Investigating the Allosteric Activation Mechanisms of the Dysregulated Epidermal Growth Factor Receptor

In these dissertation studies, I have investigated the structural role of the essential juxtamembrane region in the dysregulated epidermal growth factor receptor (EGFR). My investigations demonstrate that the juxtamembrane helical dimer reports on ligand-independent receptor activation as well as the binding of tyrosine kinase inhibitors to the ATP-binding site in mutant forms of EGFR. These observations propose that the juxtamembrane structure undergoes long-range allosteric modulation due to the presence of distal kinase domain mutations or small-molecule binding events. In this way, the flexible juxtamembrane domain acts as an essential messenger of structural perturbations from both sides of the plasma membrane.;In Chapter 1, I describe disulfide exchange experiments and circular dichroism studies used to demonstrate that the isolated juxtamembrane domain peptides exhibit only weak alpha-helicity and require the context of the full-length receptor for stable dimerization. These observations emphasize that the juxtamembrane segment possesses a highly flexible structure key to its role as a mediator of information transfer from the outside of the cell to the inside of the cell.;In Chapter 2, I describe bipartite tetracysteine display studies used to demonstrate that the juxtamembrane helical dimer reports on ligand-independent activation of oncogenic EGFR mutants L858R, T79oM, and L858R+T79oM. Additionally, the presence of the constitutively active kinase domain mutations appears to challenge the ability of extracellular growth factor binding to regulate the juxtamembrane helical structure.;In Chapter 3, I describe bipartite tetracysteine display studies used to demonstrate that covalent, ATP-competitive tyrosine kinase inhibitors selective for L858R+T79oM EGFR modulate the helical assembly of the distal juxtamembrane region in an allosteric fashion. These observations further emphasize that the helical juxtamembrane domain is an essential messenger of structural perturbations from both sides of the plasma membrane.;In Chapter 4, I describe the analysis of combination treatments using tyrosine kinase inhibitor WZ400z and various stapled peptide inhibitors of EGFR. These experiments suggest that the two molecules may demonstrate antagonism in lung cancer cells expressing L858R+T79oM EGFR. I additionally describe the preliminary design and cellular testing for a stapled peptide inhibitor targeted against L858R+T79oM EGFR.