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Applications of fluorescence and bioluminescence resonance energy transfer to drug discovery at G protein coupled receptors

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Abstract

The role of G protein coupled receptors (GPCRs) in numerous physiological processes that may be disrupted or modified in disease makes them key targets for the development of new therapeutic medicines. A wide variety of resonance energy transfer (RET) techniques such as fluorescence RET and bioluminescence RET have been developed in recent years to detect protein–protein interactions in living cells. Furthermore, these techniques are now being exploited to screen for novel compounds that activate or block GPCRs and to search for new, previously undiscovered signaling pathways activated by well-known pharmacologically classified drugs. The high resolution that can be achieved with these RET methods means that they are well suited to study both intramolecular conformational changes in response to ligand binding at the receptor level and intermolecular interactions involving protein translocation in subcellular compartments resulting from external stimuli. In this review we highlight the latest advances in these technologies to illustrate general principles.

Acceptor photobleaching of a mCerulean-mCitrine fluorescent protein tandem. Images show HEK293 cells transfected with a mCerulean-mCitrine fluorescent protein concatamer prior to selective photo-bleaching of the acceptor in the region marked by a red square. The absence of fluorescence signal in post-bleached images within the region of interest confirms that the acceptor fluorescence has been bleached and irreversibly destroyed. Once this process has taken place the donor returns to its de-quenched state that, in turn, will result in an increase in donor fluorescence intensity being detected within the bleached region.

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Abbreviations

AnFRET:

anisotropy fluorescence resonance energy transfer

apFRET:

acceptor photobleaching fluorescence resonance energy transfer

BRET:

bioluminescence resonance energy transfer

cAMP:

cyclic AMP

CFP:

cyan fluorescent protein

FlAsH:

fluorescein arsenical hairpin binder

FLIM:

fluorescence lifetime imaging microscopy

FRET:

fluorescence resonance energy transfer

GFP:

green fluorescent protein

GPCR:

guanine nucleotide binding protein coupled receptor

G protein:

guanine nucleotide binding protein

HTRF:

homogeneous time-resolved fluorescence resonance energy transfer

PA-GFP:

UV photoactivatable green fluorescent protein

PQ-FRET:

photoquenching fluorescence resonance energy transfer

RET:

resonance energy transfer

Rluc:

Renilla luciferase

SBT:

spectral bleed-through

YFP:

yellow fluorescent protein

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Acknowledgements

These studies were supported by the Biotechnology and Biosciences Research Council (grant BB/E006302/1) and the Medical Research Council (grant G0900050).

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Authors and Affiliations

  1. Molecular Pharmacology Group, Neuroscience and Molecular Pharmacology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK

    Elisa Alvarez-Curto, John D. Pediani & Graeme Milligan

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  1. Elisa Alvarez-Curto
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  2. John D. Pediani
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  3. Graeme Milligan
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Correspondence to Graeme Milligan.

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Alvarez-Curto, E., Pediani, J.D. & Milligan, G. Applications of fluorescence and bioluminescence resonance energy transfer to drug discovery at G protein coupled receptors. Anal Bioanal Chem 398, 167–180 (2010). https://doi.org/10.1007/s00216-010-3823-4

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