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| Protein interactions: EGF receptor promiscuity exposed
A genome-wide analysis of the interactions between proteins containing SH2/PTB domains and active epidermal growth factor (EGF) receptors uncovers many promiscuous relationships. Upon ligand binding, epidermal growth factor (EGF) receptors form homo- and heterodimers, allowing their intracellular kinase domains to phosphorylate specific tyrosine residues within their cytoplasmic tail. These phosphorylated residues then recruit the Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains in a variety of proteins, triggering the activation of many intracellular signaling pathways. In a new study, MacBeath and colleagues use protein microarrays to examine the binding affinity of SH2 and PTB domains to fluorescent phospho-tyrosine peptides, which act as surrogate activated EGF receptors.
Interactions between the purified SH2 and PTB binding domains of 159 proteins and 66 activated receptor peptides were measured. Using a range of peptide concentrations allowed the authors to filter for specific protein–peptide interactions. This approach identified 43 of the 65 previously reported interactions, together with 116 novel interactions. New interactions, such as that between v-crk avian sarcoma virus CT-10-homologue-like protein (CrkL) and both ErbB2 and ErbB3, were further confirmed using phosphorylation assays of EGF-stimulated cells. These quantitative interaction networks reveal interesting differences between the four members of the EGF family of receptor tyrosine kinases, best known for their roles in mitogenic signaling and cancer development. While EGFR (also called ErbB1) and ErbB3 have only two sites that bind many proteins with high affinity, ErbB2 has many high affinity binding sites that dock multiple proteins. On average, the ErbB2 sites bind 17 different proteins compared to 7.2, 8 and 2.3 for the EGFR, ErbB3 and ErbB4, respectively. Thus, ErbB2 appears to be able to induce signaling through a wider range of pathways than other EGF receptors. Lowering the binding affinity threshold in order to mimic the conditions in cells expressing high levels of EGF receptors exacerbated the differences between the receptors. In this scenario, EGFR and ErbB2 exhibited much more promiscuity than ErbB3. EGFR and ErbB2 receptors are frequently upregulated in human cancers, and these data suggest that at high levels they are able to induce signaling through alternative pathways possibly contributing to tumorigenicity. This study provides insight into the breadth of signaling that occurs through different EGF receptor dimers and furthers our understanding of EGF signaling in disease states. Monica Hoyos-Flight, Online Editor
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