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

Yang Wu1, Phillip H. Tapia2, Jonathan Jarvik3, Alan S. Waggoner3, Larry A. Sklar1

1 Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico, 2 Center for Molecular Discovery, University of New Mexico School of Medicine, Albuquerque, New Mexico, 3 Department of Biological Sciences, Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.43
DOI:  10.1002/0471142956.cy0943s67
Online Posting Date:  January, 2014
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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

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Throughput Flow Cytometry Measurement of Agonist Induced GPCR Internalization
  • Support Protocol 1: High‐Throughput Dose Response Screen for β2AR Agonists
  • Basic Protocol 2: High‐Throughput Flow Cytometry Measurement of Antagonist Stabilized Surface GPCR
  • Support Protocol 2: High‐Throughput Dose Response Screen for β2AR Antagonists
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: High‐Throughput Flow Cytometry Measurement of Agonist Induced GPCR Internalization

  Materials
  • Fluorogen TO1‐2p (Molecular Biosensor and Imaging Center, Carnegie Mellon University)
  • RPMI 1640 full medium (see recipe)
  • U937 cells
  • 20 mM Isoproterenol (ISO; Sigma‐Aldrich) dissolved in dimethyl sulfoxide (DMSO)
  • Prestwick Chemical Library (PCL) in 384 well‐format
  • FITC MESF beads (Becton‐Dickinson)
  • Shallow well 384‐well plates (Greiner Bio‐One)
  • Hemacytometer or similar cell counting device
  • Centrifuge
  • Forma Series II water‐jacketed 37°C, CO 2 incubator (Thermo Fisher)
  • BD Falcon 70‐µm cell strainers (Becton‐Dickinson)
  • Biotek Multiflow liquid dispenser (Multiflow, BioTek Instruments)
  • Biomek FXP or NXP laboratory automation work station equipped with a pin tool for compound transfer (FX or NX; Beckman Coulter)
  • Breathe‐Easy sealing membrane (Research Products International)
  • Eppendorf MixMate plate shaker
  • CyanADP flow cytometer (Beckman Coulter) or Accuri C6 flow cytometer (Becton‐Dickinson)
  • HyperCyt autosampler (IntelliCyt)

Support Protocol 1: High‐Throughput Dose Response Screen for β2AR Agonists

  Additional Materials (also see protocol 1)
  • ICI 118, 551 (ICI, Sigma‐Aldrich)
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Figures

Videos

Literature Cited

  Cole, S.W. and Sood, A.K. 2012. Molecular pathways: Beta‐adrenergic signaling in cancer. Clin. Cancer Res. 18:1201‐1206.
  Cook, D., Guyatt, G., Marshall, J., Leasa, D., Fuller, H., Hall, R., Peters, S., Rutledge, F., Griffith, L., and McLellan, A. 1998. A comparison of sucralfate and ranitidine for the prevention of upper gastrointestinal bleeding in patients requiring mechanical ventilation. N. Engl. J. Med. 338:791‐797.
  Dickinson, L., Khoo, S., and Back, D. 2010. Pharmacokinetics and drug‐drug interactions of antiretrovirals: An update. Antiviral Res. 85:176‐189.
  Fisher, G.W., Adler, S.A., Fuhrman, M.H., Waggoner, A.S., Bruchez, M.P., and Jarvik, J.W. 2010. Detection and quantification of beta 2AR internalization in living cells using FAP‐based biosensor technology. J. Biomol. Screen. 15:703‐709.
  Habashi, J.P., Judge, D.P., Holm, T.M., Cohn, R.D., Loeys, B.L., Cooper, T.K., Myers, L., Klein, E.C., Liu, G., and Calvi, C. 2006. Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome. Science 312:117‐121.
  Holleran, J., Brown, D., Fuhrman, M.H., Adler, S.A., Fisher, G.W., and Jarvik, J.W. 2010. Fluorogen‐activating proteins as biosensors of cell‐surface proteins in living cells. Cytometry A 77:776‐782.
  Hopkins, A.L. and Groom, C.R. 2002. The druggable genome. Nat. Rev. Drug Discov. 1:727‐730.
  Jarvik, J.W, Fisher, G.W., Shi, C., Hennen, L., Hauser, C., Adler, S., and Berget, P.B. 2002. In vivo functional proteomics: Mammalian genome annotation using CD‐tagging. Biotechniques 33:852‐866.
  Overington, J.P., Al‐Lazikani, B., and Hopkins, A.L. 2006. Opinion—How many drug targets are there? Nat. Rev. Drug Discov. 5:993‐996.
  Rask‐Andersen, M., Almen, M.S., and Schioth, H.B. 2011. Trends in the exploitation of novel drug targets. Nat. Rev. Drug Discov. 10:579‐590.
  Szent‐Gyorgyi, C., Schmidt, B.A., Creeger, Y., Fisher, G.W., Zakel, K.L., Adler, S., Fitzpatrick, J.A.J., Woolford, C.A., Yan, Q., Vasilev, K.V., Berget, P.B., Bruchez, M.P., Jarvik, J.W., and Waggoner, A.2008. Fluorogen‐activating single‐chain antibodies for imaging cell surface proteins. Nat. Biotechnol. 26:235‐240.
  Wu, Y., Tapia, P.H., Fisher, G.W., Simons, P.C., Strouse, J.J., Foutz, T., Waggoner, A.S., Jarvik, J.W., and Sklar, L.A. 2012. Discovery of regulators of receptor internalization by high‐throughput flow cytometry. Mol. Pharmacol. 82:645‐657.
  Wu, Y., Tapia, P.H., Fisher, G.W., Waggoner, A.S., Jarvik, J., and Sklar, L.A. 2013. High‐throughput flow cytometry compatible biosensor based on fluorogen‐activating protein technology. Cytometry A 83:220‐226.
  Zhang, J.H., Chung, T.D.Y., and Oldenburg, K.R. 1999. A simple statistical parameter for use in evaluation and validation of high‐throughput screening assays. J. Biomol. Screen. 4:67‐73.
Internet Resources
  http://nmmlsc.health.unm.edu/
  This is the homepage of UNMCMD, with detailed description of the specialties and facilities of the center. One can also find other HTFC assays performed in the center in addition to the one reported herein.
  http://www.mbic.cmu.edu/materials.html
  This Web site provides detail information regarding fluorogens Thiazole Orange (TO) and Malachite Green (MG) derivatives, as well as the FAP available in the center.
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