title: High-throughput flow cytometry compatible biosensor based on fluorogen activating protein technology.
abstract: Monitoring the trafficking of multiple proteins simultaneously in live cells is of great interest because many receptor proteins are found to function together with others in the same cell. However, existing fluorescent labeling techniques have restricted the mechanistic study of functional receptor pairs. We have expanded a hybrid system combining fluorogen-activating protein (FAP) technology and high-throughput flow cytometry to a new type of biosensor that is robust, sensitive, and versatile. This provides the opportunity to study multiple trafficking proteins in the same cell. Human beta2 adrenergic receptor (β2AR) fused with FAP AM2.2 and murine C-C chemokines receptor type 5 fused with FAP MG13 was chosen for our model system. The function of the receptor and the binding between MG13 and fluorogen MG-2p have been characterized by flow cytometry and confocal microscopy assays. The binding of fluorogen and the FAP pair is highly specific, while both FAP-tagged fusion proteins function similarly to their wild-type counterparts. The system has successfully served as a counter screen assay to eliminate false positive compounds identified in a screen against NIH Molecular Libraries Small Molecule Repository targeting regulators of the human β2AR.
authors.affiliation: UNM Center for Molecular Discovery, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA. yawu@salud.unm.edu
authors.forename: Yang
authors.initials: Y
authors.lastname: Wu
authors.forename: Phillip H
authors.initials: PH
authors.lastname: Tapia
authors.forename: Gregory W
authors.initials: GW
authors.lastname: Fisher
authors.forename: Alan S
authors.initials: AS
authors.lastname: Waggoner
authors.forename: Jonathan
authors.initials: J
authors.lastname: Jarvik
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology; D015374:Biosensing Techniques / Q000379:methods*; D018946:Chemokine CCL5 / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D006801:Humans; D020128:Inhibitory Concentration 50; D007545:Isoproterenol / Q000494:pharmacology; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis; D019713:Receptors, CCR5 / Q000819:agonists / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry*; D012394:Rosaniline Dyes / Q000737:chemistry; D020298:U937 Cells
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D018946:Chemokine CCL5; D005456:Fluorescent Dyes; D018343:Receptors, Adrenergic, beta-2; D019713:Receptors, CCR5; D011993:Recombinant Fusion Proteins; D012394:Rosaniline Dyes; C005095:malachite green; D007545:Isoproterenol