Physiological Epidermal Growth Factor Concentrations Activate High Affinity Receptors to Elicit Calcium Oscillations

Béatrice Marquèze-Pouey,Didier Marguet,Jean-Marc Goaillard,Vincent Rouger,Sébastien Mailfert,Meijia Zhang

doi:10.1371/journal.pone.0106803

Abstract

Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

Highlights

epidermal growth factor (EGF) controls key cellular processes, such as proliferation, survival, differentiation during development, tissue homeostasis, and tumorigenesis
High affinity EGF receptor (EGFR) activation elicits specific Ca2+ signaling Using sensitive Ca2+ imaging, we characterized Ca2+signals elicited through high affinity EGFRs
These signals were specific for EGFR activation as i) when buffer was applied instead of EGF, negligible Ca2+i variations were seen (Fig 1C) and ii) the increase in average Ca2+i induced by EGF was inhibited by EGFR-specific neutralizing monoclonal M225 IgGs (Fig. 2E) [12]

Summary

Introduction

EGF controls key cellular processes, such as proliferation, survival, differentiation during development, tissue homeostasis, and tumorigenesis (reviewed in [1]). Through binding to the tyrosine kinase EGF receptor (EGFR), EGF activates a wide variety of signaling cascades mostly leading to the regulation of gene transcription. EGF is synthesized as a transmembrane precursor from which a mature, diffusible form is generated by metalloproteases. Soluble EGF can activate EGFR on distant cells via an endocrine/paracrine pathway or cells of its origin via an autocrine action. Endocrine/paracrine EGF is mitogenic at picomolar concentrations. Human serum contains around 40 pM EGF [2], which is active on cell proliferation at a twenty-fold dilution [3]

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Conclusion