Mechanistic insights on the autoinhibition of the epidermal growth factor receptor (EGFR) and the formation of EGFR dimer and multimers by agonist ligand and tyrosine kinase inhibitors.

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Super-Resolution Microscopy
Sumanth Iyer (1), Sarah Needham (1), Laura Zanetti Domingues (1), Selene Roberts (1), David Clarke (1), Benjamin Davis (1), Daniel Rolfe (1), Marisa Martin-Fernandez (1)
1. UKRI: Science & Technology Facilities Council

Single molecule 


Epidermal Growth Factor Receptor

Receptor structure

Cell signalling

Abstract text


The epidermal growth factor receptor (EGFR) forms autoinhibited and ligand-activated dimers and multimers to elicit its many crucial cellular functions. Neither the mechanisms of formation nor the function of multimer complexes is yet well understood. We have developed and automated a super-resolution method that can provide population-based quantitative information of the structure of the multimers at ~4 nm resolution. Recently we obtained new insights on the mechanisms of EGFR multimer formation, and how the transition between autoinhibited and activated oligomers is catalysed by the presence on the cell surface of a different autoinhibited dimer structure. We also found how this mechanism is implicated in the resistance to some anti-cancer drugs.



A crucial step in the evolution of multi-cellular organisms was the emergence of receptor tyrosine kinases (RTKs). Trafficked to the plasma membrane, their role is to transduce signals in response to cognate growth factor ligands. EGFR was the first RTK to be cloned and this receptor is of paramount importance to cell function and human health as mutations in EGFR and gene amplification are observed in many human cancers. Indeed, cancer is the second cause of death worldwide, accounting for an estimated 9.6 million deaths in 2018. Despite ~4 decades of EGFR research, the information on EGFR structure-function relationships derived from cell-free methods still does not explain the mechanisms underpinning either its normal function, or its dysregulation in cancer. Of particular interest is the distinct roles of dimers and multimers in activation and cancer treatment autoinhibition. Determining the structure-function relationships of EGFR in cells is of paramount importance to find better cancer treatments.



As part of efforts to determine the structure of the EGFR in the physiological cell context, we developed a single molecule localisation super-resolution method, coined fluorophore localisation imaging with photobleaching (FLImP). By exploiting molecular fluorescence-photobleaching steps to localise molecules co-located within diffraction-limited spots, this method measures the lateral separation (r) between such molecules with ~4 nm resolution. Using FLImP in concert with fluorescence resonance energy transfer (FRET), to measure EGFR residue-to-plasma membrane separation (z dimension), and long-time-scale molecular dynamic simulations, we previously determined the atomic resolution structures of ligand-bound multimers, and those of ligand-free EGFR dimers and oligomers coexisting at the plasma membrane of cells (Needham et al., Nat. Commun. 2016; Zanetti-Domingues et al., Nat. Commun. 2018). These results shed new light on the mechanisms of EGFR autoinhibition and phosphorylation in both normal and dysregulated signalling. We have since automated data collection and analysis processes, and added a second spatial dimension to FLImP (from lateral r to independent measurement of x,y dimensions).


Results and Discussion

Using a palette of strategic mutations, we have found that ligand-free EGFR oligomers uses a fail-safe, autoinhibitory mechanism that relies on a triple-safe mechanism combining extracellular, transmembrane, and intracellular contacts, via which the receptor avoids serendipitous activation. We have also found that by achieving such stability, ligand-free oligomers pay a price, which is that on binding agonist ligand, autoinhibited oligomers can form ligand-bound dimers, but not the higher energy multimer structures essential for its physiological function. To achieve the latter, EGFR relies on the presence of catalytic amounts of a different inactive dimer structure, previously proposed by crystallography to be the autoinhibited dimer moiety. This structure conveyed the system with enough free energy to form ligand-bound EGFR multimers. We also found that the failure of anti-cancer drugs in the form of tyrosine kinase inhibitors is related to the mechanism of EGFR oligomerisation.



The super-resolution data at >4 nm resolution to be presented will reveal new insights into the much sought after mechanisms underpinning the formation of autoinhibited, activated and anti-cancer drug-treated EGFR multimers. By these results we are currently beginning to unravel the structure-function relationship encoding the behaviour of cancer mutants responsible for the development of therapeutic resistance, thus revealing potential new candidates for therapeutic intervention.



Needham et al., Nat. Commun. 2016

Zanetti-Domingues et al., Nat. Commun. 2018