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Using BRET to Detect Ligand-Specific Conformational Changes in Preformed Signalling Complexes

  • Nicolas Audet
  • Graciela Piñeyro
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 756)

Abstract

Bioluminescence energy transfer (BRET) has become a powerful tool to study protein–protein interactions and conformational changes among interacting proteins. In particular, BRET assays performed in living cells have revealed that heptahelical receptors (7TMRs), heterotrimeric G proteins and their proximal effectors form constitutive signalling complexes. BRET technology has also allowed us to demonstrate that these multimeric protein arrays remain intact throughout initial stages of receptor signalling, thus providing a platform for direct transmission of conformational information from activated receptors to downstream signalling partners. A clear example of the latter are the distinct intermolecular re-arrangements undergone by 7TMRs and G protein subunits following activation of the receptor by different ligands. Here we present protocols describing the type of BRET assay that has been used to reveal the existence of constitutive signalling arrays formed by 7TMRs and proximal signalling partners as well as the ability of complex components to undergo ligand-specific conformational changes.

Key words

G protein-coupled receptor Heterotrimeric guanine nucleotide-binding protein Bioluminescence resonance energy transfer Signal transduction Protein complex 

Notes

Acknowledgements

This work was supported by Discovery Grant from The National Sciences and Engineering Council of Canada (NSERC) to GP.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Nicolas Audet
    • 1
    • 2
  • Graciela Piñeyro
    • 3
    • 2
  1. 1.Department of Pharmacology, Faculty of MedicineUniversity of MontrealMontrealCanada
  2. 2.Centre de Recherche du CHU Ste-Justine, BureauMontrealCanada
  3. 3.Department of Psychiatry, Faculty of MedicineUniversity of MontrealMontrealCanada

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