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

ABSTRACT: Ligand binding to the EGF receptor (EGFR) initiates signal transduction and endocytosis of the receptor. The mechanisms of endocytosis and regulation of signaling by endocytosis 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 EGFR in response to ligand activation. Using this approach, we identified a network of signaling proteins, which remain associated with the receptor during its endocytosis 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 early/sorting endosome proximity proteome of EGFR and localized in early an sorting endosomes, and demonstrated that TFG regulates endosomal sorting of EGFR. This study provides a large-scale resource of time-dependent nanoscale environment of activated EGFR, thus opening avenues to discovering new regulatory mechanisms of membrane trafficking of receptor tyrosine kinases. EGFR-APEX2 Experiment Datasets: TMT 1-3: HEK293T cells (0, 1, 10, 30 and 60 minutes EGF treatment) TMT 4: HCT116 cells (0, 2, 4, 6 , 8 and 10 minutes EGF treatment) TMT 5: HEK293T cells (0, 2, 4, 6 , 8 and 10 minutes EGF treatment) TMT 6: HEK293T cells (0 and 10 minutes EGF treatment) TMT 7: HCT116 cells (0 and 10 minutes EGF treatment)

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tian Zhang

LAB HEAD: Steven Gygi

PROVIDER: PXD030072 | Pride | 2022-06-04

REPOSITORIES: Pride

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