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Bimolecular Fluorescence Complementation Methodology to Study G Protein-Coupled Receptor Dimerization in Living Cells

  • Doungkamol Alongkronrusmee
  • Val J. Watts
  • Richard M. van Rijn
Protocol
Part of the Neuromethods book series (NM, volume 140)

Abstract

Proteins, such as G protein-coupled receptors (GPCRs), can interact with each other to form dimeric or higher order oligomeric complexes with novel pharmacological properties. GPCRs play a crucial role in numerous physiological processes and diseases, and much research has been performed to prove the existence of GPCR heterodimerization and to investigate the physiological role of the heterodimers. GPCRs are targeted by roughly 25% of all FDA-approved drugs, but heterodimers may represent an untapped additional source of novel drug targets. However, study of GPCR heteromers is not trivial, with most methods having distinct strengths and weaknesses. One method to study GPCR dimerization in living cells is through bimolecular fluorescence complementation (BiFC). The BiFC technique is based on the complementation of two nonfluorescent fragments of a fluorescent protein that is facilitated by fusing the fragments to two interacting proteins. The advantage of BiFC over alternative resonance energy transfer techniques is a high signal-to-noise ratio due to its strong intrinsic fluorescence without exogenous fluorogenic or chromogenic agents required. Here we provide a detailed description of protocols to measure dimerization-induced BiFC in a low-throughput, high-resolution approach using confocal microscopy and in a medium-throughput, low-resolution approach using an automated cell imaging multimode plate reader (Biotek Cytation 3). In this chapter, we use mu and delta opioid receptor heterodimerization to provide a step-by-step BiFC protocol; however, the protocol can be adapted for use with other receptors as well as other confocal or automated microscopes.

Key words

Bimolecular fluorescence complementation G protein-coupled receptor Dimerization Screening Confocal microscopy 

Notes

Acknowledgements

This work was supported by funding from the National Institute on Mental Health (R33MH101673) to Dr. Watts.

