Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis
Mads Breum Larsen,
Mireia Perez Verdaguer,
Brigitte F Schmidt,
Marcel P Bruchez,
Simon C Watkins,
Alexander Sorkin
Affiliations
Mads Breum Larsen
Department of Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, United States
Mireia Perez Verdaguer
Department of Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, United States
Brigitte F Schmidt
Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, United States
Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, United States; Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, United States; Department of Chemistry, Carnegie Mellon University, Pittsburgh, United States; Sharp Edge Laboratories, Pittsburgh, United States
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.
