title: The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells.
abstract: The epidermal growth factor receptor (EGFR) is a prototypical receptor tyrosine kinase involved in cell growth and proliferation and associated with various cancers. It is commonly assumed that after activation by binding of epidermal growth factor to the extracellular domain it dimerizes, followed by autophosphorylation of tyrosine residues at the intracellular domain. However, its oligomerization state before activation is controversial. In the absence of ligands, EGFR has been found in various, inconsistent amounts of monomeric, inactive dimeric, and oligomeric forms. In addition, evidence suggests that the active conformation is not a simple dimer but contains higher oligomers. As experiments in the past have been conducted at different conditions, we investigate here the influence of cell lines (HEK293, COS-7, and CHO-K1), temperature (room temperature and 37°C), and membrane localization on the quantitation of preformed dimers using SW-FCCS, DC-FCCS, quasi PIE-FCCS, and imaging FCCS. While measurement modality, temperature, and localization on upper or lower membranes have only a limited influence on the dimerization amount observed, the cell line and location to periphery versus center of the cell can change dimerization results significantly. The observed dimerization amount is strongly dependent on the expression level of endogenous EGFR in a cell line and shows a strong cell-to-cell variability even within the same cell line. In addition, using imaging FCCS, we find that dimers have a tendency to be found at the periphery of cells compared to central positions.
authors.affiliation: Department of Chemistry, National University of Singapore, Singapore, Singapore.
authors.forename: Sibel
authors.initials: S
authors.lastname: Yavas
authors.affiliation: Department of Biological Sciences and Centre for BioImaging Sciences, National University of Singapore, Singapore, Singapore.
authors.forename: Radek
authors.initials: R
authors.lastname: Macháň
authors.affiliation: Department of Chemistry, National University of Singapore, Singapore, Singapore; Department of Biological Sciences and Centre for BioImaging Sciences, National University of Singapore, Singapore, Singapore. Electronic address: twohland@nus.edu.sg.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Wohland
mesh_terms: D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013050:Spectrometry, Fluorescence; D053778:Thiazolidines / Q000494:pharmacology
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D053778:Thiazolidines; D004815:Epidermal Growth Factor; D002784:Cholesterol; D066246:ErbB Receptors; C037067:latrunculin A