Inside-Out Signaling of Oncogenic EGFR Mutants Promotes Ligand-Independent Dimerization

Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with carcinogenesis. Specific mutations common in non-small cell lung cancer (NSCLC), including EGFR-L858R and EGFR-ΔL747-P753, lead to ligand-independent phosphorylation, however the molecular mechanism by which these mutations in the EGFR kinase domain confer constitutive activity remain unknown. Here, using multiple sub-diffraction-limit imaging modalities, we reveal the altered behavior of NSCLC-associated EGFR mutants within the plasma membrane_including altered receptor dimerization, dynamics, and structure_which collectively dysregulate receptor activity. Using multi-color single particle tracking (SPT) and Hidden Markov Model analysis, EGFR mutants are shown to form stable dimers in the absence of ligand and exhibit a slower mobility that is consistent with receptor signaling. These results were confirmed using two-color, single-molecule super-resolution microscopy (dSTORM) to visualize the spatial distribution of receptors. Receptor clustering was quantified by localization-based cross-correlation analysis to show ligand-induced aggregation of EGFR as well as ligand-independent aggregation of EGFR mutants. Since the receptor ectodomain is known to play a critical role in dimerization, live cell FRET measurements between the EGFR N-terminus and the plasma membrane was used to quantify changes in ectodomain structure. We found that unliganded EGFR mutants are more readily found in the extended conformation, similar to the ligand bound wild type receptor. Therefore, mutation within the kinase domain biases the structural equilibrium of the extracellular domain toward a dimer-competent state.Collectively, these data support a model where oncogenic signaling from NSCLC-associated EGFR mutants is a result of productive dimerization between non-ligand bound receptors. Furthermore, because these mutations are found in the kinase domain, this work introduces the concept that oncogenic EGFR signaling may be controlled in part by a form of “inside-out” signaling.