Advertisement

Dopamine pp 107-122 | Cite as

BRET Approaches to Characterize Dopamine and TAAR1 Receptor Pharmacology and Signaling

  • Stefano Espinoza
  • Bernard Masri
  • Ali Salahpour
  • Raul R. GainetdinovEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 964)

Abstract

It is evident that G protein-coupled receptors (GPCRs) such as D2 dopamine receptor and functionally related Trace Amine Associated Receptor 1 (TAAR1) can engage both in G protein-dependent (e.g., cAMP-mediated) and -independent β-arrestin-mediated signaling modalities. Both of these signaling events can be monitored in real-time and in live cells by using new biosensors based on a Bioluminescence Resonance Energy Transfer (BRET) approach. Here we discuss the practical applications of BRET to analyze dynamics of cAMP modulation via an EPAC biosensor as well as recruitment of β-arrestin2 to the D2 dopamine receptor. Combination of these approaches allows for a comparison of activity of pharmacological compounds on these signaling modalities as demonstrated for various antipsychotics as regard to D2 dopamine receptor. Furthermore, analysis of cAMP concentrations in cells expressing TAAR1 provides a simple high-throughput screening method to identify new ligands for this receptor. These BRET approaches could be applied for the characterization of pharmacology and signaling of variety of other GPCRs.

Key words

D2R TAAR1 cAMP β-arrestin2 BRET EPAC 3-Methoxytyramine β-Phenylethylamine 

Notes

Acknowledgments

Supported in part by research grants from the Michael J. Fox Foundation for Parkinson’s Research, Fondazione Compagnia di San Paolo (Torino, Italy) and research grant from F. Hoffmann-La Roche Ltd. (Basel, Switzerland) to Raul R. Gainetdinov. Bernard Masri was a recipient of a European Marie-Curie Outgoing International Fellowship (FP6—2005-Mobility-6). Ali Salahpour is supported by grants from Canadian Institute of Health Research (CIHR # 210296) and Natural Sciences and Engineering Council of Canada (NSERC # 386422).

