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. 2011 Jan 14:1:12.
doi: 10.3389/fendo.2010.00012. eCollection 2010.

Enhanced BRET Technology for the Monitoring of Agonist-Induced and Agonist-Independent Interactions between GPCRs and β-Arrestins

Martina Kocan  1 , Matthew B Dalrymple, Ruth M Seeber, Brian J Feldman, Kevin D G Pfleger
Affiliations

Enhanced BRET Technology for the Monitoring of Agonist-Induced and Agonist-Independent Interactions between GPCRs and β-Arrestins

Martina Kocan et al. Front Endocrinol (Lausanne). .

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.

Keywords: G protein-coupled receptor; arrestin; bioluminescence resonance energy transfer; luciferase; orexin; vasopressin.

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Figures

Figure 1
Figure 1
Functional validation of Rluc-tagged OxR2 compared with untagged OxR2. Total inositol phosphate production was measured after addition of increasing concentrations of Orexin A (OxA) (A) or Orexin B (OxB) (B). Data shown are mean ± SEM of three independent experiments.
Figure 2
Figure 2
Functional validation of different luciferase-tagged OxR2 BRET fusion proteins. Total inositol phosphate production was measured after addition of vehicle or OxA in HEK293FT cells transfected with one of various OxR2/luminophore constructs. Data shown are mean ± SEM of three independent experiments each carried out in triplicate. Data generated with OxA-treated cells were analyzed by one-way ANOVA and differences between cells transfected with the various OxR2/luminophore constructs were not significant (P > 0.05).
Figure 3
Figure 3
Spectral analysis comparing luminescence intensities of different luciferase-tagged OxR2 fusion proteins using coelenterazine h or DeepBlueC as luciferase substrate. HEK293FT cells were co-transfected with β-arrestin 2/Venus (A) or β-arrestin 2/GFP10 (B) and OxR2 tagged with various luciferase constructs. Emission spectra were recorded immediately after addition of coelenterazine h (A) or DeepBlueC (B). For the BRET1 system (A), the emission maximum of ~480 nm corresponds to the luciferase oxidizing its substrate coelenterazine h. For the BRET2 system (B), the emission maximum of ~420 nm corresponds to the luciferase oxidizing its substrate DeepBlueC.
Figure 4
Figure 4
Comparison of luminescence intensities of different luciferase-tagged OxR2 fusion proteins over time oxidizing different luciferase substrates: coelenterazine h and DeepBlueC. HEK293FT cells were transiently co-transfected with OxR2 tagged with various luciferase constructs and β-arrestin 2/Venus (A) or β-arrestin 2/GFP10 (B). Luciferase substrates were added immediately prior to real-time measurement at 37°C. Cells were assayed before and after treatment with vehicle (data for which are shown in this figure) or OxA (data not shown). These data were collected as part of the BRET assay and were also used for calculation of the corresponding BRET ratio presented in Figure 5. Results shown are representative of three independent experiments.
Figure 5
Figure 5
Detection of protein–protein interactions by real time BRET1 and BRET2 assays. Kinetic data comparing the BRET performance of different receptor/luminophore fusion proteins using the OxA-induced interaction between OxR2/luminophore and β-arrestin 2/Venus for BRET1 (A) or β-arrestin 2/GFP10 for BRET2 (B). Transiently co-transfected HEK293FT cells were assayed before and after treatment with OxA or vehicle. Luciferase substrates coelenterazine h for BRET1 (A) or DeepBlueC for BRET2 (B) were added immediately prior to real-time measurements at 37°C. The ligand-induced BRET ratios are calculated as described in Section “Materials and Methods.” Data shown are mean ± SEM of three independent experiments.
Figure 6
Figure 6
Detection of protein–protein interactions by real-time BRET1 assay. Kinetic data comparing V2R-wild type (A,B) and the three different mutants V2R-R137C (C,D), V2R-R137L (E,F), and V2R-R137H (G,H) tagged with Rluc8 (A,C,E,G) or Rluc (B,D,F,H) were generated by monitoring the interaction with β-arrestin 2/Venus. The luciferase substrate coelenterazine h was added immediately before real-time measurements at 37°C. The transiently co-transfected HEK293FT cells were assayed before and after treatment with AVP (final concentrations of 1, 0.1, and 0.01 μM) or vehicle. BRET ratio above wild type baseline was calculated as described in Section “Materials and Methods.” Data shown are mean ± SEM of four independent experiments. Parts of this figure (A,C,E,G) have been adapted from that published previously (Kocan et al., 2009) and are included for comparison with permission from the journal. Copyright 2009, The Endocrine Society.
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