fused gene (TFG) is associated with early and sorting endosomes • TFG regulates endosomal sorting of EGFR Perez Verdaguer et al. use time-resolved APEX labeling of the proximity proteome of EGFR upon ligand activation to provide comprehensive information about the dynamics of the EGFR-associated protein networks involved in receptor endocytosis and signaling. [ABSTRACT FROM AUTHOR]