Bioluminescence Resonance Energy Transfer Sensors Research Articles

Class-Wide Analysis of Frizzled-Dishevelled Interactions Using BRET Biosensors Reveals Functional Differences among Receptor Paralogs.

Wingless/Int-1 (WNT) signaling is mediated by WNT binding to 10 Frizzleds (FZD1-10), which propagate the signal inside the cell by interacting with different transducers, most prominently the phosphoprotein Dishevelled (DVL). Despite recent progress, questions about WNT/FZD selectivity and paralog-dependent differences in the FZD/DVL interaction remain unanswered. Here, we present a class-wide analysis of the FZD/DVL interaction using the DEP domain of DVL as a proxy in bioluminescence resonance energy transfer (BRET) techniques. Most FZDs engage in a constitutive high-affinity interaction with DEP. Stimulation of unimolecular FZD/DEP BRET sensors with different ligands revealed that most paralogs are dynamic in the FZD/DEP interface, showing distinct profiles in terms of ligand selectivity and signal kinetics. This study underlines mechanistic differences in terms of how allosteric communication between FZDs and their main signal transducer DVL occurs. Moreover, the unimolecular sensors represent the first receptor-focused biosensors to surpass the requirements for high-throughput screening, facilitating FZD-targeted drug discovery.

Glucose-1,6-bisphosphate: A new gatekeeper of cerebral mitochondrial pyruvate uptake

ObjectiveGlucose-1,6-bisphosphate (G-1,6-BP), a byproduct of glycolysis that is synthesized by phosphoglucomutase 2 like 1 (PGM2L1), is particularly abundant in neurons. G-1,6-BP is sensitive to the glycolytic flux, due to its dependence on 1,3-bisphosphoglycerate as phosphate donor, and the energy state, due to its degradation by inosine monophosphate-activated phosphomannomutase 1. Since the exact role of this metabolite remains unclear, our aim was to elucidate the specific function of G-1,6-BP in the brain. MethodsThe effect of PGM2L1 on neuronal post-ischemic viability was assessed by siRNA-mediated knockdown of PGM2L1 in primary mouse neurons. Acute mouse brain slices were used to correlate the reduction in G-1,6-BP upon ischemia to changes in carbon metabolism by 13C6-glucose tracing. A drug affinity responsive target stability assay was used to test if G-1,6-BP interacts with the mitochondrial pyruvate carrier (MPC) subunits in mouse brain protein extracts. Human embryonic kidney cells expressing a MPC bioluminescence resonance energy transfer sensor were used to analyze how PGM2L1 overexpression affects MPC activity. The effect of G-1,6-BP on mitochondrial pyruvate uptake and oxygen consumption rates was analyzed in isolated mouse brain mitochondria. PGM2L1 and a predicted upstream kinase were overexpressed in a human neuroblastoma cell line and G-1,6-BP levels were measured. ResultsWe found that G-1,6-BP in mouse brain slices was quickly degraded upon ischemia and reperfusion. Knockdown of PGM2L1 in mouse neurons reduced post-ischemic viability, indicating that PGM2L1 plays a neuroprotective role. The reduction in G-1,6-BP upon ischemia was not accompanied by alterations in glycolytic rates but we did see a reduced 13C6-glucose incorporation into citrate, suggesting a potential role in mitochondrial pyruvate uptake or metabolism. Indeed, G-1,6-BP interacted with both MPC subunits and overexpression of PGM2L1 increased MPC activity. G-1,6-BP, at concentrations found in the brain, enhanced mitochondrial pyruvate uptake and pyruvate-induced oxygen consumption rates. Overexpression of a predicted upstream kinase inhibited PGM2L1 activity, showing that besides metabolism, also signaling pathways can regulate G-1,6-BP levels. ConclusionsWe provide evidence that G-1,6-BP positively regulates mitochondrial pyruvate uptake and post-ischemic neuronal viability. These compelling data reveal a novel mechanism by which neurons can couple glycolysis-derived pyruvate to the tricarboxylic acid cycle. This process is sensitive to the glycolytic flux, the cell's energetic state, and upstream signaling cascades, offering many regulatory means to fine-tune this critical metabolic step.

