Brief Communication | Published:

Application of BRET to monitor ligand binding to GPCRs

Nature Methods volume 12, pages 661663 (2015) | Download Citation

Abstract

Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein–coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs.

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Acknowledgements

The authors thank C. Corona and P. Meinsenheimer (Promega) for providing the alprenolol-TAMRA fluorescent ligand and J. Denman (The University of Nottingham) for assistance with generating the NanoLuc-adenosine receptor constructs. This project was funded by the Australian Research Council (ARC) Linkage Grant LP130100037. The University of Nottingham, Promega Corporation and BMG Labtech Pty Ltd provided funding as partner organizations of this grant. E.K.M.J. was funded by the Richard Walter Gibbon Medical Research Scholarship from The University of Western Australia. Work in S.J.H.'s laboratory was funded by the UK Medical Research Council (G0800006). K.D.G.P. was funded by an ARC Future Fellowship (FT100100271) and subsequently a National Health and Medical Research Council of Australia RD Wright Fellowship (1085842). S.J.H. thanks the Raine Foundation for a Visiting Research Professorship to The University of Western Australia.

Author information

Author notes

    • Stephen J Hill
    •  & Kevin D G Pfleger

    These authors jointly directed this work.

Affiliations

  1. Cell Signalling Research Group, School of Life Sciences, The University of Nottingham Medical School, Nottingham, UK.

    • Leigh A Stoddart
    • , Amanda J Wheal
    • , Joëlle Goulding
    •  & Stephen J Hill
  2. Molecular Endocrinology and Pharmacology, Harry Perkins Institute of Medical Research, Nedlands, Western Australia, Australia.

    • Elizabeth K M Johnstone
    • , Stephen J Hill
    •  & Kevin D G Pfleger
  3. Centre for Medical Research, The University of Western Australia, Crawley, Western Australia, Australia.

    • Elizabeth K M Johnstone
    • , Stephen J Hill
    •  & Kevin D G Pfleger
  4. Promega Corporation, Madison, Wisconsin, USA.

    • Matthew B Robers
    • , Thomas Machleidt
    •  & Keith V Wood

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Contributions

L.A.S., E.K.M.J., A.J.W., J.G., M.B.R. and T.M. performed the research. L.A.S., E.K.M.J., M.B.R., K.V.W., S.J.H. and K.D.G.P. designed the research study. L.A.S., E.K.M.J., A.J.W., J.G. and M.B.R. analyzed the data. L.A.S., S.J.H. and K.D.G.P. wrote the paper.

Competing interests

M.B.R., T.M. and K.V.W. are employees of Promega Corporation, which has proprietary rights over the NanoBRET assay. S.J.H. is a former Director of the University of Nottingham spin-out company CellAura Technologies, which provided some of the fluorescent ligands.

Corresponding authors

Correspondence to Stephen J Hill or Kevin D G Pfleger.

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DOI

https://doi.org/10.1038/nmeth.3398

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