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Dopamine pp 95-105 | Cite as

Detection of Receptor Heteromers Involving Dopamine Receptors by the Sequential BRET-FRET Technology

  • Gemma Navarro
  • Peter J. McCormick
  • Josefa Mallol
  • Carme Lluís
  • Rafael Franco
  • Antoni Cortés
  • Vicent Casadó
  • Enric I. Canela
  • Sergi FerréEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 964)

Abstract

Until very recently, dopamine receptors, like other G-protein-coupled receptors, were believed to function as individual units on the cell surface. Now it has been described by several groups including ours that dopamine receptors not only function as homomers but also form heteromers with other receptors at the membrane level. Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) based techniques have been very useful to determine the interaction between two receptors, but to demonstrate the existence of higher-order complexes involving more than two molecules requires more sophisticated techniques. Combining BRET and FRET in the Sequential BRET-FRET (SRET) technique permits heteromers formed by three different proteins to be identified. In SRET experiments, the oxidation of a Renilla Luciferase substrate triggers acceptor excitation by BRET and subsequent energy transfer to a FRET acceptor. Using this methodology here we describe the heteromerization between adenosine A2A, dopamine D2, and cannabinoids CB1 receptors in living cells.

Key words

Dopamine receptors Dopamine receptors interacting proteins BRET FRET Sequential resonance energy transfer GPCR Receptor oligomerization Heteromer Protein–protein interaction 

Notes

Acknowledgments

Study supported by grants from Spanish Ministerio de Ciencia y Tecnología (SAF2008-00146, SAF2008-03229-E, and SAF2009-07276), grant 060110 from Fundació La Marató de TV3 and by the Intramural Funds of the National Institute on Drug Abuse.

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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Gemma Navarro
    • 1
  • Peter J. McCormick
    • 1
  • Josefa Mallol
    • 1
  • Carme Lluís
    • 1
  • Rafael Franco
    • 1
  • Antoni Cortés
    • 2
  • Vicent Casadó
    • 2
  • Enric I. Canela
    • 2
  • Sergi Ferré
    • 3
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of BarcelonaBarcelonaSpain
  2. 2.Department of Biochemistry and Molecular Biology, Faculty of BiologyUniversity of BarcelonaBarcelonaSpain
  3. 3.Department of Health and Human ServicesIntramural Research Program, National Institute on Drug Abuse, National Institutes of HealthBaltimoreUSA

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