Different Epidermal Growth Factor Receptor (EGFR) Agonists Produce Unique Signatures for the Recruitment of Downstream Signaling Proteins

Tom Ronan,Jennifer L Macdonald-Obermann,Lorel Huelsmann,Nicholas J Bessman,Kristen M Naegle,Linda J Pike

doi:10.1074/jbc.m115.710087

Tom Ronan, Jennifer L Macdonald-Obermann + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m115.710087

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Abstract

The EGF receptor can bind seven different agonist ligands. Although each agonist appears to stimulate the same suite of downstream signaling proteins, different agonists are capable of inducing distinct responses in the same cell. To determine the basis for these differences, we used luciferase fragment complementation imaging to monitor the recruitment of Cbl, CrkL, Gab1, Grb2, PI3K, p52 Shc, p66 Shc, and Shp2 to the EGF receptor when stimulated by the seven EGF receptor ligands. Recruitment of all eight proteins was rapid, dose-dependent, and inhibited by erlotinib and lapatinib, although to differing extents. Comparison of the time course of recruitment of the eight proteins in response to a fixed concentration of each growth factor revealed differences among the growth factors that could contribute to their differing biological effects. Principal component analysis of the resulting data set confirmed that the recruitment of these proteins differed between agonists and also between different doses of the same agonist. Ensemble clustering of the overall response to the different growth factors suggests that these EGF receptor ligands fall into two major groups as follows: (i) EGF, amphiregulin, and EPR; and (ii) betacellulin, TGFα, and epigen. Heparin-binding EGF is distantly related to both clusters. Our data identify differences in network utilization by different EGF receptor agonists and highlight the need to characterize network interactions under conditions other than high dose EGF.

Highlights

The phosphorylated tyrosines on the EGF receptor serve as binding sites for a large number of signaling proteins that contain SH2 and/or phosphotyrosine-binding domains (9, 10)
The cDNA for the fusion protein was transfected into a CHO cell line that stably expressed the EGF receptor C-terminally fused to the NLuc fragment (EGFR-NLuc) off a tetracycline-inducible promoter
We report here on the use of luciferase fragment complementation to study the association of downstream signaling proteins with the EGF receptor

Summary

Introduction

The phosphorylated tyrosines on the EGF receptor serve as binding sites for a large number of signaling proteins that contain SH2 and/or phosphotyrosine-binding domains (9, 10). Signaling Protein Recruitment by EGFR Agonists p66 Shc, and Shp[2] with the EGF receptor in response to increasing concentrations of all seven different EGF receptor ligands. For Grb[2] (Fig. 1D) and Shp[2] (Fig. 1H), the maximum complementation occurred at a relatively low dose of EGF, with higher doses of growth factor resulting in lower peak responses and a marked decrease at longer times.

Results

Conclusion