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J Biomol Screen. 2010 Jul;15(6):703-9. doi: 10.1177/1087057110370892. Epub 2010 May 20.

Detection and quantification of beta2AR internalization in living cells using FAP-based biosensor technology.

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1
Technology Center for Networks and Pathways, Molecular Biosensor and Imaging Center, Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

Abstract

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.

PMID:
20488980
DOI:
10.1177/1087057110370892
[Indexed for MEDLINE]
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