Single Molecule Imaging Reveals that Activating Kinase Domain Mutations Reduce EGFR Mobility and Enhance Dimerization

Abstract

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases which play an important role in a number of physiological responses and have also been identified as a common signaling component in several cancer types. Physiological signaling is initiated by ligand binding which stabilizes EGFR dimers, resulting in auto-phosphorylation of the intracellular kinase domain. Mutation of EGFR is associated with tumor progression due to constitutive activity resulting from several known kinase domain variants. Our previous work has shown that ligand-induced dimerization of wild type EGFR results in decreased receptor mobility, and that this reduction in mobility is dependent on kinase activity. Therefore, we wondered whether ligand-independent activity of EGFR mutants correlates with differences in receptor mobility or dimerization kinetics.Here, we employ multi-color, single particle tracking (SPT) of quantum dots to visualize and quantify the dynamics and interactions of individual receptors within the membrane. Using a novel, high-speed hyperspectral microscope, we are able to track up to eight spectrally distinct quantum dots for visualization and quantification of receptor interactions. From this data, we quantify receptor dimerization kinetics using a Hidden Markov Model. We find that unliganded EGFR mutants exhibit a reduced mobility and longer dimer lifetimes when compared to wild type. These results are consistent with constitutive activity of the EGFR mutants and suggest that the active kinase domain facilitates dimer formation in the absence of ligand. Moreover, we test the effects of clinical tyrosine kinase inhibitors on the single molecule behavior of EGFR mutants. We further examined the mobility and clustering of EGFR within the cellular actin cytoskeleton by hyperspectral tracking over GFP-actin, as well as two-color super-resolution microscopy.