Abstract 4915: Characterization of the EGFR interactome reveals cellular dynamics of receptor signalling and internalisation

Abstract

Abstract The epidermal growth factor receptor (EGFR) is one part of the promiscuous signaling network that regulates proliferation, growth and differentiation of mammalian cells and its overexpression, aberrant or enhanced activation are frequent events in human cancers. EGFR signaling is realized by recruiting specific adaptor proteins to the activated intracellular receptor tyrosine kinase domain. We hypothesize that the pattern of recruited adaptor proteins is characteristic for respective EGFR related cellular responses. Therefore, we developed a method to decode the EGFR adaptor protein pattern in relation to its activation state. A431 cells were stimulated with 100 ng/ml EGF for 30 minutes and the EGFR-adaptor protein-complex was isolated by immunoprecipitation after gentle lysis using the specific EGFR antibody cetuximab biotinylated and bound to magnetic beads. Controls were precipitated from lysates of unstimulated A431 cells. After pre fractionation by 1D SDS PAGE and in gel digestion, protein complexes were characterized by LC-ESI-tandem high resolution mass spectrometry (LC-MS/MS). LC-MS/MS analyses reproducibly identified 68 proteins in three replicates of precipitates from lysates of unstimulated A431 cells. 64 of these proteins were assigned to gene ontology (GO) annotations and significantly enriched domains included intracellular localisation and protein transport proteins (29 proteins) and the EGFR signaling pathway (5 proteins). The first domain contained structural proteins such as actin and tubulin subunits as well as importins, exportins and transportins. Proteins within the EGFR signaling category included growth factor receptor bound protein 2 (Grb2) and signal transducer and activator of transcription 3 (STAT3). Biogrid analyses identified an interacting EGFR network consisting of 14 known interacting proteins including several subunits of the clathrin related adaptor protein complex. Semi-quantification showed that the interactions of these proteins with the EGFR were markedly pronounced upon EGF stimulation suggesting increased receptor internalization. These data were confirmed by Western Blotting and immunofluorescence microscopy. Furthermore, several proteins were found which associations to the EGFR are as yet unknown such as Hook homolog 2, MON2 homolog or FRG1 proteins. Quantification of dynamic changes will further be carried out by stable isotope labeling (SILAC) technology. As proof of principle, we were able to identify proteins which are part of the EGFR signaling pathway as well as proteins that relate to cellular localization and protein trafficking and which abundances changed upon EGFR stimulation. Using this approach will provide further insights into mechanisms of EGFR signal activation from a systems biology point of view. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4915. doi:1538-7445.AM2012-4915