Abstract The EGF receptor (EGFR) is a cell surface receptor tyrosine kinase that is frequently mutated or over- expressed in a variety of tumors. Stimulation of the receptor by EGF leads to the activation of its intracellular tyrosine kinase which autophosphorylates multiple tyrosines on its intrinsically-disordered C-terminal tail. These phosphotyrosines serve as binding sites for the SH2 and PTB domain-containing proteins that mediate the downstream effects of EGF. SH2 and PTB domains recognize their binding sites via a short linear motif in which the pTyr is embedded. In preliminary experiments, we have found that regions well outside of these short motifs contribute to the binding of SH2 and PTB domain proteins. Further evidence for the importance of more distant sequences in the binding of SH2 domains to the EGFR tail was derived from our preliminary computer modeling studies that show that when the Grb2 SH2 domain is bound to Tyr-1068, the sequence surrounding Tyr-1148, the major site of autophosphorylation of the EGFR, interacts with a secondary binding surface on the SH2 domain. This interaction limits the availability of Tyr-1148 to bind to its partner proteins. We therefore hypothesize that regions of the tail distant from the pTyr motif participate in the binding of SH2 domain proteins and that these extended interactions modify the conformational ensemble of the tail and alter its ability to interact with other partners in a form of ensemble allostery. The EGFR can be activated by any one of seven different ligands, which can elicit different long-term biological effects when applied to the same cell. Stimulation of the same cell with two different ligands can give rise to two completely different biological outcomes. We have demonstrated differences in the ability of different ligands to induce receptor homo- and hetero-dimerization and to bind downstream signaling proteins. In preliminary studies, we have also documented differences in their ability to stimulate glycolytic flux, a key metabolic feature of rapidly growing cells. Different tumors are known to be preferentially transformed by structurally different forms of the EGFR and to express different EGFR ligands. In many instances, clinical outcome has been shown to correlate with which ligand is expressed. We hypothesize that the structurally diverse oncogenic EGFRs are functionally different and that certain combinations of EGFR variant and ligand are more transforming than others. The goal of my laboratory is to elucidate the fundamental principles underlying signaling via receptor tyrosine kinases and to apply this to understand how mutant EGFRs induce tumors. The specific aims of this proposal are to: 1. Determine how inter-tyrosine sequences modulate the binding of SH2 domain proteins to the C-terminal tail of the EGFR; and, 2) Identify functional differences among oncogenic EGFRs and their response to EGFR agonists that are relevant to how transformation is established...