Issue 7, 2013

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.

Graphical abstract: Exploring higher-order EGFR oligomerisation and phosphorylation—a combined experimental and theoretical approach

Supplementary files

Article information

Article type
Paper
Submitted
20 Feb 2013
Accepted
19 Apr 2013
First published
30 Apr 2013

Mol. BioSyst., 2013,9, 1849-1863

Exploring higher-order EGFR oligomerisation and phosphorylation—a combined experimental and theoretical approach

N. Kozer, D. Barua, S. Orchard, E. C. Nice, A. W. Burgess, W. S. Hlavacek and A. H. A. Clayton, Mol. BioSyst., 2013, 9, 1849 DOI: 10.1039/C3MB70073A

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