A Vector Nanoplatform for the Bioimaging of Deep-Seated Tumors.

Today, in preclinical studies, optical bioimaging based on luminescence and fluorescence is indispensable in studying the development of neoplastic transformations, the proliferative activity of the tumor, its metastatic potential, as well as the therapeutic effect of antitumor agents. In order to expand the capabilities of optical imaging, sensors based on the bioluminescence resonance energy transfer (BRET) mechanism and, therefore, independent of an external light source are being developed. A targeted nanoplatform based on HER2-specific liposomes whose internal environment contains a genetically encoded BRET sensor was developed in this study to visualize deep-seated tumors characterized by overexpression of human epidermal growth factor receptor type 2 (HER2). The BRET sensor is a hybrid protein consisting of the highly catalytic luciferase NanoLuc (an energy donor) and a LSSmKate1 red fluorescent protein with a large Stokes shift (an energy acceptor). During the bioimaging of disseminated intraperitoneal tumors formed by HER2-positive SKOV3.ip1cells of serous ovarian cystadenocarcinoma, it was shown that the developed system is applicable in detecting deep-seated tumors of a certain molecular profile. The developed system can become an efficient platform for optimizing preclinical studies of novel targeted drugs.

Highly Sensitive and Selective Detection of Inorganic Phosphates in the Water Environment by Biosensors Based on Bioluminescence Resonance Energy Transfer.

The accurate detection of phosphate in water is very important to prevent water eutrophication and ensure the health of water quality. However, traditional phosphomolybdenum blue spectrophotometry is not sensitive, is time-consuming, and demands large amounts of chemical reagents. Therefore, highly sensitive, rapid, and environmentally friendly Pi detection methods are urgently needed. Here, we developed a bioluminescence resonance energy transfer (BRET)-based biosensor, which can detect Pi in water quickly, highly sensitively, and highly selectively. The NanoLuc and the Venus fluorescent protein were selected as the bioluminescence donor and energy acceptor, respectively. The best-performing BRET sensor variant, VenusΔC10-PΔC12-ΔN4Nluc, was identified by Pi-specific binding protein (PiBP) screening and systematic truncation. Single-factor experiments optimized the key parameters affecting the detection performance of the sensor. Under the optimal detection conditions, the detection limit of this method was 1.3 μg·L-1, the detection range was 3.3-434 μg·L-1, and it had excellent selectivity, repeatability, and stability. This low-cost and environment-friendly BRET sensor showed a good application prospect in real water quality detection.

Modular and genetically encodable RNA-based bioluminescence resonance energy transfer (BRET) sensors for cellular target detection.

Genetically encodable bioluminescent sensors have been broadly used for highly sensitive, low-background, low-phototoxicity bioanalysis and imaging in living biological systems. To achieve real-time quantitative measurement and high-throughput screening, powerful bioluminescence resonance energy transfer (BRET) sensors have been designed, especially based on interactions between a luciferase/luciferin donor and a fluorescent protein acceptor. While fluorescent protein-based BRET sensors have been widely applied in cellular and in vivo studies, more modular and easy-to-engineer RNA-based BRET sensors have never been reported. In this project, we report, for the first time, genetically encoded fluorogenic RNA-based BRET sensors for cellular imaging and quantification of different target analytes and RNA-protein interactions. Fluorogenic RNAs are single-stranded RNA aptamers that can bind specific small-molecule fluorophores and activate the fluorescence signals. By coupling a NanoLuc luciferase donor with different types of fluorogenic RNA acceptor, without reliance on external light, sensitive BRET signals are generated in response to target metabolite, signaling molecule, or RNA levels inside living cells. We envision that this novel genetically encoded RNA-based BRET system can be widely used in bioanalysis and nanomedicine.

Conformational GPCR BRET Sensors Based on Bioorthogonal Labeling of Noncanonical Amino Acids.

