Unit

UNIT 9.43 Real-Time Detection of Protein Trafficking with High-Throughput Flow Cytometry (HTFC) and Fluorogen-Activating Protein (FAP) Base Biosensor

  1. Yang Wu1,2,
  2. Phillip H. Tapia1,
  3. Jonathan Jarvik3,
  4. Alan S. Waggoner3,
  5. Larry A. Sklar1,2

Published Online: 2 JAN 2014

DOI: 10.1002/0471142956.cy0943s67

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Wu, Y., Tapia, P. H., Jarvik, J., Waggoner, A. S. and Sklar, L. A. 2014. Real-Time Detection of Protein Trafficking with High-Throughput Flow Cytometry (HTFC) and Fluorogen-Activating Protein (FAP) Base Biosensor. Current Protocols in Cytometry. 67:9.43:9.43.1–9.43.11.

Author Information

  1. 1

    Center for Molecular Discovery, University of New Mexico School of Medicine, Albuquerque, New Mexico

  2. 2

    Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico

  3. 3

    Department of Biological Sciences, Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania

Publication History

  1. Published Online: 2 JAN 2014

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. Curr. Protoc. Cytom. 67:9.43.1-9.43.11. © 2014 by John Wiley & Sons, Inc.

Keywords:

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