title: Real-time detection of protein trafficking with high-throughput flow cytometry (HTFC) and fluorogen-activating protein (FAP) base biosensor.
abstract: We combined fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G protein-coupled receptors, receptor tyrosine kinases, and ion channels, which were previously not suitable for high-throughput screening by flow cytometry. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ∼ 1200 off-patent drugs was screened against cells expressing FAP-tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a nontraditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification.
authors: Center for Molecular Discovery, University of New Mexico School of Medicine, Albuquerque, New Mexico.|Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico.
authors: Yang
authors: Y
authors: Wu
authors: Center for Molecular Discovery, University of New Mexico School of Medicine, Albuquerque, New Mexico.
authors: Phillip H
authors: PH
authors: Tapia
authors: Department of Biological Sciences, Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania.
authors: Jonathan
authors: J
authors: Jarvik
authors: Department of Biological Sciences, Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania.
authors: Alan S
authors: AS
authors: Waggoner
authors: Center for Molecular Discovery, University of New Mexico School of Medicine, Albuquerque, New Mexico.|Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico.
authors: Larry A
authors: LA
authors: Sklar
mesh_terms: D015374:Biosensing Techniques; D003199:Computer Systems; D004705:Endocytosis; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D020298:U937 Cells
chemical_list: D005456:Fluorescent Dyes; D043562:Receptors, G-Protein-Coupled
keywords: G protein-coupled receptor; fluorogen-activating protein; high-throughput flow cytometer; live-cell assay; receptor trafficking