Here we describe the application of genetic code expansion and site-specific incorporation of noncanonical amino acids that serve as anchor points for fluorescent labeling to generate bioluminescence resonance energy transfer (BRET)-based conformational sensors. Using a receptor with an N-terminal NanoLuciferase (Nluc) and a fluorescently labeled noncanonical amino acid in the receptor's extracellular part allows to analyze receptor complex formation, dissociation, and conformational rearrangements over time and in living cells. These BRET sensors can be used to investigate ligand-induced intramolecular (cysteine-rich domain [CRD] dynamics), but also intermolecular (dimer dynamics) receptor rearrangements. With the design of BRET conformational sensors based on the minimally invasive bioorthogonal labeling procedure, we describe a method that can be used in a microtiter plate format and can be easily adopted to investigate ligand-induced dynamics in various membrane receptors.

Predicting response to platinum and non-platinum drugs through bioluminescence resonance energy transfer (BRET) based bio-molecular interactions in platinum resistant epithelial ovarian cancer

Therapy induced rewiring of signalling networks often lead to acquirement of platinum-resistance, thereby necessitating the use of non-platinum agents as second-line treatment particularly for epithelial ovarian cancer (EOC). A prior subject-specific assessment can guide the choice of optimal non-platinum agent/s and possible targeted therapeutic/s. Assessment of protein-protein interactions are superior to simple cytotoxicity assays to determine therapeutic efficacy and associated molecular responses. Utilizing improved PIP3-AKT and ERK1/2 activation Bioluminescence Resonance Energy Transfer (BRET) sensors, we report chemotherapy-induced ERK1/2 activation predominantly in cisplatin-paclitaxel resistant EOC cells and increased activation of both ERK1/2 and AKT in malignant ascites derived cancer cells from platinum-resistant patients but not from treatment-naive or platinum-sensitive relapse patients. Further, majority of the non-platinum drugs except irinotecan increased ERK1/2 activation in platinum-taxol resistant cells as observed by live-cell BRET assessment which were associated with p90RSK1/2 and BAD activation along with upregulation of multidrug transporter gene ABCC1 and cell survival genes like cyclin D1 and Bcl2. Interestingly, only irinotecan was able to sensitize these resistant cells. Altogether, this first report of BRET based sensing of molecular pathway activations in platinum resistant cell lines and patient's derived cancer cells highlight the clinical potential of BRET sensors in management of therapy resistant cancer.

Thread-Based Bioluminescent Sensor for Detecting Multiple Antibodies in a Single Drop of Whole Blood.

Antibodies are important biomarkers in clinical diagnostics in addition to being increasingly used for therapeutic purposes. Although numerous methods for their detection and quantification exist, they predominantly require benchtop instruments operated by specialists. To enable the detection of antibodies at point-of-care (POC), the development of simple and rapid assay methods independent of laboratory equipment is of high relevance. In this study, we demonstrate microfluidic thread-based analytical devices (μTADs) as a new platform for antibody detection by means of bioluminescence resonance energy-transfer (BRET) switching sensor proteins. The devices consist of vertically assembled layers including a blood separation membrane and a plastic film with a sewn-in cotton thread, onto which the BRET sensor proteins together with the substrate furimazine have been predeposited. In contrast to intensity-based signaling, the BRET mechanism enables time-independent, ratiometric readout of bioluminescence signals with a digital camera in a darkroom or a smartphone camera with a 3D-printed lens adapter. The device design allows spatially separated deposition of multiple bioluminescent proteins on a single sewn thread, enabling quantification of multiple antibodies in 5 μL of whole blood within 5 min. The bioluminescence response is independent of the applied sample volume within the range of 5-15 μL. Therefore, μTADs in combination with BRET-based sensor proteins represent user-friendly analytical tools for POC quantification of antibodies without any laboratory equipment in a finger prick (5 μL) of whole blood.

Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor.

