Identification of Heteroreceptors Complexes and Signal Transduction Events Using Bioluminescence Resonance Energy Transfer (BRET)

Abstract

Detecting protein-protein interactions by co-immunoprecipitation provided a major advancement in the immunology research field. In the G-protein-coupled receptors (GPCRs) research field, colocalization and co-immunoprecipitation were used to detect interactions, but doubts arose due to specificity of the antibodies (monoclonal in the case of receptors related to immunology and polyclonal in the case of GPCRs) and due to the possibility of false positive due to the potential occurrence of bridging proteins. Accordingly, new methodological approaches were needed, and energy transfer techniques have been instrumental to detect direct protein-protein, protein-receptor or receptor-receptor interactions. Of the two most relevant methods (Förster, or fluorescence resonance energy transfer: FRET and Bioluminescence energy transfer: BRET), the protocol for BRET is here presented. BRET has been instrumental to detect direct interactions between GPCRs and has contributed to demonstrate that GPCR dimers/oligomer functionality is different from that exerted by individual receptors. Advantages outweigh those of FRET as no fluorescence source is needed. Interestingly, BRET is not only useful to validate interactions detected by other means or hypothesized in the basis of indirect evidence, but to measure signal transduction events. In fact, BRET may, for instance, be used to assess β-arrestin recruitment to activated GPCRs.

Keywords: BRET; Bioluminescence; Energy transfer; Fluorescence; G-protein-coupled receptors; GPCR; Heteromers; Protein-protein interactions.

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.

Figure 2.. Cartoon representation of the fusion proteins containing Rluc or YFP at the C terminus of each receptor

Figure 3.. Example of positive BRET.

When two receptors form heteromers, BRET assay results in a saturable curve. B_max and K_D for the curve are provided by non-linear-regression. For this curve, BRET_max= 220 ± 10 mBU and BRET50= 43 ± 7 (data are mean ± SEM).

Figure 4.. Example of negative BRET.

In the case of two receptors that do not form heteromers, data fit to a linear regression.