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Curr Protoc Cytom. 2014 Jan 2;67:Unit 9.43.. doi: 10.1002/0471142956.cy0943s67.

Real-time detection of protein trafficking with high-throughput flow cytometry (HTFC) and fluorogen-activating protein (FAP) base biosensor.

Author information

1
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.

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.

KEYWORDS:

G protein-coupled receptor; fluorogen-activating protein; high-throughput flow cytometer; live-cell assay; receptor trafficking

PMID:
24510772
PMCID:
PMC3961059
DOI:
10.1002/0471142956.cy0943s67
[Indexed for MEDLINE]
Free PMC Article
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