Luciferase-based reporters provide a key measurement approach in a broad range of applications, from in vitro high-throughput screening to whole animal imaging. For example, luminescence intensity is widely used to measure promoter activity, protein expression levels, and cell growth. However, luminescence intensity measurements are subject to quantitative irregularities caused by luminescence decay and variation in reporter expression level. In contrast, bioluminescence resonance energy transfer (BRET) sensors provide the advantages of luciferase-based reporters but overcome the aforementioned irregularities because of the inherently ratiometric readout. Here, we generated a new ratiometric BRET sensor of ATP (ARSeNL—ATP detection with a Ratiometric mScarlet-NanoLuc sensor), and we demonstrated that it provides a stable and robust readout across protein, cell, and whole animal tissue contexts. The ARSeNL sensor was engineered by screening a color palette of sensors utilizing variants of the high photon flux NanoLuc luciferase as donors and a panel of red fluorescent proteins as acceptors. We found that the novel combination of NanoLuc and mScarlet exhibited the largest dynamic range, with a 5-fold change in the BRET ratio upon saturation with ATP. Importantly, the NanoLuc-mScarlet BRET pair provided a large spectral separation between luminescence emission channels that is compatible with green and red filter sets extensively used in typical biological microscopes and animal imaging systems. Using this new sensor, we showed that the BRET ratio was independent of luminescence intensity decay and sensor expression level, and the BRET ratio faithfully reported differences in live-cell energy metabolism whether in culture or within mouse tissue. In particular, BRET analyte sensors have not been used broadly in tissue contexts, and thus, in principle, our sensor could provide a new tool for in vivo imaging of metabolic status.

Integration of Nanomaterials and Bioluminescence Resonance Energy Transfer Techniques for Sensing Biomolecules.

Bioluminescence resonance energy transfer (BRET) techniques offer a high degree of sensitivity, reliability and ease of use for their application to sensing biomolecules. BRET is a distance dependent, non-radiative energy transfer, which uses a bioluminescent protein to excite an acceptor through the resonance energy transfer. A BRET sensor can quickly detect the change of a target biomolecule quantitatively without an external electromagnetic field, e.g., UV light, which normally can damage tissue. Having been developed quite recently, this technique has evolved rapidly. Here, different bioluminescent proteins have been reviewed. In addition to a multitude of bioluminescent proteins, this manuscript focuses on the recent development of BRET sensors by utilizing quantum dots. The special size-dependent properties of quantum dots have made the BRET sensing technique attractive for the real-time monitoring of the changes of target molecules and bioimaging in vivo. This review offers a look into the basis of the technique, donor/acceptor pairs, experimental applications and prospects.

Luciferase-induced photoreductive uncaging of small-molecule effectors

Bioluminescence resonance energy transfer (BRET) is extensively used to study dynamic systems and has been utilized in sensors for studying protein proximity, metabolites, and drug concentrations. Herein, we demonstrate that BRET can activate a ruthenium-based photocatalyst which performs bioorthogonal reactions. BRET from luciferase to the ruthenium photocatalyst is used to uncage effector molecules with up to 64 turnovers of the catalyst, achieving concentrations >0.6 μM effector with 10 nM luciferase construct. Using a BRET sensor, we further demonstrate that the catalysis can be modulated in response to an analyte, analogous to allosterically controlled enzymes. The BRET-induced reaction is used to uncage small-molecule drugs (ibrutinib and duocarmycin) at biologically effective concentrations in cellulo.

A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics

G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the α2A-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning β2-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands.

A novel BRET biosensor for Gαq‐GTP reveals unique properties of cancer‐associated GNAQ mutants

