Abstract

The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR. The catalytic activity of EGFR is switched on through allosteric activation of one kinase domain by another, and we show that if this is restricted to dimers, then sites in the tail that are proximal to the kinase domain are phosphorylated in only one subunit. We propose a structural model for EGFR multimerization through self-association of ligand-bound dimers, in which the majority of kinase domains are activated cooperatively, thereby boosting tail phosphorylation.

Article and author information

Author details

  1. Yongjian Huang

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  2. Shashank Bharill

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  3. Deepti Karandur

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  4. Seana M Peterson

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  5. Morgan Marita

    Department of Chemistry, University of Akron, Akron, United States
    Competing interests
    No competing interests declared.
  6. Xiaojun Shi

    Department of Chemistry, University of Akron, Akron, United States
    Competing interests
    No competing interests declared.
  7. Megan J Kaliszewski

    Department of Chemistry, University of Akron, Akron, United States
    Competing interests
    No competing interests declared.
  8. Adam W Smith

    Department of Chemistry, University of Akron, Akron, United States
    Competing interests
    No competing interests declared.
  9. Ehud Y Isacoff

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
  10. John Kuriyan

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    jkuriyan@mac.com
    Competing interests
    John Kuriyan, Senior editor, eLife.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Our Xenopus protocol has been approved by Institutional Animal Care and Use Committee (IACUC) of the University of California.

Copyright

© 2016, Huang et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 9,815
    views
  • 2,275
    downloads
  • 163
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Yongjian Huang
  2. Shashank Bharill
  3. Deepti Karandur
  4. Seana M Peterson
  5. Morgan Marita
  6. Xiaojun Shi
  7. Megan J Kaliszewski
  8. Adam W Smith
  9. Ehud Y Isacoff
  10. John Kuriyan
(2016)
Molecular basis for multimerization in the activation of the epidermal growth factor receptor
eLife 5:e14107.
https://doi.org/10.7554/eLife.14107

Share this article

https://doi.org/10.7554/eLife.14107

Further reading

    1. Biochemistry and Chemical Biology
    2. Computational and Systems Biology
    A Sofia F Oliveira, Fiona L Kearns ... Adrian J Mulholland
    Short Report

    The spike protein is essential to the SARS-CoV-2 virus life cycle, facilitating virus entry and mediating viral-host membrane fusion. The spike contains a fatty acid (FA) binding site between every two neighbouring receptor-binding domains. This site is coupled to key regions in the protein, but the impact of glycans on these allosteric effects has not been investigated. Using dynamical nonequilibrium molecular dynamics (D-NEMD) simulations, we explore the allosteric effects of the FA site in the fully glycosylated spike of the SARS-CoV-2 ancestral variant. Our results identify the allosteric networks connecting the FA site to functionally important regions in the protein, including the receptor-binding motif, an antigenic supersite in the N-terminal domain, the fusion peptide region, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected link between different allosteric sites. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spike revealed that glycans do not qualitatively change the internal allosteric pathways but can facilitate the transmission of the structural changes within and between subunits.

    1. Biochemistry and Chemical Biology
    2. Genetics and Genomics
    Conor J Howard, Nathan S Abell ... Nathan B Lubock
    Research Article

    Deep Mutational Scanning (DMS) is an emerging method to systematically test the functional consequences of thousands of sequence changes to a protein target in a single experiment. Because of its utility in interpreting both human variant effects and protein structure-function relationships, it holds substantial promise to improve drug discovery and clinical development. However, applications in this domain require improved experimental and analytical methods. To address this need, we report novel DMS methods to precisely and quantitatively interrogate disease-relevant mechanisms, protein-ligand interactions, and assess predicted response to drug treatment. Using these methods, we performed a DMS of the melanocortin-4 receptor (MC4R), a G-protein-coupled receptor (GPCR) implicated in obesity and an active target of drug development efforts. We assessed the effects of >6600 single amino acid substitutions on MC4R’s function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated Gαs- and Gαq-signaling pathways, which could be used to design drugs that selectively bias MC4R’s activity. We also identified pathogenic variants that are likely amenable to a corrector therapy. Finally, we functionally characterized structural relationships that distinguish the binding of peptide versus small molecule ligands, which could guide compound optimization. Collectively, these results demonstrate that DMS is a powerful method to empower drug discovery and development.