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

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.

The ERBB3 receptor in cancer and cancer gene therapy

“…105 A proliferative response of NIH3T3 cells to NRG, dependent on expression of ERBB3, involved association of PI3K with ERBB3. 134 Early studies indicated that a prominent association between ERBB3 and p85 is a unique feature of ERBB3, 135 and this was confirmed by the assays of Jones et al, 133 which found that both SH2 groups in each of the three PI3KR isoforms bound with moderate or high affinity at multiple sites in ERBB3, as previously reported. 104 However, EGFR and ERBB2 (but not ERBB4) did bind to p85 at a limited number of sites.…”

“…104 However, EGFR and ERBB2 (but not ERBB4) did bind to p85 at a limited number of sites. 133 Each of the p85 sites contributes to ERBB3 signaling, as demonstrated by their one-at-a-time mutation and restoration, and cooperation among p85-binding sites was observed. 136 These interactions involve the N-terminal SH2 domain of p85, with the two phosphotyrosine-binding sites in this domain each interacting preferentially with certain phosphotyrosyl peptides from ERBB3.…”

“…A remarkable accomplishment was recently reported by Jones et al: 133 all of the SRC homology 2 (SH2) and phosphotyrosine-binding (PTB) domains encoded in the human genome were measured for binding to each of the phosphopeptides from the ERBB receptors. This included 106 SH2 domains and 41 PTB domains, and for ERBB3, 10 phosphotyrosinecontaining peptides and their non-phosphorylated counterparts.…”

Act Globally, Think Locally: Systems Biology Addresses the PDZ Domain

“…This report follows closely that of another from the same laboratory that mapped the interaction profile of human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains (13). As with this predecessor study, the PDZ domain investigation uses the same methodological blueprint, although the scope is smaller and more in keeping with the nature of a pilot study.…”

A Lock on Phosphotyrosine Signaling

“…In the human genome, 109 SH2 domains have been identified (10), and many are believed to mediate sequence-specific protein-protein interactions that require pTyr. SH2-pTyr interactions can stimulate (e.g., SHP-2) (11) or inhibit (e.g., Src) enzymatic activity in an intramolecular fashion (12).…”