Heterotrimeric G proteins are critical signal transducers and dysregulation of their activity is emerging a critical driver of cancer. In fact, the mutational landscape of heterotrimeric G protein signaling networks in cancer has expanded dramatically in the recent years through large scale sequencing campaigns. However, the rate at which novel mutations are identified clearly outpaces the ability to analyze their functional consequences in a systematic and reliable manner, making evident the need for developing novel tools for their investigation. Optical reporters that directly monitor G protein activity in intact cells are ideally suited for this purpose as they allow the rapid characterization of signaling mechanisms with high sensitivity and temporal resolution. Despite that nucleotide exchange in the α-subunit is the event that defines G protein activation, all reporters described to date rely on measuring the dissociation and/or rearrangement of Gα-Gβγ complexes as an indirect readout of G protein activation. Here we describe a Bioluminescence Resonance Energy Transfer (BRET)-based biosensor that specifically detects GTP-bound Gαq in cells with sub-second resolution and its implementation to characterize cancer-associated Gαq mutants. Using this novel biosensor, we show that Gαq activation by GPCR stimulation leads to a robust increase in BRET that is rapidly reserved by receptor antagonists. This sensor can also detect modulation of the amplitude and kinetics of Gαq responses by GTPase Activating Proteins (GAPs). By combining this Gαq-GTP BRET sensor with other approaches, we dissected the mechanisms of action for several cancer-associated Gαq mutants to make novel and, in some cases, unexpected observations. For example, we have identified mutants that display normal basal activity and GPCR-mediated activation, but deactivate slowly due to GAP-insensitivity, thereby suggesting a novel cancer-promoting mechanism. Strikingly, we found that Gαq Q209L and Q209P, the two most frequent mutations in uveal melanoma, lead to signaling hyperactivation via different molecular mechanisms. Our data suggests that this difference arises from unique structural features of the Q209P mutant, which might represent a novel vulnerability of the 20–25% uveal melanomas that bear this mutation. In summary, we have validated a first-in-class biosensor for the direct measurement of the bona fide active species of Gα and demonstrated its implementation to gain further insights into the properties of G protein mutants in cancer. Support or Funding Information NIH grants R01GM112631 and R01GM108733, American Cancer Society grants RSG-13-362-01-TBE and IRG-72-001-36, and the Karin Grunebaum Cancer Research Foundation. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reporter-Based BRET Sensors for Measuring Biological Functions In Vivo.

Genetic reporter systems provide a good alternative to monitor cellular functions in vitro and in vivo and are contributing immensely in experimental research. Reporters like fluorescence and bioluminescence genes, which support optical measurements, provide exquisite sensitivity to the assay systems. In recent years several activatable strategies have been developed, which can relay specialized molecular functions from inside the cells. The application of bioluminescence resonance energy transfer (BRET) is one such strategy that has been proved to be extremely valuable as an in vitro or in vivo assay to measure dynamic events such as protein-protein interactions (PPIs).The BRET assay using RLuc-YFP was introduced in biological research in the late 1990s and demonstrated the interaction of two proteins involved in circadian rhythm. Since then, BRET has become a popular genetic reporter-based assay for PPI studies due to several inherent attributes that facilitate high-throughput assay development such as rapid and fairly sensitive ratio-metric measurement, the assessment of PPI irrespective of protein location in cellular compartment and cost effectiveness. In BRET-based screening, within a defined proximity range of 10-100Å, the excited energy state of the luminescent molecule excites the acceptor fluorophore in the form of resonance energy transfer, causing it to emit at its characteristic emission wavelength. Based on this principle, several such donor-acceptor pairs, using Renilla luciferase or its mutants as donor and either GFP2, YFP, mOrange, TagRFP or TurboFP as acceptor, have been reported for use.In recent years, the applicability of BRET has been greatly enhanced by the adaptation of the assay to multiple detection devices such as a luminescence plate reader, a bioluminescence microscope and a small animal optical imaging platform. Apart from quantitative measurement studies of PPIs and protein dimerization, molecular spectral imaging has expanded the scope for fast screening of pharmacological compounds that modulate PPIs by unifying in vitro, live cell and in vivo animal/plant measurement, all using one assay. Using examples from the literature, we will describe methods to perform in vitro and in vivo BRET imaging experiments and some of its applications.

