In-cell structural dynamics of an EGF receptor during ligand-induced dimer-oligomer transition.

The epidermal growth factor receptor (EGFR) is a membrane protein that regulates cell proliferation, differentiation and survival, and is a drug target for cancer therapy. Ligand-induced activation of the EGFR kinase is generally regarded to require ligand-bound-dimers, while phosphorylation and down-stream signalling is modulated by oligomers. Recent work has unveiled changes in EGFR dynamics from ligand-induced dimerization in membranes extracted from cells, however, less is known about the changes in EGFR dynamics that accompany the ligand-induced oligomerization in a live cell environment. Here, we determine the dynamics of a c-terminal GFP tag attached to EGFR in the unliganded dimer and in the liganded oligomers. By means of the single-frequency polarized phasor ellipse approach we extracted two correlation times on the sub-nanosecond and super-nanosecond timescales, respectively. EGF binding to the EGFR-GFP dimer lengthened the sub-nanosecond correlation time (from 0.1 to 1.3 ns) and shortened the super-nanosecond correlation time (from 210 to 56 ns) of the c-terminal GFP probe. The sub-nanosecond depolarization processes were assigned to electronic energy migration between proximal GFPs in the EGFR dimer or oligomer, while the super-nanosecond correlation times were assigned to nanosecond fluctuations of the GFP probe in the EGFR complex. Accordingly, these results show that ligand binding increased the average separation between the c-terminal tags and increased their rotational mobility. We propose that the dynamics are linked to an inhibitory function of the c-terminal tail in the un-liganded dimer and to the requirement of facile stochastic switching between kinase activation and cytoplasmic adaptor/effector binding in the active oligomers.