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Molecular Pharmacology

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Research ArticleArticle

Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry

Yang Wu, Phillip H. Tapia, Gregory W. Fisher, Peter C. Simons, J. Jacob Strouse, Terry Foutz, Alan S. Waggoner, Jonathan Jarvik and Larry A. Sklar
Molecular Pharmacology October 2012, 82 (4) 645-657; DOI: https://doi.org/10.1124/mol.112.079897
Yang Wu
Department of Pathology (Y.W., L.A.S.) and Center for Molecular Discovery (Y.W., P.H.T., P.C.S., J.J.S., T.F., L.A.S.), School of Medicine, University of New Mexico, Albuquerque, New Mexico; Department of Biological Science (A.S.W, J.J.) and Technology Center of Networks and Pathways (G.W.F., A.S.W., J.J.), Carnegie Mellon University, Pittsburgh, Pennsylvania
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Phillip H. Tapia
Department of Pathology (Y.W., L.A.S.) and Center for Molecular Discovery (Y.W., P.H.T., P.C.S., J.J.S., T.F., L.A.S.), School of Medicine, University of New Mexico, Albuquerque, New Mexico; Department of Biological Science (A.S.W, J.J.) and Technology Center of Networks and Pathways (G.W.F., A.S.W., J.J.), Carnegie Mellon University, Pittsburgh, Pennsylvania
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Gregory W. Fisher
Department of Pathology (Y.W., L.A.S.) and Center for Molecular Discovery (Y.W., P.H.T., P.C.S., J.J.S., T.F., L.A.S.), School of Medicine, University of New Mexico, Albuquerque, New Mexico; Department of Biological Science (A.S.W, J.J.) and Technology Center of Networks and Pathways (G.W.F., A.S.W., J.J.), Carnegie Mellon University, Pittsburgh, Pennsylvania
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Peter C. Simons
Department of Pathology (Y.W., L.A.S.) and Center for Molecular Discovery (Y.W., P.H.T., P.C.S., J.J.S., T.F., L.A.S.), School of Medicine, University of New Mexico, Albuquerque, New Mexico; Department of Biological Science (A.S.W, J.J.) and Technology Center of Networks and Pathways (G.W.F., A.S.W., J.J.), Carnegie Mellon University, Pittsburgh, Pennsylvania
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J. Jacob Strouse
Department of Pathology (Y.W., L.A.S.) and Center for Molecular Discovery (Y.W., P.H.T., P.C.S., J.J.S., T.F., L.A.S.), School of Medicine, University of New Mexico, Albuquerque, New Mexico; Department of Biological Science (A.S.W, J.J.) and Technology Center of Networks and Pathways (G.W.F., A.S.W., J.J.), Carnegie Mellon University, Pittsburgh, Pennsylvania
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Terry Foutz
Department of Pathology (Y.W., L.A.S.) and Center for Molecular Discovery (Y.W., P.H.T., P.C.S., J.J.S., T.F., L.A.S.), School of Medicine, University of New Mexico, Albuquerque, New Mexico; Department of Biological Science (A.S.W, J.J.) and Technology Center of Networks and Pathways (G.W.F., A.S.W., J.J.), Carnegie Mellon University, Pittsburgh, Pennsylvania
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Alan S. Waggoner
Department of Pathology (Y.W., L.A.S.) and Center for Molecular Discovery (Y.W., P.H.T., P.C.S., J.J.S., T.F., L.A.S.), School of Medicine, University of New Mexico, Albuquerque, New Mexico; Department of Biological Science (A.S.W, J.J.) and Technology Center of Networks and Pathways (G.W.F., A.S.W., J.J.), Carnegie Mellon University, Pittsburgh, Pennsylvania
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Jonathan Jarvik
Department of Pathology (Y.W., L.A.S.) and Center for Molecular Discovery (Y.W., P.H.T., P.C.S., J.J.S., T.F., L.A.S.), School of Medicine, University of New Mexico, Albuquerque, New Mexico; Department of Biological Science (A.S.W, J.J.) and Technology Center of Networks and Pathways (G.W.F., A.S.W., J.J.), Carnegie Mellon University, Pittsburgh, Pennsylvania
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Larry A. Sklar
Department of Pathology (Y.W., L.A.S.) and Center for Molecular Discovery (Y.W., P.H.T., P.C.S., J.J.S., T.F., L.A.S.), School of Medicine, University of New Mexico, Albuquerque, New Mexico; Department of Biological Science (A.S.W, J.J.) and Technology Center of Networks and Pathways (G.W.F., A.S.W., J.J.), Carnegie Mellon University, Pittsburgh, Pennsylvania
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Abstract

We developed a platform combining 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 facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the β2-adrenergic receptor (β2AR) system. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged β2ARs, all 33 known β2AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway.

Footnotes

  • This research was supported by the National Institutes of Health National Institute of Mental Health [Grants 1U54-MH084690-02, 5U54-MH084690-03, 1R03-MH093192]; and the National Institutes of Health National Center for Research Resources [Grant 5U54-RR022241].

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    http://dx.doi.org/10.1124/mol.112.079897.

  • ABBREVIATIONS:

    GPCR
    G protein-coupled receptor
    FAP
    fluorogen-activating protein
    β2AR
    β2-adrenergic receptor
    αAR
    α-adrenergic receptor
    MCF
    median channel fluorescence
    ISO
    isoproterenol
    HCS
    high-content screening
    HTS
    high-throughput screening
    HTFC
    high- throughput flow cytometry
    PCL
    Prestwick Chemical Library
    TO1-2p
    sulfonated thiazole orange coupled to diethylene glycol diamine
    ICI 118,551
    3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol
    RV
    response value
    DHA
    dihydroalprenolol
    GFP
    green fluorescent protein
    DMSO
    dimethylsulfoxide
    HPSM
    HEPES-potassium-sodium-magnesium
    ALP
    alprenolol
    PRO
    propranolol
    LEVON
    levonordefrin
    ANI
    anisomycin
    NAF
    naftopidil
    DOM
    domperidone
    PIZ
    pizotifen
    BUC
    bucindolol
    CAV
    carvedilol
    CYC
    cycloheximide
    AC
    adenylate cyclase
    ERK
    extracellular signal-regulated kinase
    MAPK
    mitogen-activated protein kinase.

  • Received May 8, 2012.
  • Accepted July 5, 2012.
  • Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 82 (4)
Molecular Pharmacology
Vol. 82, Issue 4
1 Oct 2012
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Research ArticleArticle

Discovery of Receptor Internalization Regulators

Yang Wu, Phillip H. Tapia, Gregory W. Fisher, Peter C. Simons, J. Jacob Strouse, Terry Foutz, Alan S. Waggoner, Jonathan Jarvik and Larry A. Sklar
Molecular Pharmacology October 1, 2012, 82 (4) 645-657; DOI: https://doi.org/10.1124/mol.112.079897

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Research ArticleArticle

Discovery of Receptor Internalization Regulators

Yang Wu, Phillip H. Tapia, Gregory W. Fisher, Peter C. Simons, J. Jacob Strouse, Terry Foutz, Alan S. Waggoner, Jonathan Jarvik and Larry A. Sklar
Molecular Pharmacology October 1, 2012, 82 (4) 645-657; DOI: https://doi.org/10.1124/mol.112.079897
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