System-level studies of receptor tyrosine kinase signaling networks

Receptor tyrosine kinases are transmembrane proteins that activate intracellular signaling pathways in response to extracellular ligands. Upon activation, receptor tyrosine kinases recruit proteins containing SH2 and PTB domains to sites oftyrosine phosphorylation on the receptors, which is the initial step in intracellular signaling. Despite the fact that RTKs induce diverse biological responses including proliferation, migration and differentiation, they often recruit similar sets of SH2- and PTB-containing proteins and activate many of the same signaling pathways. In order to assess how seemingly similar receptors induce distinct biological outcomes, a protein microarray platform was developed to assess the global recruitment of SH2- and PTB-containing proteins to receptor tyrosine kinases. These arrays consisted of purified SH2 and PTB domains printed in wells of a microtiter plate and were probed with a dilution series of fluorescently labeled phosphotyrosine-containing peptides. This format was used to generate quantitative interaction maps for the four ErbB [epidermal growth factor receptor (EGFR) family] receptors. To extend these studies to cellular events, six diverse receptor tyrosine kinases were expressed in a common cellular background, and the phosphorylation of intracellular proteins induced by each receptor was measured by immunoblotting. Each receptor induced a unique phosphorylation pattern based on intrinsic differences in signaling properties. Using global recruitment profiles for the six receptors determined with protein microarrays, it was found that the phosphorylation of upstream signaling proteins can be modeled as a linear function of recruitment events. Finally, as physiological concentrations of extracellular ligands vary, the pathway activation induced by a single receptor, EGFR, was assessed in response to diverse ligand concentrations. Low ligand concentrations were found to fully activate most canonical signaling proteins, while much higher ligand concentrations were required to activate a subset of proteins. The ligand concentrations needed to activate both sets of proteins implicate the involvement of high- and low-affinity EGFR in distinct pathways, and suggest, for the first time, a role for low-affinity receptors in autocrine and paracrine signaling...