Background

Fluorimeters have been in research laboratories for decades and still are. Also, cell biology took advantage of fluorescent detection of molecules using antibodies conjugated to small fluorescent molecules. A next stage was reached upon the discovery and further development of big fluorescent molecules. A fluorescent protein may be fused to a protein of interest to detect it even in living cells; now it is even possible to follow proteins in tissues of transgenic animals engineered to express the fluorescent fusion protein. Here are now available a variety of proteins with different fluorometric properties, i.e., with different wavelengths of absorption and/or emission. The Nobel Prize in Chemistry in 2008 was awarded to O. Shimomura, M. Chalfie and R. Y. Tsien: “for the discovery and development of the green fluorescent protein, GFP”. The press note indicated: “By using DNA technology, researchers can now connect GFP to other interesting, but otherwise invisible, proteins. This glowing marker allows them to watch the movements, positions and interactions of the tagged proteins” (https://www.nobelprize.org/prizes/chemistry/2008/press-release/).

Energy transfer protocols are now instrumental to detect molecular interactions, which were previously suspected by colocalization and “confirmed” by co-immunoprecipitation, i.e. by bringing down one protein using a monoclonal antibody against the “interacting” partner (Burger et al., 1986; Springer et al., 1986; Dianzani et al., 1999; Serrador et al., 1999). Unfortunately, it was not (still is not) easy to develop monoclonal antibodies against G-protein-coupled receptors (GPCRs). The first one produced to detect a GPCR, the adenosine A_2A receptor, is probably still the best one (Rosin et al., 1998). But the majority of “good” anti-GPCR antibodies are polyclonal and the first ones appearing in the literature for class A GPCRs recognized adenosine receptors (Ciruela et al., 1995; Jimenez et al., 2008). Many of the antibodies against GPCRs are polyclonal and were used for coimmunoprecipitation (Ginés et al., 2000). Critiques arrived soon from two points of view: doubts on the specificity of polyclonal antibodies and the possibility that in detergent-treated cells a (third) bridging protein could lead to artifacts.

Energy transfer between proteins can only occur if the donor and the acceptor are at a distance of < 10 nm. The first option is using two proteins of interest each fused to a different fluorescence protein. Such approach is known as Förster resonance energy transfer: FRET (Förster, 1948; Perkins et al., 1984). In parallel Bioluminescence resonance energy transfer: BRET, was developed in the basis of a bioluminescent donor and a fluorescent acceptor (Xu et al., 1999). This is the technique whose protocol will be detailed here. Conceptually, the technique requires one protein fused to an enzyme (Renilla reniformis luciferase; a protein of circa 36 kDa) that degrades a substrate and emits light that in turns excite another protein fused, for instance, to the yellow fluorescent protein (YFP) (Figure 1). Two possible BRETs exist but we will detail BRET¹, as the protocol would be quite similar with BRET² (the main two differences are the substrate of Rluc and the fluorescent acceptor). BRET can also be used for signal transduction measurements. Probably the best example in the field of GPCRs is the measurement of β-arrestin binding to activated GPCRs. In that case the β-arrestin is fused to Rluc and acts as donor of the fluorescent protein fused to the GPCR of interest. BRET is repeated at different times to get time-response curves of β-arrestin recruitment or at a given time but with different doses of the GPCR activator to get dose-response curves. Reviews on the BRET technique (including the so-called nano-BRET) and on different possibilities that it offers in different fields and/or to answer scientific questions can be elsewhere found (Pfleger and Eidne, 2005; Breton et al., 2010; Schann et al., 2013; Beautrait et al., 2014; Sun et al., 2016; Dale et al., 2019).


Figure 1. Cartoon representation of the BRET assay between a fusion protein constituted by R1 and Rluc and another fusion protein constituted by R2 and YFP. A. When both receptors (R1 and R2) are in close proximity (distance ≤ 100 A) there is energy transfer from Rluc-derived bioluminescence to YFP. B. When receptors are at a higher distance, no energy transfer can be detected. Rluc: Renilla luciferase. Col H: coelenterazine H, a substrate of Rluc. YFP: Yellow fluorescent protein. R1: cell surface receptor number 1. R1: cell surface receptor number 2.