Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis

  1. Mads Breum Larsen
  2. Mireia Perez Verdaguer
  3. Brigitte F Schmidt
  4. Marcel P Bruchez
  5. Simon C Watkins  Is a corresponding author
  6. Alexander Sorkin  Is a corresponding author
  1. University of Pittsburgh, United States
  2. Carnegie Mellon University, United States

Abstract

Previously we used gene-editing to label endogenous EGF receptor (EGFR) with GFP and demonstrate that picomolar concentrations of EGFR ligand drive signaling and endocytosis of EGFR in tumors in vivo (Pinilla-Macua et al, 2017). We now use gene-editing to insert a fluorogen activating protein (FAP) in the EGFR extracellular domain. Binding of the tandem dye pair MG-Bis-SA to FAP-EGFR provides a ratiometric pH-sensitive model with dual fluorescence excitation and a single far-red emission. The excitation ratio of fluorescence intensities was demonstrated to faithfully report the fraction of FAP-EGFR located in acidic endosomal/lysosomal compartments. Coupling native FAP-EGFR expression with the high method sensitivity has allowed development of a high-throughput assay to measure the rates of clathrin-mediated FAP-EGFR endocytosis stimulated with physiological EGF concentrations. The assay was utilized to screen a phosphatase siRNA library. These studies highlight the utility of endogenous pH-sensitive FAP-receptor chimeras in high-throughput analysis of endocytosis.

Data availability

All source data from library screening are provided in supplemental materials

Article and author information

Author details

  1. Mads Breum Larsen

    Department of Cell Biology, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    No competing interests declared.
  2. Mireia Perez Verdaguer

    Department of Cell Biology, University of Pittsburgh, Pittsburgh, United States
    Competing interests
    No competing interests declared.
  3. Brigitte F Schmidt

    Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, United States
    Competing interests
    No competing interests declared.
  4. Marcel P Bruchez

    Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, United States
    Competing interests
    Marcel P Bruchez, founder of Sharp Edge Laboratories, a company that licensed the FAP technology from CMU. The author has no other competing interests to declare.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7370-4848
  5. Simon C Watkins

    Department of Cell Biology, University of Pittsburgh, Pittsburgh, United States
    For correspondence
    swatkins@pitt.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4092-1552
  6. Alexander Sorkin

    Department of Cell Biology, University of Pittsburgh, Pittsburgh, United States
    For correspondence
    sorkin@pitt.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4446-1920

Funding

National Institutes of Health (GM124186)

  • Simon C Watkins
  • Alexander Sorkin

National Institutes of Health (R01EB017268)

  • Marcel P Bruchez
  • Simon C Watkins

National Institutes of Health (R01GM114075)

  • Marcel P Bruchez
  • Simon C Watkins

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. John Kuriyan, University of California, Berkeley, United States

Version history

  1. Received: February 18, 2019
  2. Accepted: May 7, 2019
  3. Accepted Manuscript published: May 8, 2019 (version 1)
  4. Version of Record published: May 23, 2019 (version 2)

Copyright

© 2019, Larsen 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.

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  1. Mads Breum Larsen
  2. Mireia Perez Verdaguer
  3. Brigitte F Schmidt
  4. Marcel P Bruchez
  5. Simon C Watkins
  6. Alexander Sorkin
(2019)
Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis
eLife 8:e46135.
https://doi.org/10.7554/eLife.46135

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https://doi.org/10.7554/eLife.46135

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