Spatial control of EGF receptor activation by reversible dimerization on living cells (original) (raw)

Nature volume 464, pages 783–787 (2010)Cite this article

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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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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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Authors and Affiliations

  1. Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA ,
    Inhee Chung, Robert Akita, Richard Vandlen & Ira Mellman
  2. Department of Cell Biology,,
    Derek Toomre
  3. Deparment of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA ,
    Joseph Schlessinger

Authors

  1. Inhee Chung
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  2. Robert Akita
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  3. Richard Vandlen
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  4. Derek Toomre
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  5. Joseph Schlessinger
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  6. Ira Mellman
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Corresponding author

Correspondence toIra Mellman.

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Competing interests

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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Editorial Summary

A time and a place for EGFR

Here, the dimerization dynamics of individual epidermal growth factor receptor (EGFR) molecules have been determined in living cells in real time using a quantum-dot-based approach. Signalling by EGFR, a type I receptor kinase that has been implicated in a number if human carcinomas, is preceded by receptor dimerization. It has been widely assumed that ligand binding is required to trigger conformation changes in EGFR, which in turn lead to dimer formation and kinase activation. However, this new work shows that dimerization is a continuous and reversible process, with ligand binding serving to stabilize receptor dimers by decreasing their rate of dissociation, thereby increasing the stable dimer population in a given cell. In addition, the location of the receptor is important, with spontaneous dimer formation being more prevalent at the cell margins than at the cell centre.