References

  1. 1.
    Beaulieu JM, Gainetdinov RR (2011) The physiology, signaling and pharmacology of dopamine receptors. Pharmacol Rev 63: 182–217PubMedGoogle Scholar
  2. 2.
    Beaulieu JM, Gainetdinov RR, Caron MG (2007) The Akt-GSK-3 signaling cascade in the actions of dopamine. Trends Pharmacol Sci 28:166–172PubMedCrossRefGoogle Scholar
  3. 3.
    Lefkowitz RJ, Shenoy SK (2005) Transduction of receptor signals by beta-arrestins. Science 308:512–517PubMedCrossRefGoogle Scholar
  4. 4.
    Beaulieu JM, Gainetdinov RR, Caron MG (2009) Akt/GSK3 signaling in the action of psychotropic drugs. Annu Rev Pharmacol Toxicol 49:327–347PubMedCrossRefGoogle Scholar
  5. 5.
    Masri B, Salahpour A, Didriksen M, Ghisi V, Beaulieu JM, Gainetdinov RR, Caron MG (2008) Antagonism of dopamine D2 receptor/beta-arrestin 2 interaction is a common property of clinically effective antipsychotics. Proc Natl Acad Sci U S A 105:13656–13661PubMedCrossRefGoogle Scholar
  6. 6.
    Bunzow JR, Sonders MS, Arttamangkul S, Harrison LM, Zhang G, Quigley DI, Darland T, Suchland KL, Pasumamula S, Kennedy JL, Olson SB, Magenis RE, Amara SG, Grandy DK (2001) Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor. Mol Pharmacol 60:1181–1188PubMedGoogle Scholar
  7. 7.
    Borowsky B, Adham N, Jones KA, Raddatz R, Artymyshyn R, Ogozalek KL, Durkin MM, Lakhlani PP, Bonini JA, Pathirana S, Boyle N, Pu X, Kouranova E, Lichtblau H, Ochoa FY, Branchek TA, Gerald C (2001) Trace amines: identification of a family of mammalian G protein-coupled receptors. Proc Natl Acad Sci U S A 98:8966–8971PubMedCrossRefGoogle Scholar
  8. 8.
    Lindemann L, Meyer CA, Jeanneau K, Bradaia A, Ozmen L, Bluethmann H, Bettler B, Wettstein JG, Borroni E, Moreau JL, Hoener MC (2008) Trace amine-associated receptor 1 modulates dopaminergic activity. J Pharmacol Exp Ther 324:948–956PubMedCrossRefGoogle Scholar
  9. 9.
    Bradaia A, Trube G, Stalder H, Norcross RD, Ozmen L, Wettstein JG, Pinard A, Buchy D, Gassmann M, Hoener MC, Bettler B (2009) The selective antagonist EPPTB reveals TAAR1-mediated regulatory mechanisms in dopaminergic neurons of the mesolimbic system. Proc Natl Acad Sci U S A 106:20081–20086PubMedGoogle Scholar
  10. 10.
    Barak LS, Salahpour A, Zhang X, Masri B, Sotnikova TD, Ramsey AJ, Violin JD, Lefkowitz RJ, Caron MG, Gainetdinov RR (2008) Pharmacological characterization of ­membrane-expressed human trace amine-­associated receptor 1 (TAAR1) by a bioluminescence resonance energy transfer cAMP biosensor. Mol Pharmacol 74:585–594PubMedCrossRefGoogle Scholar
  11. 11.
    Sotnikova TD, Beaulieu JM, Espinoza S, Masri B, Zhang X, Salahpour A, Barak LS, Caron MG, Gainetdinov RR (2010) The dopamine metabolite 3-methoxytyramine is a neuromodulator. PLoS One 5:e13452PubMedCrossRefGoogle Scholar
  12. 12.
    Ayoub MA, Pfleger KD (2010) Recent advances in bioluminescence resonance energy transfer technologies to study GPCR heteromerization. Curr Opin Pharmacol 10:44–52PubMedCrossRefGoogle Scholar
  13. 13.
    Marullo S, Bouvier M (2007) Resonance energy transfer approaches in molecular pharmacology and beyond. Trends Pharmacol Sci 28:362–365PubMedCrossRefGoogle Scholar
  14. 14.
    Ni Q, Titov DV, Zhang J (2006) Analyzing protein kinase dynamics in living cells with FRET reporters. Methods 40:279–286PubMedCrossRefGoogle Scholar
  15. 15.
    Pfleger KD, Seeber RM, Eidne KA (2006) Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions. Nat Protoc 1:337–345PubMedCrossRefGoogle Scholar
  16. 16.
    Pfleger KD, Eidne KA (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 3:165–174PubMedCrossRefGoogle Scholar
  17. 17.
    Salahpour A, Espinoza S, Masri B, Lam V, Barak LS, Gainetdinov RR (2012) BRET biosensors to study GPCR biology, pharmacology, and signal transduction. Front Endocrinol (Lausanne) 3:105PubMedCrossRefGoogle Scholar
  18. 18.
    Espinoza S, Salahpour A, Masri B, Sotnikova TD, Messa M, Barak LS, Caron MG, Gainetdinov RR (2011) Functional interaction between Trace Amine Associated Receptor 1 (TAAR1) and dopamine D2 receptor. Mol Pharmacol 80:416–425PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Stefano Espinoza
    • 1
  • Bernard Masri
    • 2
  • Ali Salahpour
    • 3
  • Raul R. Gainetdinov
    • 1
    Email author
  1. 1.Department of Neuroscience and Brain TechnologiesItalian Institute of TechnologyGenoaItaly
  2. 2.Cancer Research Center of ToulouseINSERM U1037 - Université Paul Sabatier Toulouse III, CHU RangueilToulouseFrance
  3. 3.Department of Pharmacology and ToxicologyUniversity of TorontoTorontoCanada

Personalised recommendations