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

Abstract

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 crosstalk with signaling are poorly understood. Here, we combined 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 identified a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We showed that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, was enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes, and demonstrated 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.

Time-Resolved Interactome Profiling Deconvolutes Secretory Protein Quality Control Dynamics

“…These methods have been powerful for mapping steady-state proteostasis interactions to disease states, yet most lack the ability to measure interaction dynamics over time. Proximity labeling mass spectrometry (BioID & APEX-MS) has had limited use to spatiotemporally resolve protein-protein interactions only following protein maturation, as synchronization of newly synthesized protein populations is challenging to achieve (Lobingier et al, 2017;Perez Verdaguer et al, 2022). Unnatural amino acid incorporation methods, such as biorthogonal non-canonical amino acid tagging (BONCAT), pulsed azidohomoalanine (PALM), or heavy isotope labeled azidohomoalanine (HILAQ) can identify newly synthesized proteins but have not focused on a single endogenously expressed protein or group of proteins in the context of disease (Bagert et al, 2014;van Bergen et al, 2021;Dieterich et al, 2006;Howden et al, 2013;Ma et al, 2017Ma et al, , 2018McClatchy et al, 2015).…”