Using Bioluminescence Resonance Energy Transfer (BRET) to Characterize Agonist‐Induced Arrestin Recruitment to Modified and Unmodified G Protein‐Coupled Receptors

Prashant Donthamsetti1, Jose Rafael Quejada1, Jonathan A. Javitch1, Vsevolod V. Gurevich2, Nevin A. Lambert3

1 Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, 2 Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, 3 Department of Pharmacology and Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.14
DOI:  10.1002/0471141755.ph0214s70
Online Posting Date:  September, 2015
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Abstract

G protein‐coupled receptors (GPCRs) represent ∼25% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which are involved in differential signaling pathways. Because functionally selective or biased ligands activate one of these two pathways, they may be superior medications for certain diseases states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for two bioluminescence resonance energy transfer (BRET)‐based assays used to monitor arrestin recruitment to GPCRs. One assay requires modification of GPCRs by fusion to a BRET donor or acceptor moiety, whereas the other can detect arrestin recruitment to unmodified GPCRs. © 2015 by John Wiley & Sons, Inc.

Keywords: G protein‐coupled receptors (GPCRs); arrestin; bioluminescence resonance energy transfer (BRET)

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Receptor‐Arrestin BRET Assay to Measure Ligand‐Induced Recruitment of Arrestin to Receptors
  • Alternate Protocol 1: Arrestin Translocation BRET Assay to Measure Ligand‐Induced Recruitment of Arrestin to Plasma Membrane
  • Support Protocol 1: Optimization of Polyethylenimine (PEI) Concentration for Transfection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Receptor‐Arrestin BRET Assay to Measure Ligand‐Induced Recruitment of Arrestin to Receptors

  Materials
  • HEK293T cells (ATCC, cat. no. CRL‐3216), fully confluent 10‐cm plate
  • HEK293T culture medium (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS; Cellgro, cat. no. 21‐031‐CV)
  • Trypsin (Cellgro, cat. no. 25‐052‐Cl)
  • Mammalian expression plasmids (plasmids that are not commercially available can be obtained from the authors for non‐profit use):
    • Plasmid encoding donor‐fused GPCR, e.g., pcDNA3.1‐D2R‐linker‐Rluc8
    • Plasmid encoding acceptor‐fused arrestin, e.g., pIRES‐puro‐Venus‐linker‐arrestin‐3
    • Plasmid encoding GRK, e.g., pcDNA3.1‐GRK2
    • Empty vector plasmid, e.g., pcDNA3.1 (Life Technologies)
  • DMEM (Gibco, cat. no. 11965‐092)
  • 1 μg/μl polyethyleimine (PEI; see recipe)
  • Agonist stock: e.g., 22 mM dopamine in dH 2O (dopamine hydrochloride available from Sigma‐Aldrich, cat. no. H8502)
  • Antagonist stock: e.g., 44 mM sulpiride in DMSO (S‐(−)‐sulpiride available from Sigma‐Aldrich, cat. no. S7771)
  • DPBS with 40 mg/L sodium bisulfite, pH 7.4, used to reduce dopamine oxidation (required only when using dopamine)
  • 5 mM glucose in DPBS, pH 7.4
  • 5 mM coelenterazine H in absolute ethanol (see recipe)
  • 10‐cm tissue‐culture plates (BD Falcon, cat. no. 353003)
  • Compound plate, 96‐well V‐bottom plates (Greiner Bio‐One, cat. no. 651101)
  • BRET assay plate, white 96‐well flat bottom plates (Greiner Bio‐One, cat. no. 655075) or Black/White 96‐well Isoplate (PerkinElmer, cat. no. 6005030)
  • 12‐Channel multichannel pipettor, 50‐300 μl (Labsystems Finnpipette, cat. no. Z368989) or 5‐50 μl (Labsystems Finnpipette, cat. no. Z678031)
  • Pipette basin (USA Scientific, cat. no. 2320‐2620)
  • Repeater Plus Pipettor (Eppendorf, cat. no. 022260201)
  • Plate reader for luminescence, fluorescence, and BRET detection (e.g., BMG Labtech, Pherastar FS, or Tecan, Infinite F500
  • Software for data analysis (e.g., Microsoft Excel and GraphPad Prism)
NOTE: This protocol can be employed for either a single dopamine agonist curve or a sulpiride antagonist curve at a single dose of dopamine. The procedure can be scaled up, depending on the number of compounds for screening.NOTE: All mammalian tissue culture must be conducted using aseptic techniques in a laminar flow hood. Cells should be maintained in an incubator at 37°C at 5% CO 2.

Alternate Protocol 1: Arrestin Translocation BRET Assay to Measure Ligand‐Induced Recruitment of Arrestin to Plasma Membrane

  Additional Materials (also see protocol 1Basic Protocol)
  • Mammalian expression plasmids:
    • Plasmid encoding for GPCR (e.g., pcDNA3.1‐D2R)
    • Plasmid encoding for Rluc8‐Arrestin‐3‐Sp1
    • Plasmid encoding for mem‐linker‐citrine‐SH3

Support Protocol 1: Optimization of Polyethylenimine (PEI) Concentration for Transfection

  Materials
  • See protocol 1Basic Protocol
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Figures

Videos

Literature Cited

Literature Cited
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