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Measuring GPCR Stoichiometry Using Types-1, -2, and -3 Bioluminescence Resonance Energy Transfer-Based Assays

  • James H. FelceEmail author
  • John R. JamesEmail author
  • Simon J. DavisEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1947)

Abstract

How G protein-coupled receptors are assembled is a matter of considerable interest owing in large part to their remarkable pharmacological importance. For determining receptor stoichiometry, resonance energy transfer-based methods offer considerable advantages insofar as they provide the necessary spatial resolution, and because measurements can be made in situ, relatively easily. This chapter describes three complementary stoichiometric assays that rely on measurements of bioluminescence resonance energy transfer. These quantitative approaches make it possible to identify true protein–protein interactions from non-specific associations that inevitably result from constraining proteins in cellular membranes. In our experience, concordant data obtained in two or more of these assays, benchmarked with suitable controls, strongly predict receptor stoichiometry.

Key words

G protein-coupled receptors Membrane proteins Bioluminescence resonance energy transfer (BRET) Stoichiometry Oligomerization Luciferase GFP 

Notes

Acknowledgements

This work was supported by the Wellcome Trust (098274/Z/12/Z to S.J.D., 107375/Z/15/Z to J.H.F.).

References

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Copyright information

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

Authors and Affiliations

  1. 1.Kennedy Institute of RheumatologyUniversity of OxfordOxfordUK
  2. 2.Warwick Medical SchoolUniversity of WarwickCoventryUK
  3. 3.Medical Research Council Human Immunology Unit, Radcliffe Department of Clinical Medicine, Weatherall Institute of Molecular MedicineUniversity of OxfordOxfordUK

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