Fluorescence Localisation Imaging with Photobleaching at 5 Nm Resolution Reveals the Architecture of Basal EGFR Complexes and Mechanisms of Autoinhibition and Activation

Epidermal growth factor (EGF) receptor (EGFR, or HER1/ErbB1) is a cell-surface receptor tyrosine kinase that plays a fundamental signalling role in regulation of cellular metabolism, growth and differentiation. We have developed a super-resolution method based on fluorescence localisation imaging with photobleaching (FLImP) that probes in detail the architecture of dimers and oligomers of the EGFR with a ∼5 nm resolution. Using a fluorescent derivative of EGF, we showed that growth factor-induced EGFR oligomerization is essential to signal regulation of signalling, mediated extracellularly by unoccupied ligand-binding sites, and that it organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. Understanding of the mechanisms by which EGF induces formation of EGFR oligomers lags well behind. Currently founded on ligand-induced interactions between inactive monomers, mounting evidence suggests that besides a monomer cohort, ligand-free EGFRs forms dimers and possibly oligomers whose autoinhibition mechanisms and signalling role are unclear. A key obstacle is that the nature of the ligand-free EGFR species populating the basal state is not well understood. Replacing EGF with antagonist anti-EGFR Affibody or nanobody and combining FLImP with fluorescent resonance energy transfer and single particle tracking we have found two multimeric EGFR basal states that exploit distinct means of suppressing kinase domain function. Ligand-free EGFRs form dimers with tethered ectodomains structurally coupled across the plasma membrane to asymmetric tyrosine kinase domain dimers, autoinhibited when forming part of linear tethered polymer structures. Ligand-free EGFRs also form extended dimers coupled to inactive symmetric kinase dimers. Upon ligand-binding, the ectodomains of the ligand-free tethered polymers become extended and singly-bound dimers sandwich ligand-free ones. The two dynamically linked mechanisms allow face-to-face interactions to mount signals reactive to stimulus strength.