Bright Bioluminescent BRET Sensor Proteins for Measuring Intracellular Caspase Activity

FRET-based caspase activity probes have become important tools to monitor apoptotic cell signaling. However, their dependence on external illumination is incompatible with light sensitive cells and hampers applications that suffer from autofluorescence and light scattering. Here we report the development of three caspase sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) that retain the advantages of genetically encoded, ratiometric optical probes but do not require external illumination. These sensors consist of the bright and stable luciferase NanoLuc and the fluorescent protein mNeonGreen, fused together via a linker containing a recognition site for caspase-3, -8, or -9. In vitro characterization showed that each caspase sensor displayed a robust 10-fold decrease in BRET ratio upon linker cleavage, with modest caspase specificity. Importantly, whereas scattering and background fluorescence precluded FRET-based detection of intracellular caspase activity in plate-reader assays, such measurements could be easily performed using our caspase BRET sensors in a high throughput format. The brightness of the BRET sensors also enabled long-term single-cell imaging, allowing BRET-based recording of cell heterogeneity in caspase activity in a heterogenic cell population.

Dual Readout BRET/FRET Sensors for Measuring Intracellular Zinc.

Genetically encoded FRET-based sensor proteins have significantly contributed to our current understanding of the intracellular functions of Zn2+. However, the external excitation required for these fluorescent sensors can give rise to photobleaching and phototoxicity during long-term imaging, limits applications that suffer from autofluorescence and light scattering, and is not compatible with light-sensitive cells. For these applications, sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) would provide an attractive alternative. In this work, we used the bright and stable luciferase NanoLuc to create the first genetically encoded BRET sensors for measuring intracellular Zn2+. Using a new sensor approach, the NanoLuc domain was fused to the Cerulean donor domain of two previously developed FRET sensors, eCALWY and eZinCh-2. In addition to preserving the excellent Zn2+ affinity and specificity of their predecessors, these newly developed sensors enable both BRET- and FRET-based detection. While the dynamic range of the BRET signal for the eCALWY-based BLCALWY-1 sensor was limited by the presence of two competing BRET pathways, BRET/FRET sensors based on the eZinCh-2 scaffold (BLZinCh-1 and -2) yielded robust 25–30% changes in BRET ratio. In addition, introduction of a chromophore-silencing mutation resulted in a BRET-only sensor (BLZinCh-3) with increased BRET response (50%) and an unexpected 10-fold increase in Zn2+ affinity. The combination of robust ratiometric response, physiologically relevant Zn2+ affinities, and stable and bright luminescence signal offered by the BLZinCh sensors allowed monitoring of intracellular Zn2+ in plate-based assays as well as intracellular BRET-based imaging in single living cells in real time.

Surface‐Tunable Bioluminescence Resonance Energy Transfer via Geometry‐Controlled ZnO Nanorod Coordination

The use of ZnO nanorods (NRs) as an effective coordinator and biosensing platform to create bioluminescence resonance energy transfer (BRET) is reported. Herein, a hydrothermal approach is applied to obtain morphologically controlled ZnO NRs, which are directly bound to luciferase (Luc) and carboxy-modified quantum dot (QD) acting as a donor-acceptor pair for BRET. BRET efficiency varies significantly with the geometry of ZnO NRs, which modulates the coordination between hexahistidine-tagged Luc (Luc-His6 ) and QD, owing to the combined effect of the total surface area consisting of (001) and (100) planes and their surface polarities. Unlike typical QD-BRET reactions with metal ions (e.g., zinc ions), a geometry-controlled ZnO NR platform can facilitate the design of surface-initiated BRET sensors without being supplemented by copious metal ions: the geometry-controlled ZnO NR platform can therefore pave the way for nanostructure-based biosensors with enhanced analytical performance.

