Bioluminescence Resonance Energy Transfer Assay to Characterize Gi‐Like G Protein Subtype‐Dependent Functional Selectivity

Hideaki Yano1, Marta Sánchez‐Soto1, Sergi Ferré1

1 Integrative Neurobiology Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.33
DOI:  10.1002/cpns.38
Online Posting Date:  October, 2017
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G protein–coupled receptors (GPCRs) comprise the single most targeted protein class in pharmacology. G protein signaling transduces extracellular stimuli such as neurotransmitters into cellular responses. Although preference for a specific GPCR among different G protein families (e.g., Gs‐, Gi‐, or Gq‐like proteins) is often well studied, preference for a specific G protein subtype (e.g., Gi1, Gi2, Gi3, Go1, and Go2) has received little attention. Due to tissue expression differences and potentially exploitable functional differences, G protein subtype‐dependent functional selectivity is an attractive framework to expand GPCR drug development. Herein we present a bioluminescence resonance energy transfer (BRET)‐based method to characterize functional selectivity among Gi‐like protein subtypes. © 2017 by John Wiley & Sons, Inc.

Keywords: GPCR; G protein subtype; BRET; catecholamine; functional selectivity

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Table of Contents

  • Introduction
  • Basic Protocol 1: G Protein Activation BRET Assay
  • Support Protocol 1: Optimization of Transfection Conditions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: G Protein Activation BRET Assay

  • HEK293T cells (ATCC #CRL‐3216)
  • HEK293T culture medium: supplemented DMEM (see recipe)
  • Non‐supplemented DMEM (e.g., Gibco, cat. no. 11960044)
  • Dulbecco's phosphate‐buffered saline (DBPS; e.g., Gibco, cat. no. 14130144)
  • Trypsin/EDTA solution (e.g., Gibco, cat. no. 25300054)
  • Mammalian expression plasmids encoding:
    • Unfused receptor of choice (e.g., pcDNA3.1‐α 2A)
    • Donor‐fused Gαi‐like subunit (e.g., pcDNA3.1‐Gαi1‐Rluc8)
    • Unfused Gβ subunit (e.g., pcDNA3.1‐Gβ1)
    • Acceptor‐fused Gγ subunit (e.g., pcDNA3.1‐Gγ2‐Venus)
  • 1 µg/µl polyethylenimine (PEI; see recipe)
  • Agonist and antagonist appropriate for the receptor of interest
  • Dimethyl sulfoxide (DMSO)
  • DPBS BRET buffer (see recipe)
  • 5 mM coelenterazine H (see recipe)
  • 10‐cm tissue culture plates (e.g., USA Scientific, cat. no. CC7682‐3614)
  • 15‐ml conical tubes
  • Centrifuge
  • Hemocytometer
  • Microscope
  • Cell culture incubator, 37°C, 5% CO 2
  • 1.5‐ml microcentrifuge tubes
  • Vortex
  • Compound plate (for agonist): 96‐well clear U‐bottom plates (e.g., Greiner Bio‐One, cat. no. 650101)
  • Compound plate (for antagonist): 96‐well clear V‐bottom plates (e.g., Greiner Bio‐One, cat. no. 651101)
  • BRET assay plate: white 96‐well flat bottom plates (e.g., Greiner Bio‐One, cat. no. 655075)
  • Fluorescence plate: black 96‐well flat bottom plates (e.g., Greiner Bio‐One, cat. no. 655076)
  • Manual repeater pipette (e.g., Eppendorf, cat. no. EPR‐1000R)
  • 12‐channel multichannel pipette, 10 to 100 µl (e.g., USA scientific, cat. no. 7112‐1100) or 30 to 300 µl (e.g., USA scientific, cat. no. 7112‐3300)
  • Table top centrifuge with 96‐well plate adaptor (e.g., Thermo, Sorvall Legend XTR)
  • Pipette basin (USA scientific, cat. no. 2330‐2220)
  • Plate reader for luminescence, fluorescence, and BRET (e.g., Berthold Technologies Mithras LB 940)
  • Software for data analysis (e.g., Microsoft Excel and GraphPad Prism)
NOTE: All mammalian tissue culture must be conducted using aseptic techniques in a laminar flow hood. Cells should be maintained in a humidified incubator at 37°C with 5% CO 2.NOTE: This protocol entails the transfection of five 10‐cm plates with α 2A adrenergic receptor and one of the Gαi/o subtypes for each plate. For analysis of how to compare different receptors and Gαi/o protein subtypes, see Critical Parameters and Troubleshooting.
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Literature Cited

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