Monitoring Protein‐Protein Interactions in Living Cells by Bioluminescence Resonance Energy Transfer (BRET)

Fadi F. Hamdan1, Yann Percherancier1, Billy Breton1, Michel Bouvier1

1 University of Montreal, Montreal, Quebec
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.23
DOI:  10.1002/0471142301.ns0523s34
Online Posting Date:  February, 2006
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Bioluminescence resonance energy transfer (BRET) allows monitoring of protein‐protein interactions in real time in living cells. One candidate interacting protein is fused to a luminescent energy donor, such as Renilla luciferase, and the other to a fluorescent energy acceptor, such the green fluorescent protein (GFP), and the two are then coexpressed in the same cells. If the two proteins interact, their close proximity allows nonradiative energy transfer (BRET) between the luciferase and the GFP. BRET does not occur if the two proteins are separated by more than 100 Å, making the technique ideal for monitoring protein‐protein interactions in biological systems. This unit describes the use of BRET to study constitutive and agonist‐promoted interactions among signaling molecules, as illustrated by the homodimerization of the CXCR4 receptor and the recruitment of β‐arrestin2 to agonist‐activated G‐protein‐coupled receptors. This noninvasive and homogeneous assay provides a robust and sensitive proteomic platform with applications for basic science research and drug discovery.

Keywords: protein‐protein interaction; GFP; energy transfer; luminescence

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Table of Contents

  • Basic Protocol 1: Detection of Constitutive Protein‐Protein Interactions by BRET
  • Alternate Protocol 1: Measurement of Dynamic Protein‐Protein Interactions by BRET: Example Of β‐Arrestin2 Recruitment to Agonist‐Activated GPCRs as a Functional Readout of Receptor Activation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Detection of Constitutive Protein‐Protein Interactions by BRET

  • CXCR4 template DNA (entire coding sequence)
  • Control template DNA: coding sequence of γ‐aminobutyric acid type B (GBR2) receptor
  • Coding sequence of EYFP
  • Mammalian expression plasmids:
    • Plasmids encoding a codon‐humanized R. reniformis luciferase (hRluc): phRluc‐N (allows cloning of candidate proteins upstream of hRluc) and phRluc‐C (allows cloning of candidate proteins downstream of hRluc); available in all translation frames from Perkin‐Elmer; humanized Rluc plasmids are also available from Promega
    • Plasmids encoding EYFP: pEYFP‐N (to clone upstream of EYFP) and pEYFP‐C (to clone downstream of EYFP), available from BD Biosciences
  • HEK293T cells (ATCC #CRL 11554)
  • HEK293T cell culture medium (see recipe)
  • Transfection reagent (also see appendix 11), e.g., Fugene 6 (Roche Applied Science)
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A) containing 0.5 mM MgCl 2
  • PBS, pH 7.4 ( appendix 2A) containing 5 mM EDTA
  • PBS, pH 7.4 ( appendix 2A) containing 0.5 mM MgCl 2
  • PBS, pH 7.4 ( appendix 2A) containing 0.5 mM MgCl 2 and 0.1% (w/v) glucose
  • 50 µM coelenterazine‐h (Nanolight) in PBS, pH 7.4 ( appendix 2A); prepare fresh from 1 mM stock (see recipe)
  • Test compound (e.g., receptor agonist or antagonist)
  • 6‐well tissue culture plates
  • White opaque‐bottom polystyrene 96‐well plates (for luminescence and BRET measurement; Costar or Perkin‐Elmer)
  • White clear‐bottom polystyrene 96‐well plates (for fluorescence measurements; Costar or Perkin‐Elmer)
  • Plate reader for luminescence, fluorescence, and BRET detection (see discussion of instrumentation in Critical Parameters and Troubleshooting)
  • Software for data analysis; Microsoft Excel or Graph Pad Prism (unit 7.5)
  • Additional reagents and equipment for PCR (CPMB UNIT ), cloning (CPMB Chapter 3), mammalian cell culture ( appendix 3B), and cell counting ( appendix 3B) or protein assay (CPMB UNIT )

Alternate Protocol 1: Measurement of Dynamic Protein‐Protein Interactions by BRET: Example Of β‐Arrestin2 Recruitment to Agonist‐Activated GPCRs as a Functional Readout of Receptor Activation

  • V2R, β2AR, and β‐arrestin2 coding sequences
  • 8‐arginine‐vasopressin (8‐AVP)
  • Isoproterenol
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Literature Cited

Literature Cited
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