Exploring Higher-Order EGFR Oligomerisation and Phosphorylation -- A Combined Experimental and Theoretical Approach

Abstract

The epidermal growth factor receptor (EGFR) kinase is generally considered to be activated by either ligand-induced dimerisation or a ligand-induced conformational change within pre-formed dimers. Ligand-induced higher-order EGFR oligomerisation or clustering has been reported but it is not clear how EGFR oligomers, as distinct from EGFR dimers, influence signaling outputs. To address this question, we combined measures of receptor clustering (microscopy; image correlation spectroscopy) and phosphorylation (Western blots) with modelling of mass-action chemical kinetics. A stable BaF/3 cell-line that contains a high proportion ( > 90%) of inactive dimers of EGFR-eGFP but no secreted ligand and no other detectable ErbB receptors was used as the model cell system. EGF at concentrations of greater than 1 nM was found to cluster EGFR-eGFP dimers into higher-order complexes and cause parallel increases in EGFR phosphorylation. The kinetics of EGFR clustering and phosphorylation were both rapid, plateauing within 2 minutes after stimulation with 30 nM EGF. A rule-based model was formulated to interpret the data. This model took into account ligand binding, ligand-induced conformational changes in the cytosolic tail, monomer-dimer-trimer-tetramer transitions via ectodomain- and kinase-mediated interactions, and phosphorylation. The model predicts that cyclic EGFR tetramers are the predominant phosphorylated species, in which activated receptor dimers adopt a cyclic side-by-side orientation, and that receptor kinase activation is stabilised by the intramolecular interactions responsible for cyclic tetramerization.

Department(s)

Chemical and Biochemical Engineering

Keywords and Phrases

Epidermal Growth Factor; Epidermal Growth Factor Receptor; Ligand; Epidermal Growth Factor Receptor; Protein Binding, Animal; Article; Biological Model; Cell Line; Chemistry; Computer Simulation; Kinetics; Metabolism; Mouse; Phosphorylation; Protein Binding; Protein Domain; Protein Multimerization; Chemistry, Animals; Cell Line; Computer Simulation; Epidermal Growth Factor; Kinetics; Ligands; Mice; Models, Biological; Phosphorylation; Protein Binding; Protein Interaction Domains And Motifs; Protein Multimerization; Receptor, Epidermal Growth Factor, Animals; Cell Line; Computer Simulation; Epidermal Growth Factor; Kinetics; Ligands; Mice; Models, Biological; Phosphorylation; Protein Binding; Protein Interaction Domains And Motifs; Protein Multimerization; Receptor, Epidermal Growth Factor

International Standard Serial Number (ISSN)

1742-206X

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2013 Royal Society of Chemistry, All rights reserved.

Publication Date

01 Jul 2013

PubMed ID

23629589

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