Detection and Quantification of beta 2AR Internalization in Living Cells Using FAP-Based Biosensor Technology

作者：Fisher Gregory W; Adler Sally A; Fuhrman Margaret H; Waggoner Alan S; Bruchez Marcel P; Jarvik Jonathan W^*

来源：Journal of Biomolecular Screening, 2010, 15(6): 703-709.

DOI：10.1177/1087057110370892

• G-protein-coupled receptors (GPCRs)
• cell-based assays
• flow cytometry
• imaging
• reporter gene assays

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 (beta 2AR) 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. (Journal of Biomolecular Screening 2010: 703-709)

• 出版日期2010-7