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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.