Complementing the EGFR dynamic interactome using live-cell proximity labeling

Abstract

AbstractThe epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family (RTK) of transmembrane receptors, known to regulate many key cellular processes, including growth, proliferation, and differentiation. Its expression, activation, trafficking, and degradation have been extensively studied, as dysregulation of EGFR activation has been linked to a vast number of cancers. Activation of EGFR by different ligands results in distinct cellular responses, and the relative distribution of EGFR in different endosome pools in a process called endosomal sorting, leading to lysosomal degradation, or cell surface recycling, respectively, is considered a fundamental process in EGFR stimulation outcome. The EGFR interactome is therefore an essential element in the study of RTK functional selectivity. Here, we aimed to complement the existing EGFR interactome with spatio-temporal information on EGFR, its interactors, and phosphorylation state. We identified and quantified EGFR stable and transient interactions at different time points after stimulation using an EGFR-APEX2 fusion construct expressed in HEK293T cells and were able to use bystander proteins to map EGFR subcellular location at each time point. Utilizing the fast and concise biotinylation of proximity proteins by APEX2, we were able to detect slight differences in early signaling kinetics between TGF-α and EGF, thereby increasing our knowledge on RTK signaling and differential trafficking.