TY - JOUR T1 - EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation JF - bioRxiv DO - 10.1101/305292 SP - 305292 AU - Charlotte Fournier AU - Adam J. M. Wollman AU - Isabel Llorente-Garcia AU - Oliver Harriman AU - Djamila Ouarat AU - Jenny Wilding AU - Walter Bodmer AU - Mark C. Leake Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/04/20/305292.abstract N2 - Epidermal growth factor (EGF) signalling regulates cell growth, differentiation and proliferation in epithelium and EGF receptor (EGFR) overexpression has been reported in several carcinoma types. Structural and biochemical evidence suggests EGF binding stimulates EGFR monomer-dimer transitions, activating downstream signalling. However, mechanistic details of ligand binding to functional receptors in live cells remain contentious. We report real time single-molecule TIRF of human epithelial carcinoma cells with negligible native EGFR expression, transfected with GFP-tagged EGFR, before and after receptor activation with TMR-labelled EGF ligand. Fluorescently labelled EGFR and EGF are simultaneously tracked to 40nm precision to explore stoichiometry and spatiotemporal dynamics upon EGF binding. Using inhibitors that block binding to EGFR directly, or indirectly through HER2, our results indicate that pre-activated EGFR consists of preformed homoclusters, while larger heteroclusters including HER2 form upon activation. The relative stoichiometry of EGFR to EGF after binding peaks at 2, indicating negative cooperativity of EGFR activation. ER -