Detection and quantification of beta2AR internalization in living cells using FAP-based biosensor technology.
Ligand-dependent receptor internalization is a feature of numerous signaling systems. In this article, the authors describe a new kind of live-cell biosensor of receptor internalization that takes advantage of fluorogen-activating protein (FAP) technology. Recombinant genes that express the human beta2 adrenergic receptor (beta2AR) with FAP domains at their extracellular N-termini were transduced into mammalian cells. Exposure of the cells to membrane-impermeant fluorogens led to a strong fluorescent signal from the cell surface. Agonist-dependent translocation of the receptor from the surface to the cell interior was readily observed and quantified by fluorescence microscopy or flow cytometry in a homogeneous format without wash or separation steps. The approach described here is generalizable to other receptors and cell surface proteins and is adaptable to a variety of fluorescence-based high-throughput screening platforms.
Fisher
Gregory W
GW
Technology Center for Networks and Pathways, Molecular Biosensor and Imaging Center, Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Adler
Sally A
SA
Fuhrman
Margaret H
MH
Waggoner
Alan S
AS
Bruchez
Marcel P
MP
Jarvik
Jonathan W
JW
D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D000818:Animals; D001681:Biological Assay; D015374:Biosensing Techniques / Q000379:methods*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D041681:NIH 3T3 Cells; D011433:Propranolol / Q000494:pharmacology; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D013194:Staining and Labeling; D013499:Surface Properties / Q000187:drug effects; D013997:Time Factors
D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D005456:Fluorescent Dyes; D018343:Receptors, Adrenergic, beta-2; D011433:Propranolol; D007545:Isoproterenol