Abstract
AbstractReceptor proteins, such as epidermal growth factor receptor (EGFR), interact with other proteins in the extracellular region of the cell membrane to drive intracellular signalling. Therefore, analysis of extracellular protein-protein interactions (exPPIs) is important for understanding the biological function of receptor proteins. Here, we present an approach using a proximity biotinylation enzyme (AirID) fusion fragment of antigen binding (FabID) to analyse the proximity exPPIs of EGFR. AirID was C-terminally fused to the Fab fragment against EGFR (EGFR-FabID), which could then biotinylate the extracellular region of EGFR in several cell lines. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis indicated that many known EGFR interactors were identified as proximity exPPIs, along with many unknown candidate interactors, using EGFR-FabID. Interestingly, these proximity exPPIs were influenced by treatment with EGF ligand and its specific kinase inhibitor, gefitinib. These results indicate that FabID provides accurate proximity exPPI analysis of target receptor proteins on cell membranes with ligand and drug responses.
Funder
Japan Agency for Medical Research and Development
MEXT | Japan Society for the Promotion of Science
Takeda Science Foundation
Joint Usage and Joint Research Programs of the Institute of Advanced Medical Sciences, Tokushima University
Publisher
Springer Science and Business Media LLC
Subject
General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry,Multidisciplinary