Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Thursday Speaker Abstracts

Single-Molecule Visualisation of the Mechanisms of Autoinhibition and Dysregulation of the EGFR on Living Cell Membranes Laura C. Zanetti Domingues 1 , Sarah R. Needham 1 , Christopher J. Tynan 1 , Dimitrios Korovesis 1 , Selene K. Roberts 1 , David T. Clarke 1 , Michela Perani 2 , Peter J. Parker 2,3 , Daniel J. Rolfe 1 , Marisa Martin-Fernandez 1 . 1 STFC, Didcot, United Kingdom, 2 King's College London, London, United Kingdom, 3 London Research Institute, London, United Kingdom. EGFR family receptors are involved in a variety of epithelial tumours. While the solution structure is well-known, the mechanisms that regulate its activity at the membrane level are less well understood. In particular, the conformational linkage between extracellular and intracellular domains and the effect of inhibitors on full-length structure are yet to be fully elucidated. By combining multicolour Single-Particle Tracking (SPT), fluorescence localisation imaging with photobleaching (FLImP) at a resolution of <7 nm, and fluorescent resonance energy transfer (FRET) on a stably transfected CHO cell model, we have probed the geometry and the ability of EGFR to oligomerise and activate under perturbations that disrupt regulation mechanisms proposed by studies of isolated EGFR domains. We have observed conformational coupling across the membrane in intact cells, and confirmed a role for the extended conformation activation, as suggested by crystallographic studies. Moreover, we have confirmed the existence of ECD-mediated quasi-dimers in the basal state, and the inhibitory role of plasma-membrane/kinase domain interactions. Finally, we have uncovered an intermediate ligand-independent conformation in which the kinase domains and C-terminus associate to reduce the energy of activation, which results in hyper-activation in presence of EGF. This conformation is achieved by blocking protein-lipid interactions or inhibitor treatment. In summary, our investigations have managed to assemble a coherent view of how intact EGFRs are regulated in their native membrane environment. Furthermore, we have identified an intermediate oligomeric association driven by tyrosine kinase inhibitors, which might be relevant for resistance mechanisms.

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