Comprehensive Binary Interaction Mapping of SH2 Domains via Fluorescence Polarization Reveals Novel Functional Diversification of ErbB Receptors

Abstract

First-generation interaction maps of Src homology 2 (SH2) domains with receptor tyrosine kinase (RTK) phosphosites have previously been generated using protein microarray (PM) technologies. Here, we developed a large-scale fluorescence polarization (FP) methodology that was able to characterize interactions between SH2 domains and ErbB receptor phosphosites with higher fidelity and sensitivity than was previously achieved with PMs. We used the FP assay to query the interaction of synthetic phosphopeptides corresponding to 89 ErbB receptor intracellular tyrosine sites against 93 human SH2 domains and 2 phosphotyrosine binding (PTB) domains. From 358,944 polarization measurements, the affinities for 1,405 unique biological interactions were determined, 83% of which are novel. In contrast to data from previous reports, our analyses suggested that ErbB2 was not more promiscuous than the other ErbB receptors. Our results showed that each receptor displays unique preferences in the affinity and location of recruited SH2 domains that may contribute to differences in downstream signaling potential. ErbB1 was enriched versus the other receptors for recruitment of domains from RAS GEFs whereas ErbB2 was enriched for recruitment of domains from tyrosine and phosphatidyl inositol phosphatases. ErbB3, the kinase inactive ErbB receptor family member, was predictably enriched for recruitment of domains from phosphatidyl inositol kinases and surprisingly, was enriched for recruitment of domains from tyrosine kinases, cytoskeletal regulatory proteins, and RHO GEFs but depleted for recruitment of domains from phosphatidyl inositol phosphatases. Many novel interactions were also observed with phosphopeptides corresponding to ErbB receptor tyrosines not previously reported to be phosphorylated by mass spectrometry, suggesting the existence of many biologically relevant RTK sites that may be phosphorylated but below the detection threshold of standard mass spectrometry procedures. This dataset represents a rich source of testable hypotheses regarding the biological mechanisms of ErbB receptors.

Do low-affinity ErbB receptor protein interactions represent the base of a cell signaling iceberg?

“…Therefore, best-fit consensus motifs are definitely not required for SH2 domain containing proteins to function in a physiological context. In our study [1], we empirically demonstrated that EGFR does not contain a single high-affinity recruitment site for Src family kinase SH2 domains. The highest affinity EGFR recruitment site had a dissociation constant of approximately 2 µM, much less than the 3 nM dissociation constants originally described for the pYEEI consensus peptide [20].…”

“…We recently described one of the most comprehensive and quantitative assessments of human ErbB receptor recruitment potential for SH2 domains to date using a high-throughput fluorescence polarization methodology [1]. This study uncovered 1405 unique interactions, 83% of which had not been previously described and the majority of which were of low affinity relative to previously described best-fit interaction motifs for SH2 domains.…”

Analytical reduction of combinatorial complexity arising from multiple protein modification sites

“…To reduce the number of potential binding proteins, we relied on two sets of experimental data. First, we required that binding proteins have less than a 3 mM K d to any EGFR tyrosine phosphorylation site [13]. Second, we required that binding proteins have detectable concentrations as measured in NIH3T3 cells [16].…”

“…On the epidermal growth factor receptor (EGFR), phosphorylation of Y1068 is correlated with longer survival in non-small cell lung cancer patients, whereas phosphorylation of Y1173 is correlated with shorter survival [12]. There are multiple proteins, such as Grb2, PI3K, SHP2, PLCg1, RasA1 ( p120RasGAP) and Shc1, which bind to these and the other approximately 10 tyrosine phosphorylation sites on EGFR in both unique and overlapping manners with varying affinities [13]. The relative recruitment levels of these various proteins determine the strength of downstream signalling to pathways such as Akt, ERK or PKC, which subsequently dictate biological responses such as proliferation, migration, differentiation or apoptosis [14].…”

“…The relative recruitment levels of these various proteins determine the strength of downstream signalling to pathways such as Akt, ERK or PKC, which subsequently dictate biological responses such as proliferation, migration, differentiation or apoptosis [14]. However, predicting the recruitment levels of these binding proteins without considering site-specific effects, and therefore combinatorial complexity, is challenging: each EGFR tyrosine is phosphorylated at different rates [15], and the host of binding proteins has distinct affinities for the various EGFR phosphotyrosines [13] as well as different expression levels [16]. Incorporating tens of thousands or even millions of differential equations into a model to account for all these site-specific phenomena in a theoretically precise manner is problematic for several reasons.…”