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A quantitative protein interaction network for the ErbB receptors using protein microarrays

Nature volume 439, pages 168–174 (2006)Cite this article

Abstract

Although epidermal growth factor receptor (EGFR; also called ErbB1) and its relatives initiate one of the most well-studied signalling networks, there is not yet a genome-wide view of even the earliest step in this pathway: recruitment of proteins to the activated receptors. Here we use protein microarrays comprising virtually every Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain encoded in the human genome to measure the equilibrium dissociation constant of each domain for 61 peptides representing physiological sites of tyrosine phosphorylation on the four ErbB receptors. This involved 77,592 independent biochemical measurements and provided a quantitative protein interaction network that reveals many new interactions, including ones that fall outside of our current view of domain selectivity. By slicing through the network at different affinity thresholds, we found surprising differences between the receptors. Most notably, EGFR and ErbB2 become markedly more promiscuous as the threshold is lowered, whereas ErbB3 does not. Because EGFR and ErbB2 are overexpressed in many human cancers, our results suggest that the extent to which promiscuity changes with protein concentration may contribute to the oncogenic potential of receptor tyrosine kinases, and perhaps other signalling proteins as well.

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Figure 1: Measuring the binding affinity of SH2/PTB domains for phosphopeptides derived from the ErbB receptors using protein microarrays.
Figure 2: Quantitative protein interaction networks for the four human ErbB receptors.
Figure 3: Venn diagrams for 11 of the 15 SH2/PTB domains available in Scansite 2.0.
Figure 4: A system-level view of EGFR, ErbB2 and ErbB3 at different affinity thresholds.

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Acknowledgements

We thank B. Schoeberl for comments on the manuscript, J. Grudzien for development work on the silicone gaskets, and the Bauer Center for Genomics Research at Harvard University for support with instrumentation and automation. This work was supported by awards from the W. M. Keck Foundation and the Arnold and Mabel Beckman Foundation. R.B.J. is the recipient of a Ruth L. Kirschstein National Research Service Award (NIH), A.G. is the recipient of an NSF Graduate Research Fellowship, and J.A.K. is the recipient of a Howard Hughes Medical Institute Predoctoral Fellowship in the Biological Sciences.

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  1. Richard B. Jones and Andrew Gordus: *These authors contributed equally to this work

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology,

    Richard B. Jones, Andrew Gordus, Jordan A. Krall & Gavin MacBeath

  2. Program in Biophysics, Harvard University, Massachusetts, 02138, Cambridge, USA

    Andrew Gordus

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Correspondence to Gavin MacBeath.

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Supplementary information

Supplementary Figure 1

This figure shows multiple sequence alignments for human SH2 domains (a) and human PTB domains (b). (RTF 241 kb)

Supplementary Figure 2

This figure shows SDS-polyacrylamide gels of purified SH2 and PTB domains. (PDF 313 kb)

Supplementary Figure 3

This figure shows western blots of cells stimulated with EGF or HRGβ1. (PDF 84 kb)

Supplementary Table 1

This table provides a list of the SH2 domains (a) and PTB domains (b) used in this study. (RTF 156 kb)

Supplementary Table 2

This table provides an estimate of the percentage of each purified SH2 or PTB domain that was monomeric as judged by analytical size exclusion column chromatography. (RTF 104 kb)

Supplementary Table 3

This table provides a list of the peptides used in this study. (RTF 52 kb)

Supplementary Table 4

This table provides equilibrium dissociation constants for SH2/PTB interactions with peptides derived from the four human ErbB receptors. (XLS 46 kb)

Supplementary Table 5

This table provides a comparison of the interactions observed on the protein microarrays with previously reported interactions. (RTF 176 kb)

Supplementary Table 6

This table provides a list of the domains that bound to EGFR, ErbB2, and ErbB3 at four different affinity thresholds. (RTF 49 kb)

Supplementary Methods

This file contains technical details of experimental methods used in this study. (RTF 29 kb)

Supplementary Notes

Additional notes on the Supplementary Figures and Tables. (RTF 6 kb)

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Jones, R., Gordus, A., Krall, J. et al. A quantitative protein interaction network for the ErbB receptors using protein microarrays. Nature 439, 168–174 (2006). https://doi.org/10.1038/nature04177

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