Comment on “The Use of BRET to Study Receptor-Protein Interactions”

This Research Topic assembles for the first time a comprehensive selection of articles (mini review, review articles, original research, and opinion articles) on the bioluminescence resonance energy transfer (BRET) technology. BRET is a natural phenomenon known in several marine organisms. It relies on the non-radiative transfer of energy from an appropriate energy donor to an energy acceptor, provided that both are located at a distance lower than 10 nm [see Ref. (1) for a basic and theoretical introduction]. BRET has been first applied to the detection of protein–protein interactions (PPIs) in 1999 (2). Since then, the technology constantly evolved by combining new donor and acceptor couples and developing various BRET assay formats as discussed in the Mini Review article of De et al. (3). Many BRET assays have been developed to study the oligomerization of seven-transmembrane-spanning G protein-coupled receptors (GPCRs). The BRET technology provides an attractive way to study this phenomenon in intact cells without the need of solubilization of receptors from their natural membrane environment. The overwhelming majority of BRET-based studies conclude that GPCRs do indeed exist as dimers or higher-order oligomers when transfected into cells at physiological levels. Three of the articles of this Research Topic discuss BRET assays that have been developed to properly address the specificity of BRET signals obtained upon expression of different GPCRs (1, 4, 5). The current consensus confirms that several different BRET assays are needed to evaluate the specificity of BRET signals. The precise role of each of these assays remains a source of controversy in the field (4). Further refinement of BRET control experiments and application of new techniques like single-molecule measurements and functional in vivo studies are likely to provide new insights to the existence and physiological relevance of GPCR oligomerization. Not surprisingly, GPCR oligomerization is the main issue of four articles of this Research Topic (4–7) ranging from studies on class A and B GPCR homo- and heteromers using BRET or alternative approaches like time-resolved FRET measurements. Apart from performing the proper control experiments, another important issue in the BRET field concerns the proper interpretation of stimulus-induced BRET signals. In the context of GPCR oligomerization, agonist-induced BRET signals can be generated by an agonist-driven oligomerization or agonist-induced conformational changes within preassembled oligomers. Discrimination between these two possibilities is not trivial but has become possible due to the development of BRET donor saturation experiments in the absence and presence of receptor stimulation (8, 9). This issue, to which BRET has made a significant contribution, is obviously of general importance for the field of PPIs. This is illustrated by the articles describing the interaction of the protease-activated receptor 1 and 2 with its cognate G proteins as described by Ayoub and Pin (10) and Ayoub et al. (11). The BRET technology has been extended toward other receptor families like tyrosine kinase receptors and cytokine receptors. This diversification demonstrates the general feature of this technique. These studies did not only address the question of receptor oligomerization but also monitored the real-time interaction of receptors with various effector molecules such as Grb2, PTP1-B, PLC-γ1, etc. This important aspect is discussed in the review article of Siddiqui et al. (12). Receptor–effector interactions have been also monitored by BRET for two privileged GPCR interacting partners, heterotrimeric G proteins and β-arrestins, as documented in articles of this Research Topic (6, 10, 11, 13). A more recent application of BRET concerns the development of biosensors to monitor downstream events of cellular signaling like the generation of second messengers and activation of intracellular kinases. These sensors are typically composed of the energy donor and acceptor separated by an assay-specific domain that changes its conformation upon second messenger binding or phosphorylation, thus modifying the position of the donor and acceptor and consequently the BRET signal. Similar sensors have been developed for FRET applications, which served as source of inspiration for the development of the BRET sensors. The articles from Salahpour et al. (13) and Xu et al. (2) describe the design and validation of cAMP and ERK sensors. The high reproducibility of BRET and the robustness of the measurements make BRET an interesting option for the design of high throughput screening assays. Two applications are discussed in this Research Topic. The first concerns the design of an assay for the identification of compounds that specifically activate GPCR heteromers (6) based on the recruitment of β-arrestin to GPCR heteromers. The second case concerns the identification of PPI inhibitors (14). Taken together, this Research Topic provides an illustrative overview of the principles and applications of the BRET technology that should be of interest for any scientist interested in monitoring PPI in intact cells.

Buffer enhanced bioluminescence resonance energy transfer sensor based on Gaussia luciferase for in vitro detection of protease

Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.

Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway

Regulation of intracellular cyclic adenosine 3 ',5 '-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed G(s)-dependent receptors for isoproterenol and prostaglandin E(2). Whereas two ligands, uridine 5 '-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via G(q)/calcium and G(i), the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for G(s)-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P(2) receptor and the heterotrimeric G(13) protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.