4.7 Review

Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding

Journal

BRITISH JOURNAL OF PHARMACOLOGY
Volume 173, Issue 20, Pages 3028-3037

Publisher

WILEY
DOI: 10.1111/bph.13316

Keywords

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Funding

  1. Medical Research Council [G0800006]
  2. Australian Research Council (ARC) [LP130100037]
  3. University of Nottingham
  4. Promega Corporation
  5. BMG Labtech Pty Ltd
  6. ARC [FT100100271]
  7. National Health and Medical Research Council (NHMRC) of Australia [1085842]
  8. MRC [G0800006] Funding Source: UKRI
  9. Medical Research Council [G0800006] Funding Source: researchfish
  10. Australian Research Council [FT100100271, LP130100037] Funding Source: Australian Research Council
  11. National Health and Medical Research Council of Australia [1085842] Funding Source: NHMRC

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Ligand binding is a vital component of any pharmacologist's toolbox and allows the detailed investigation of how a molecule binds to its receptor. These studies enable the experimental determination of binding affinity of labelled and unlabelled compounds through kinetic, saturation (K-d) and competition (K-i) binding assays. Traditionally, these studies have used molecules labelled with radioisotopes; however, more recently, fluorescent ligands have been developed for this purpose. This review will briefly cover receptor ligand binding theory and then discuss the use of fluorescent ligands with some of the different technologies currently employed to examine ligand binding. Fluorescent ligands can be used for direct measurement of receptor-associated fluorescence using confocal microscopy and flow cytometry as well as in assays such as fluorescence polarization, where ligand binding is monitored by changes in the free rotation when a fluorescent ligand is bound to a receptor. Additionally, fluorescent ligands can act as donors or acceptors for fluorescence resonance energy transfer (FRET) with the development of assays based on FRET and time-resolved FRET (TR-FRET). Finally, we have recently developed a novel b resonance energy transfer (BRET) ligand binding assay utilizing a small (19kDa), super-bright luciferase subunit (NanoLuc) from a deep sea shrimp. In combination with fluorescent ligands, measurement of RET now provides an array of methodologies to study ligand binding. While each method has its own advantages and drawbacks, binding studies using fluorescent ligands are now a viable alternative to the use of radioligands. This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit Linked Articles

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