Combining SRET2 and BiFC to Study GPCR Heteromerization and Protein–Protein Interactions

  • Amina M. Bagher
  • Melanie E. M. Kelly
  • Eileen M. Denovan-WrightEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1947)


G protein-coupled receptors (GPCRs) are the target for many drugs. Evidence continues to accumulate demonstrating that multiple receptors form homo- and heteromeric complexes, which in turn dynamically couple with G proteins, and other interacting proteins. Here, we describe a method to simultaneously determine the identity of up to four distinct constituents of GPCR complexes using a combination of sequential bioluminescence resonance energy transfer 2—fluorescence resonance energy transfer (SRET2) with bimolecular fluorescence complementation (BiFC). The method is amenable to moderate throughput screening of changes in response to ligands and time-course analysis of protein–protein oligomerization.

Key words

BiFC Bimolecular fluorescence complementation BRET2, Bioluminescence energy transfer 2 FRET, Fluorescence resonance energy transfer SRET2, Sequential BRET2-FRET 


  1. 1.
    Rios CD, Jordan BA, Gomes I et al (2001) G-protein-coupled receptor dimerization: Modulation of receptor function. Pharmacol Ther 92:71–87CrossRefGoogle Scholar
  2. 2.
    Milligan G (2004) G protein-coupled receptor dimerization: function and ligand pharmacology. Mol Pharmacol 66:1–7CrossRefGoogle Scholar
  3. 3.
    Milligan G (2009) G protein-coupled receptor hetero-dimerization: contribution to pharmacology and function. Br J Pharmacol 158:5–14CrossRefGoogle Scholar
  4. 4.
    Ferré S, Casadó V, Devi LA et al (2014) G protein-coupled receptor oligomerization revisited: functional and pharmacological perspectives. Pharmacol Rev 66:413–434CrossRefGoogle Scholar
  5. 5.
    Ferré S (2015) The GPCR heterotetramer: challenging classical pharmacology. Trends Pharmacol Sci 36:145–152CrossRefGoogle Scholar
  6. 6.
    Franco R, Martínez-Pinilla E, Lanciego JL et al (2016) Basic pharmacological and structural evidence for class A G-protein-coupled receptor heteromerization. Front Pharmacol 7:76CrossRefGoogle Scholar
  7. 7.
    Gaitonde SA, González-Maeso J (2017) Contribution of heteromerization to G protein-coupled receptor function. Curr Opin Pharmacol 32:23–31CrossRefGoogle Scholar
  8. 8.
    Gomes I, Ayoub MA, Fujita W et al (2016) G protein-coupled receptor heteromers. Annu Rev Pharmacol Toxicol 56:403–425CrossRefGoogle Scholar
  9. 9.
    James JR, Oliveira MI, Carmo AM et al (2006) A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer. Nat Methods 3:1001–1006CrossRefGoogle Scholar
  10. 10.
    Pfleger KD, Seeber RM, Eidne KA (2006) Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions. Nat Protoc 1:337–345CrossRefGoogle Scholar
  11. 11.
    Marullo S, Bouvier M (2007) Resonance energy transfer approaches in molecular pharmacology and beyond. Trends Pharmacol Sci 28:362–365CrossRefGoogle Scholar
  12. 12.
    Carriba P, Navarro G, Ciruela F et al (2008) Detection of heteromerization of more than two proteins by sequential BRET-FRET. Nat Methods 5:727–733CrossRefGoogle Scholar
  13. 13.
    Navarro G, McCormick PJ, Mallol J et al (2013) Detection of receptor heteromers involving dopamine receptors by the sequential BRET-FRET technology. Methods Mol Biol 964:95–105CrossRefGoogle Scholar
  14. 14.
    Hu CD, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9:789–798CrossRefGoogle Scholar
  15. 15.
    Vidi PA, Przybyla JA, Hu CD et al (2010) Visualization of G protein-coupled receptor (GPCR) interactions in living cells using bimolecular fluorescence. Curr Protoc Neurosci. Chapter 5:Unit 5.29Google Scholar
  16. 16.
    Shyu YJ, Liu H, Deng X et al (2006) Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. Biotech 40:61–66CrossRefGoogle Scholar
  17. 17.
    Bagher AM, Laprairie RB, Toguri J et al (2017) Bidirectional allosteric interactions between cannabinoid receptor type 1 (CB1) and the dopamine receptor type 2 (D2) agonists mediated through CB1/D2L heterotetramers (2017). Eur J Pharmacol 813:66–83CrossRefGoogle Scholar
  18. 18.
    Bagher AM, Laprairie RB, Kelly MEM et al (2018) Methods to quantify cell signaling and GPCR receptor ligand bias: characterization of drugs that target the endocannabinoid receptors in Huntington’s disease. Methods Mol Biol 1780:549–571CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Amina M. Bagher
    • 1
    • 2
  • Melanie E. M. Kelly
    • 1
    • 3
  • Eileen M. Denovan-Wright
    • 1
    Email author
  1. 1.Department of PharmacologyDalhousie UniversityHalifaxCanada
  2. 2.Department of Pharmacology and ToxicologyKing AbdulAziz UniversityJeddahSaudi Arabia
  3. 3.Department of Ophthalmology and Visual SciencesDalhousie UniversityHalifaxCanada

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