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Spatial control of EGF receptor activation by reversible dimerization on living cells

Abstract

Epidermal growth factor receptor (EGFR) is a type I receptor tyrosine kinase, the deregulation of which has been implicated in a variety of human carcinomas1,2,3,4. EGFR signalling is preceded by receptor dimerization, typically thought to result from a ligand-induced conformational change in the ectodomain that exposes a loop (dimerization arm) required for receptor association. Ligand binding may also trigger allosteric changes in the cytoplasmic domain of the receptor that is crucial for signalling5,6,7. Despite these insights, ensemble-averaging approaches have not determined the precise mechanism of receptor activation in situ. Using quantum-dot-based optical tracking of single molecules8,9,10,11 combined with a novel time-dependent diffusivity analysis, here we present the dimerization dynamics of individual EGFRs on living cells. Before ligand addition, EGFRs spontaneously formed finite-lifetime dimers kinetically stabilized by their dimerization arms12,13,14. The dimers were primed both for ligand binding and for signalling, such that after EGF addition they rapidly showed a very slow diffusivity state that correlated with activation. Although the kinetic stability of unliganded dimers was in principle sufficient for EGF-independent activation, ligand binding was still required for signalling. Interestingly, dimers were enriched in the cell periphery in an actin- and receptor-expression-dependent fashion, resulting in a peripheral enhancement of EGF-induced signalling that may enable polarized responses to growth factors.

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Figure 1: Reversible dimerization of unliganded EGFR: dimerization arm contributes to kinetic and structural stabilities of the dimer state.
Figure 2: EGFR dimers exhibit an EGF-induced very slow diffusion state correlated with kinase activation.
Figure 3: Increased dimerization frequency of EGFR in the cell periphery depends on receptor expression levels.
Figure 4: Peripheral enrichment of EGFR dimers that are primed for EGF binding and ligand-induced activation.

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Acknowledgements

This work was supported in part by NIH grants AR 051448 (to J.S.), AR 051886 (to J.S.), P50 AR 054086 (to J.S.), and the Brown-Coxe Fellowship (to I.C.). We thank S. Marsters for assisting with the cDNA constructs, and K. Schroeder, S. Lee, G. Schaffer, M. Eliott, L. Shao and I. Lax for technical help. M. Sliwkowski, M. Lemmon and the Mellman laboratory provided advice and discussions.

Author Contributions I.C. performed the Fab-QD labelling, preparing for cell transfectants, the imaging and computational analyses. R.A. and R.V. were responsible for the biochemical analyses and antibody preparations. D.T. provided initial direction about TIRFM. J.S. provided essential reagents and critical insight into data interpretation. I.M. was responsible for overseeing all studies and, with I.C., for planning and interpreting all experiments. I.M. and I.C. were primarily responsible for preparing the manuscript, but all authors assisted.

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Correspondence to Ira Mellman.

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I.C., R.A., R.V. and I.M. are full-time employees of Genentech, Inc.

Supplementary information

Supplementary Information

This file contains Supplementary Information sections 1-13 including 9 figures and 1 table, legends to Supplementary Movies 1-2, and Supplementary References. (PDF 955 kb)

Supplementary Movie 1

This movie shows 10Hz continuous acquisition of WT EGFR:Fab-QDs fluorescence images by TIRFM. (MOV 3890 kb)

Supplementary Movie 2

This movie shows time sequence of Grb2-eGFP recruitment to the live A431 cell periphery after the addition of EGF. (MOV 2442 kb)

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Chung, I., Akita, R., Vandlen, R. et al. Spatial control of EGF receptor activation by reversible dimerization on living cells. Nature 464, 783–787 (2010). https://doi.org/10.1038/nature08827

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