EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

Abstract

Epidermal growth factor (EGF) signalling regulates cell growth, differentiation and proliferation 19 in epithelium and EGF receptor (EGFR) overexpression has been reported in several carcinoma types. 20 Structural and biochemical evidence suggests EGF binding stimulates EGFR monomer-dimer transitions, 21 activating downstream signalling. However, mechanistic details of ligand binding to functional receptors in 22 live cells remain contentious. We report real time single-molecule TIRF of human epithelial carcinoma 23 cells with negligible native EGFR expression, transfected with GFP-tagged EGFR, before and after 24 receptor activation with TMR-labelled EGF ligand. Fluorescently labelled EGFR and EGF are 25 simultaneously tracked to 40nm precision to explore stoichiometry and spatiotemporal dynamics upon 26 EGF binding. Using inhibitors that block binding to EGFR directly, or indirectly through HER2, our 27 results indicate that pre-activated EGFR consists of preformed homoclusters, while larger heteroclusters 28 including HER2 form upon activation. The relative stoichiometry of EGFR to EGF after binding peaks at 29 2, indicating negative cooperativity of EGFR activation. 30 31Main Text: 35 The epidermal growth factor receptor (EGFR) is essential for normal growth and development of epithelial 36 tissues and is a key component in several signaling pathways 1 . Aberrant signal transduction is a primary 37 driver of many epithelial cancers, EGFR upregulation implicated in formation and progression of several 38 carcinomas 2 . Human EGFR or ERBB1, (also denoted 'ErB1'or 'HER1') is a 1,186 amino acid (aa) residue 39 170 kDa molecular weight protein 3 belonging to a family of receptor tyrosine kinase (RTK) receptors with 40 three additional members: ERBB2 ('ErbB2', 'HER2' or 'neu'), ERBB3 ('ErbB3' or 'HER3') and ERBB4 41 ('ErbB4' or 'HER4') expressed predominantly in the plasma membrane of epithelial cells 4 . EGFR has a 42 621aa extracellular region, divided into subdomains I-IV 5 . Domains I and III directly participate in ligand 43 binding 6 , connected via a 23aa hydrophobic transmembrane α-helix to a 542aa cytoplasmic domain 44 containing a 300aa tyrosine kinase 7 . 45 EGFR activation requires ligand binding, receptor-receptor interactions, and full activation of the 46 tyrosine kinase 8 . At least 11 different ligands bind to the EGFR family, four to EGFR including EGF 47 itself 9 . Prior to ligand binding the tyrosine kinase has low catalytic activity. Ligand binding results in full 48 kinase activation through c-lobe interaction of an 'activator' and n-lobe 'receiver' 10 . Subsequent 49 autophosphorylation of intracellular tyrosine residues 11 initiates intracellular reactions ultimately 50 stimulating cellular growth, differentiation and proliferation 12 , terminated by internalization and 51 proteolytic degradation of the receptor-ligand complex 13 . 52The field has detailed insights concerning extracellular and intracellular interactions that contribute 53 to signal transduction, however, there remai...