Trends in Pharmacological Sciences
Research updateThe use of resonance energy transfer in high-throughput screening: BRET versus FRET
Section snippets
Bioluminescence resonance energy transfer
BRET is a natural phenomenon, observed in marine organisms, in which an energy transfer occurs between luminescent donor and fluorescent acceptor proteins. Oxidation of cœlenterazine by Renilla luciferase produces light with a wavelength of 480 nm. However, in the sea pansy Renilla, the close proximity of a green fluorescent protein (Renilla GFP) allows a non-radiative energy transfer that results in light emission at 509 nm by the GFP 1., 2.. Resonance energy transfer occurs when part of the
BRET versus FRET
Before the development of BRET in 1999, fluorescence resonance energy transfer (FRET) was used widely to study protein–protein interactions or to monitor conformational changes within a given protein. In the FRET methodology, luciferase is replaced by a fluorophore that is excited using monochromatic light at the appropriate wavelength 12., 13., 14..
A major advantage of FRET is to permit, under microscopic observation, visualization in a single living cell of protein–protein interactions at the
Possible application of BRET for drug discovery
The potential of BRET as a tool in drug discovery is illustrated below by recent work showing how it can be used to monitor the activation state of the insulin receptor.
Insulin plays a crucial role in the regulation of energy metabolism. Pathological states such as diabetes and obesity are associated with insulin deficiency and/or insulin resistance. Given the increasing worldwide prevalence of these pathologies, the discovery of new treatments represents a major public health goal for the next
Concluding remarks
Three years after the first description of BRET [5], this technique has already been applied to the study of various types of receptors, both in vitro and in intact cells. This methodology will certainly develop considerably in the near future, with new generations of luciferase, luciferase substrates and fluorescent protein variants specifically designed for BRET applications [17]. Indeed, BRET is a highly sensitive technique that allows the study of a very large number of experimental
Acknowledgements
Our work was supported the Centre National de la Recherche Scientifique, the Association pour la Recherche sur le Cancer, the Ligue contre le Cancer and by a Roche-Pharma-ALFEDIAM (Association de Langue Française d’Etude du Diabète et des Maladies Métaboliques) research grant.
References (23)
- et al.
Resonance energy transfer: methods and applications
Anal. Biochem.
(1994) Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta-opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy
J. Biol. Chem.
(2001)Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer
J. Biol. Chem.
(2001)- et al.
Agonist-dependent dissociation of oligomeric complexes of G protein-coupled cholecystokinin receptors demonstrated in living cells using bioluminescence resonance energy transfer
J. Biol. Chem.
(2001) Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer
J. Biol. Chem.
(2002)Understanding, improving and using green fluorescent proteins
Trends Biochem. Sci.
(1995)Fluorescence techniques: shedding light on ligand–receptor interactions
Trends Pharmacol. Sci.
(2000)Recent advances in technology for measuring and manipulating cell signals
Curr. Opin. Neurobiol.
(2000)Imaging biochemistry inside cells
Trends Cell Biol.
(2001)Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer
Anal. Biochem.
(2001)