Inhibition of Receptor Dimerization as a Novel Negative Feedback Mechanism of EGFR Signaling.

Banafshe Larijani,Malgorzata Kluba,Yves Engelborghs,Hideaki Mizuno,Johan Hofkens

doi:10.1371/journal.pone.0139971

Abstract

Dimerization of the epidermal growth factor receptor (EGFR) is crucial for initiating signal transduction. We employed raster image correlation spectroscopy to continuously monitor the EGFR monomer-dimer equilibrium in living cells. EGFR dimer formation upon addition of EGF showed oscillatory behavior with a periodicity of about 2.5 min, suggesting the presence of a negative feedback loop to monomerize the receptor. We demonstrated that monomerization of EGFR relies on phospholipase Cγ, protein kinase C, and protein kinase D (PKD), while being independent of Ca2+ signaling and endocytosis. Phosphorylation of the juxtamembrane threonine residues of EGFR (T654/T669) by PKD was identified as the factor that shifts the monomer-dimer equilibrium of ligand bound EGFR towards the monomeric state. The dimerization state of the receptor correlated with the activity of an extracellular signal-regulated kinase, downstream of the EGFR. Based on these observations, we propose a novel, negative feedback mechanism that regulates EGFR signaling via receptor monomerization.

Highlights

Epidermal growth factor receptor (EGFR) signaling plays a role in cell growth, differentiation, survival and proliferation [1]
All measurements and analyses were performed on selected cells with moderate expression, in which eGFP-EGFR localized in the plasma membrane
Using CHO cells as a model system, we demonstrated that EGFR displayed periodical changes of diffusion coefficient in the presence of epidermal growth factor (EGF)

Summary

Introduction

Epidermal growth factor receptor (EGFR) signaling plays a role in cell growth, differentiation, survival and proliferation [1]. EGFR consists of a ligand binding ectodomain followed by a transmembrane single-helix, a juxtamembrane (JM) segment and an intracellular kinase domain with a regulatory C-terminus (Fig 1D) [6, 7]. When epidermal growth factor (EGF) binds to the monomeric receptor, steric constraints are removed to expose the “dimerization arm” enabling the association of two EGFR units [8]. Subsequent association of the two transmembrane domains and an antiparallel interaction between the N-terminal juxtamembrane helices (JM-A) promotes EGFR activation [9]. The C-lobe of the kinase domain of one EGFR, the activator, interacts with the N-lobe of the PLOS ONE | DOI:10.1371/journal.pone.0139971. The C-lobe of the kinase domain of one EGFR, the activator, interacts with the N-lobe of the PLOS ONE | DOI:10.1371/journal.pone.0139971 October 14, 2015

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Conclusion