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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Detection of Constitutive Protein‐Protein Interactions by BRET

  Materials
  • 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
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Angers, S, Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., and Bouvier, M. 2000. Detection of beta 2‐adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. U.S.A. 97:3684‐3689.
   Arai, R., Nakagawa, H., Tsumoto, K., Mahoney, W., Kumagai, I., Ueda, H., and Nagamune, T. 2001. Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer. Anal. Biochem. 289:77‐81.
   Bertrand, L., Parent, S., Caron, M., Legault, M., Joly, E., Angers, S., Bouvier, M., Brown, M., Houle, B., and Menard, L. 2002. The BRET2/arrestin assay in stable recombinant cells: A platform to screen for compounds that interact with G protein‐coupled receptors (GPCRS). J. Recept. Signal Transduct. Res. 22:533‐541.
   Boute, N., Boubekeur, S., Lacasa, D., and Issad, T. 2003. Dynamics of the interaction between the insulin receptor and protein tyrosine‐phosphatase 1B in living cells. EMBO Rep. 4:313‐319.
   Boute, N., Pernet, K., and Issad, T. 2001. Monitoring the activation state of the insulin receptor using bioluminescence resonance energy transfer. Mol. Pharmacol. 60:640‐645.
   Charest, P.G., Terrillon, S., and Bouvier, M. 2005. Monitoring agonist‐promoted conformational changes of beta‐arrestin in living cells by intramolecular BRET. EMBO Rep. 6:334‐340.
   Eidne, K.A., Kroeger, K.M., and Hanyaloglu, A.C. 2002. Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells. Trends Endocrinol. Metab. 13:415‐421.
   Gales, C., Rebois, R.V., Hogue, M., Trieu, P., Breit, A., Hebert, T.E., and Bouvier, M. 2005. Real‐time monitoring of receptor and G‐protein interactions in living cells. Nat. Methods 2:177‐184.
   Germain‐Desprez, D., Bazinet, M., Bouvier, M., and Aubry, M. 2003. Oligomerization of transcriptional intermediary factor 1 regulators and interaction with ZNF74 nuclear matrix protein revealed by bioluminescence resonance energy transfer in living cells. J. Biol. Chem. 278:22367‐22373.
   Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A., and Tsien, R.Y. 2001. Reducing the environmental sensitivity of yellow fluorescent protein: Mechanism and applications. J. Biol. Chem. 276:29188‐29194.
   Hamdan, F.F., Audet, M., Garneau, P., Pelletier, J., and Bouvier, M. 2005. High‐throughput screening of G protein‐coupled receptors antagonists using a BRET1‐based β‐arrestin2 recruitment assay. J Biomol. Screen. 10:463‐475.
   Hu, C.D., Chinenov, Y., and Kerppola, T.K. 2002. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9:789‐798.
   Kaihara, A., Kawai, Y., Sato, M., Ozawa, T., and Umezawa, Y. 2003. Locating a protein‐protein interaction in living cells via split Renilla luciferase complementation. Anal. Chem. 75:4176‐4181.
   Mercier, J.F., Salahpour, A., Angers, S., Breit, A., and Bouvier, M. 2002. Quantitative assessment of beta 1‐ and beta 2‐adrenergic receptor homo‐ and heterodimerization by bioluminescence resonance energy transfer. J. Biol. Chem. 277:44925‐44931.
   Michelini, E., Mirasoli, M., Karp, M., Virta, M., and Roda, A. 2004. Development of a bioluminescence resonance energy‐transfer assay for estrogen‐like compound in vivo monitoring. Anal. Chem. 76:7069‐7076.
   Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K., and Miyawaki, A. 2002. A variant of yellow fluorescent protein with fast and efficient maturation for cell‐biological applications. Nat. Biotechnol. 20:87‐90.
   Oakley, R.H., Laporte, S.A., Holt, J.A., Caron, M.G., and Barak, L.S. 2000. Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein‐coupled receptors delineate two major classes of receptors. J. Biol. Chem. 275:17201‐17210.
   Otsuji, T., Okuda‐Ashitaka, E., Kojima, S., Akiyama, H., Ito, S., and Ohmiya, Y. 2004. Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase. Anal. Biochem. 329:230‐237.
   Percherancier, Y., Berchiche, Y.A., Slight, I., Volkmer‐Engert, R., Tamamura, H., Fujii, N., Bouvier, M., and Heveker, N. 2005. Bioluminescence resonance energy transfer reveals ligand‐induced conformational changes in CXCR4 homo‐ and heterodimers. J. Biol. Chem. 280:9895‐9903.
   Perroy, J., Pontier, S., Charest, P.G., Aubry, M., and Bouvier, M. 2004. Real‐time monitoring of ubiquitination in living cells by BRET. Nat. Methods 1:203‐208.
   Pfleger, K.D. and Eidne, K.A. 2004. Monitoring the formation of dynamic G‐protein coupled receptor/protein complexes in living cells. Biochem. J. 385:625‐637.
   Subramanian, C., Kim, B.H., Lyssenko, N.N., Xu, X., Johnson, C.H., and von Arnim, A.G. 2004. The Arabidopsis repressor of light signaling, COP1, is regulated by nuclear exclusion: Mutational analysis by bioluminescence resonance energy transfer. Proc. Natl. Acad. Sci. U.S.A. 101:6798‐6802.
   Terrillon, S., Durroux, T., Mouillac, B., Breit, A., Ayoub, M.A., Taulan, M., Jockers, R., Barberis, C., and Bouvier, M. 2003. Oxytocin and vasopressin V1a and V2 receptors form constitutive homo‐ and heterodimers during biosynthesis. Mol. Endocrinol. 17:677‐691.
   Verhaegent, M. and Christopoulos, T.K. 2002. Recombinant Gaussia luciferase: Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal. Chem. 74:4378‐4385.
   Vrecl, M., Jorgensen, R., Pogacnik, A., and Heding, A. 2004. Development of a BRET2 screening assay using beta‐arrestin 2 mutants. J. Biomol. Screen. 9:322‐333.
   Wilson, T. and Hastings, J.W. 1998. Bioluminescence. Annu. Rev. Cell. Dev. Biol. 14:197‐230.
   Xu, Y., Piston, D.W., and Johnson, C.H. 1999. A bioluminescence resonance energy transfer (BRET) system: Application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. U.S.A. 96:151‐156.
   Zacharias, D.A., Violin, J.D., Newton, A.C., and Tsien, R.Y. 2002. Partitioning of lipid‐modified monomeric GFPs into membrane microdomains of live cells. Science 296:913‐916.
   Zapata‐Hommer, O. and Griesbeck, O. 2003. Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP. BMC Biotechnol. 3:5.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library