Abstract
The bioluminescence resonance energy transfer (BRET) technique has become extremely valuable for the real-time monitoring of protein–protein interactions in live cells. This method is highly amenable to the detection of G protein-coupled receptor (GPCR) interactions with proteins critical for regulating their function, such as β-arrestins. Of particular interest to endocrinologists is the ability to monitor interactions involving endocrine receptors, such as orexin receptor 2 or vasopressin type II receptor. The BRET method utilizes heterologous co-expression of fusion proteins linking one protein of interest (GPCR) to a bioluminescent donor enzyme, a variant of Renilla luciferase, and a second protein of interest (β-arrestin) to an acceptor fluorophore. If in close proximity, energy resulting from oxidation of the coelenterazine substrate by the donor will transfer to the acceptor, which in turn fluoresces. Using novel luciferase constructs, we were able to monitor interactions not detectable using less sensitive BRET combinations in the same configuration. In particular, we were able to show receptor/β-arrestin interactions in an agonist-independent manner using Rluc8-tagged mutant receptors, in contrast to when using Rluc. Therefore, the enhanced BRET methodology has not only enabled live cell compound screening as we have recently published, it now provides a new level of sensitivity for monitoring specific transient, weak or hardly detectable protein–protein complexes, including agonist-independent GPCR/β-arrestin interactions. This has important implications for the use of BRET technologies in endocrine drug discovery programs as well as academic research.
Highlights
Bioluminescence resonance energy transfer (BRET) is gaining increasing recognition as an advantageous approach for detecting protein–protein interactions in living cells and real time (Xu et al, 2003; Pfleger and Eidne, 2006; Pfleger et al, 2006a; Kocan et al, 2008)
If the luminophore and fluorophore are less than 10 nm apart (Dacres et al, 2010), energy resulting from the rapid oxidation of a cell-permeable coelenterazine substrate by the luminophore will transfer to the fluorophore, which in turn fluoresces at a longer wavelength
We have recently demonstrated advances in the bioluminescence resonance energy transfer (BRET) technology enabling its application for live cell compound screening as exemplified using the thyrotropin-releasing hormone receptor (TRHR) interaction with β-arrestin (Kocan et al, 2008)
Summary
Bioluminescence resonance energy transfer (BRET) is gaining increasing recognition as an advantageous approach for detecting protein–protein interactions in living cells and real time (Xu et al, 2003; Pfleger and Eidne, 2006; Pfleger et al, 2006a; Kocan et al, 2008). Energy transfer implies that these molecules are in close proximity, and the proteins of interest (e.g., GPCR and β-arrestin) fused to the donor and acceptor interact directly or as part of a complex (Pfleger and Eidne, 2006; Pfleger et al, 2006a,b; Kocan et al, 2008). Light emission from the corresponding acceptor is observed at longer wavelengths following energy transfer (e.g., peaking at ∼510 nm for enhanced green fluorescent protein (EGFP) or ∼530 nm for yellow fluorescent proteins such as Venus). GFP10 or GFP2 is used as acceptor, with light emission peaking at ∼510 nm following energy transfer
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