References

  1. 1.
    Maggio R, Novi F, Scarselli M, Corsini GU (2005) The impact of G-protein-coupled receptor hetero-oligomerization on function and pharmacology. FEBS J 272(12):2939–2946CrossRefPubMedGoogle Scholar
  2. 2.
    Fuxe K, Canals M, Torvinen M, Marcellino D, Terasmaa A, Genedani S, Leo G, Guidolin D, Diaz-Cabiale Z, Rivera A, Lundstrom L, Langel U, Narvaez J, Tanganelli S, Lluis C, Ferre S, Woods A, Franco R, Agnati LF (2007) Intramembrane receptor-receptor interactions: a novel principle in molecular medicine. J Neural Transm (Vienna) 114(1):49–75CrossRefGoogle Scholar
  3. 3.
    Gomes I, Ayoub MA, Fujita W, Jaeger WC, Pfleger KD, Devi LA (2016) G protein-coupled receptor Heteromers. Annu Rev Pharmacol Toxicol 56:403–425. https://doi.org/10.1146/annurev-pharmtox-011613-135952 CrossRefPubMedGoogle Scholar
  4. 4.
    Pin JP, Neubig R, Bouvier M, Devi L, Filizola M, Javitch JA, Lohse MJ, Milligan G, Palczewski K, Parmentier M, Spedding M (2007) International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the recognition and nomenclature of G protein-coupled receptor heteromultimers. Pharmacol Rev 59(1):5–13. https://doi.org/10.1124/pr.59.1.5 CrossRefPubMedGoogle Scholar
  5. 5.
    Vidi PA, Ejendal KF, Przybyla JA, Watts VJ (2011) Fluorescent protein complementation assays: new tools to study G protein-coupled receptor oligomerization and GPCR-mediated signaling. Mol Cell Endocrinol 331(2):185–193. https://doi.org/10.1016/j.mce.2010.07.011 CrossRefPubMedGoogle Scholar
  6. 6.
    Truong K, Ikura M (2001) The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo. Curr Opin Struct Biol 11(5):573–578CrossRefPubMedGoogle Scholar
  7. 7.
    Pfleger KD, Eidne KA (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 3(3):165–174CrossRefPubMedGoogle Scholar
  8. 8.
    Koch S, Helbing I, Bohmer SA, Hayashi M, Claesson-Welsh L, Soderberg O, Bohmer FD (2016) In situ proximity ligation assay (in situ PLA) to assess PTP-protein interactions. Methods Mol Biol 1447:217–242. https://doi.org/10.1007/978-1-4939-3746-2_13 CrossRefPubMedGoogle Scholar
  9. 9.
    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(4):789–798CrossRefPubMedGoogle Scholar
  10. 10.
    Kodama Y, Hu CD (2012) Bimolecular fluorescence complementation (BiFC): a 5-year update and future perspectives. BioTechniques 53(5):285–298CrossRefPubMedGoogle Scholar
  11. 11.
    Shyu YJ, Hu CD (2008) Fluorescence complementation: an emerging tool for biological research. Trends Biotechnol 26(11):622–630. https://doi.org/10.1016/j.tibtech.2008.07.006 CrossRefPubMedGoogle Scholar
  12. 12.
    Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40(3):428–438. https://doi.org/10.1111/j.1365-313X.2004.02219.x CrossRefPubMedGoogle Scholar
  13. 13.
    Sung MK, Huh WK (2007) Bimolecular fluorescence complementation analysis system for in vivo detection of protein-protein interaction in Saccharomyces cerevisiae. Yeast 24(9):767–775CrossRefPubMedGoogle Scholar
  14. 14.
    Kerppola TK (2008) Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu Rev Biophys 37:465–487CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Shyu YJ, Suarez CD, Hu CD (2008) Visualization of ternary complexes in living cells by using a BiFC-based FRET assay. Nat Protoc 3(11):1693–1702CrossRefPubMedGoogle Scholar
  16. 16.
    Duffraisse M, Hudry B, Merabet S (2014) Bimolecular fluorescence complementation (BiFC) in live drosophila embryos. Methods Mol Biol 1196:307–318. https://doi.org/10.1007/978-1-4939-1242-1_19 CrossRefPubMedGoogle Scholar
  17. 17.
    Shyu YJ, Liu H, Deng X, Hu CD (2006) Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. BioTechniques 40(1):61–66CrossRefPubMedGoogle Scholar
  18. 18.
    Kodama Y, Hu CD (2010) An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio. BioTechniques 49(5):793–805CrossRefPubMedGoogle Scholar
  19. 19.
    Miller KE, Kim Y, Huh WK, Park HO (2015) Bimolecular fluorescence complementation (BiFC) analysis: advances and recent applications for genome-wide interaction studies. J Mol Biol 427(11):2039–2055. https://doi.org/10.1016/j.jmb.2015.03.005 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Ciruela F, Vilardaga JP, Fernandez-Duenas V (2010) Lighting up multiprotein complexes: lessons from GPCR oligomerization. Trends Biotechnol 28(8):407–415. https://doi.org/10.1016/j.tibtech.2010.05.002 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Vidi PA, Chen J, Irudayaraj JM, Watts VJ (2008) Adenosine a(2A) receptors assemble into higher-order oligomers at the plasma membrane. FEBS Lett 582(29):3985–3990. https://doi.org/10.1016/j.febslet.2008.09.062 CrossRefPubMedGoogle Scholar
  22. 22.
    Vidi PA, Chemel BR, Hu CD, Watts VJ (2008) Ligand-dependent oligomerization of dopamine D(2) and adenosine a(2A) receptors in living neuronal cells. Mol Pharmacol 74(3):544–551. https://doi.org/10.1124/mol.108.047472 CrossRefPubMedGoogle Scholar
  23. 23.
    Kodama Y, Hu CD (2013) Bimolecular fluorescence complementation (BiFC) analysis of protein-protein interaction. how to calculate signal-to-noise ratio Methods Cell Biol 113:107–121. https://doi.org/10.1016/B978-0-12-407239-8.00006-9 CrossRefPubMedGoogle Scholar
  24. 24.
    Yost EA, Mervine SM, Sabo JL, Hynes TR, Berlot CH (2007) Live cell analysis of G protein beta5 complex formation, function, and targeting. Mol Pharmacol 72(4):812–825. https://doi.org/10.1124/mol.107.038075 CrossRefPubMedGoogle Scholar
  25. 25.
    Gomes I, Gupta A, Filipovska J, Szeto HH, Pintar JE, Devi LA (2004) A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia. Proc Natl Acad Sci U S A 101(14):5135–5139. https://doi.org/10.1073/pnas.0307601101 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Milan-Lobo L, Enquist J, van Rijn RM, Whistler JL (2013) Anti-analgesic effect of the mu/delta opioid receptor heteromer revealed by ligand-biased antagonism. PLoS One 8(3):e58362CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    O’Dowd BF, Ji X, O’Dowd PB, Nguyen T, George SR (2012) Disruption of the mu-delta opioid receptor heteromer. Biochem Biophys Res Commun 422(4):556–560CrossRefPubMedGoogle Scholar
  28. 28.
    He SQ, Zhang ZN, Guan JS, Liu HR, Zhao B, Wang HB, Li Q, Yang H, Luo J, Li ZY, Wang Q, Lu YJ, Bao L, Zhang X (2011) Facilitation of mu-opioid receptor activity by preventing delta-opioid receptor-mediated codegradation. Neuron 69(1):120–131CrossRefPubMedGoogle Scholar
  29. 29.
    Vidi PA, Przybyla JA, Hu CD, Watts VJ (2010) Visualization of G protein-coupled receptor (GPCR) interactions in living cells using bimolecular fluorescence complementation (BiFC). Current protocols in neuroscience Chapter 5:Unit 5 29Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Doungkamol Alongkronrusmee
    • 1
  • Val J. Watts
    • 1
  • Richard M. van Rijn
    • 1
  1. 1.Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University Institute for Integrative NeurosciencePurdue UniversityWest LafayetteUSA

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