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

Mads Breum Larsen,Alexander Sorkin,Mireia Perez Verdaguer,Marcel P Bruchez,Brigitte F Schmidt,Simon C Watkins

doi:10.7554/elife.46135

Mads Breum Larsen, Alexander Sorkin + Show 4 more

Open Access

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

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Journal: eLife	Publication Date: May 8, 2019
Citations: 7	License type: CC BY 4.0

Affiliation: University of Pittsburgh, Carnegie Mellon University

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.

Highlights

Ligand binding to EGF receptor (EGFR) at the cell surface leads to activation of the receptor tyrosine kinase activity, initiation of multiple signaling cascades and cell proliferation, differentiation or oncogenic transformation
We have previously developed fluorogen activating protein (FAP)-tagged antibodies and nanobodies to EGFR to study its expression and endocytosis in cells with high receptor levels (Ackerman et al, 2018; Tan et al, 2017; Wang et al, 2017; Wang et al, 2015)
To generate the pH-sensitive EGFR, FAP was inserted at the amino-terminus of the EGFR molecule in the EGFR gene locus downstream of the sequence encoding the signal peptide and upstream of the sequence of the mature EGFR using CRISPR/Cas9 gene-editing method (Figure 1A)

Summary

Introduction

Ligand binding to EGFR at the cell surface leads to activation of the receptor tyrosine kinase activity, initiation of multiple signaling cascades and cell proliferation, differentiation or oncogenic transformation (reviewed in Lemmon and Schlessinger, 2010). The use of overexpressed recombinant EGFR and high ligand concentrations favor EGFR internalization through clathrin-independent rather than CME pathways To resolve these experimental inconsistences, we were prompted to renew our efforts to systematically analyze proteins and processes involved in fundamental mechanisms underlying EGFR trafficking. Our recent study using endogenously expressed GFP-tagged EGFR allowed the demonstration that low concentrations of EGFR ligands are sufficient to drive EGFR-dependent growth of mouse tumor xenografts and that EGFR endocytosis in tumors in vivo is clathrin-mediated (PinillaMacua et al, 2017). These results inspired us to develop a more sophisticated endocytosis assay that would allow a quantitative analysis of the receptor traffic under physiological conditions. Combining the MG-Bis-SA/FAP-based methodology and cells expressing endogenous EGFR with FAP at the amino-terminus allowed the development of an experimental model system that is amenable to a simple and highly sensitive single-cell readout, in a high-throughput assay format, such that we can monitor and measure constitutive or ligand-induced EGFR endocytosis under physiological conditions, for example using low EGFR ligand concentrations

Results and discussion

Materials and methods

Funding Funder National Institutes of Health