Bioluminescence Resonance Energy Transfer Technique Research Articles

An In vitro dimerization assay for the adverse outcome pathway approach in risk assessment of human estrogen receptor α-mediated endocrine-disrupting chemicals

The adverse outcome pathway (AOP) has been recently proposed as an effective framework for chemical risk assessment. The AOP framework offers the advantage of effectively integrating individual in vitro studies and in silico prediction models. Thus, the development of an effective testing method to measure key events caused by chemicals is essential for chemical risk assessment through a fully developed AOP framework. We developed a human cell-based estrogen receptor α (ERα) dimerization assay using the bioluminescence resonance energy transfer (BRET) technique and evaluated the ERα dimerization activities of 72 chemicals. Fifty-one chemicals were identified to mediate dimerization of ERα, and the BRET-based ERα dimerization assay could effectively measure the events that mediated dimerization of ERα by the estrogenic chemicals. These results were compared with the results of pre-existing assay to determine whether the BRET-based ERα dimerization assay could be employed as an in vitro test method to provide scientific information for explaining key events as a part of the AOP framework. Consequently, we propose that the BRET-based ERα dimerization assay is suitable for measuring the chemical-mediated dimerization of ERα, a key event in the AOP framework for cellular-level risk assessment of estrogenic chemicals.

Ghrelin and Cannabinoid Functional Interactions Mediated by Ghrelin/CB1 Receptor Heteromers That Are Upregulated in the Striatum From Offspring of Mice Under a High-Fat Diet.

There is evidence of ghrelinergic-cannabinoidergic interactions in the central nervous system (CNS) that may impact on the plasticity of reward circuits. The aim of this article was to look for molecular and/or functional interactions between cannabinoid CB1 and ghrelin GHS-R1a receptors. In a heterologous system and using the bioluminescence resonance energy transfer technique we show that human versions of cannabinoid CB1 and ghrelin GHS-R1a receptors may form macromolecular complexes. Such receptor heteromers have particular properties in terms of CB1/Gi-mediated signaling and in terms of GHS-R1a-Gq-mediated signaling. On the one hand, just co-expression of CB1R and GHS-R1a led to impairment of cannabinoid signaling. On the other hand, cannabinoids led to an increase in ghrelin-derived calcium mobilization that was stronger at low concentrations of the CB1 receptor agonist, arachidonyl-2’-chloroethylamide (ACEA). The expression of CB1-GHS-R1a receptor complexes in striatal neurons was confirmed by in situ proximity ligation imaging assays. Upregulation of CB1-GHS-R1a- receptor complexes was found in striatal neurons from siblings of pregnant female mice on a high-fat diet. Surprisingly, the expression was upregulated after treatment of neurons with ghrelin (200 nM) or with ACEA (100 nM). These results help to better understand the complexities underlying the functional interactions of neuromodulators in the reward areas of the brain.

Correction to "High-Throughput Screening of Transient Receptor Potential Channel 1 Ligands in the Light of the Bioluminescence Resonance Energy Transfer Technique".

In the above article [Chappe Y, Michel P, Joushomme A, Barbeau S, Pierredon S, Baron L, Garenne A, De Gannes FP, Hurtier A, Mayer S, Lagroye I, Quignard J-F, Ducret T, Compan V, Franchet C, and Percherancier Y (2021) Mol Pharmacol 100:[237–257][1]; DOI: <https://doi.org/10.1124/molpharm.121.000271

High-Throughput Screening of Transient Receptor Potential Channel 1 Ligands in the Light of the Bioluminescence Resonance Energy Transfer Technique.

Ion channels are attractive drug targets for many therapeutic applications. However, high-throughput screening (HTS) of drug candidates is difficult and remains very expensive. We thus assessed the suitability of the bioluminescence resonance energy transfer (BRET) technique as a new HTS method for ion-channel studies by taking advantage of our recently characterized intra- and intermolecular BRET probes targeting the transient receptor potential vanilloid type 1 (TRPV1) ion channel. These BRET probes monitor conformational changes during TRPV1 gating and subsequent coupling with calmodulin, two molecular events that are intractable using reference techniques such as automated calcium assay (ACA) and automated patch-clamp (APC). We screened the small-sized Prestwick chemical library, encompassing 1200 compounds with high structural diversity, using either intra- and intermolecular BRET probes or ACA. Secondary screening of the detected hits was done using APC. Multiparametric analysis of our results shed light on the capability of calmodulin inhibitors included in the Prestwick library to inhibit TRPV1 activation by capsaicin. BRET was the lead technique for this identification process. Finally, we present data exemplifying the use of intramolecular BRET probes to study other transient receptor potential (TRP) channels and non-TRPs ion channels. Knowing the ease of use of BRET biosensors and the low cost of the BRET technique, these assays may advantageously be included for extending ion-channel drug screening. SIGNIFICANCE STATEMENT: This study screened a chemical library against TRPV1 ion channel using bioluminescence resonance energy transfer (BRET) molecular probes and compared the results with the ones obtained using reference techniques such as automated calcium assay and automated patch-clamp. Multiparametric analysis of our results shed light on the capability of calmodulin antagonists to inhibit chemical activation of TRPV1 and indicates that BRET probes may advantageously be included in ion channel drug screening campaigns.

BRET-based assay to specifically monitor β2AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation.

The β-adrenergic receptors (βARs) are members of G protein-coupled receptor (GPCR) family and have been one of the most important GPCRs for studying receptor endocytosis and signaling pathway. Agonist binding of βARs leads to an activation of G proteins and their canonical effectors. In a parallel way, βAR stimulation triggers the termination of its signals by receptor desensitization. This termination process is initiated by G protein-coupled receptor kinase (GRK)-induced βAR phosphorylation that promotes the recruitment of β-arrestins to phosphorylated βAR. The uncoupled βARs which formed a complex with GRK and β-arrestin subsequently internalize into the cytosol. In addition, GRKs and β-arrestins also act as scaffolding proteins and signal transducers in their own functions to modulate various downstream effectors. Upon translocation to the βAR, β-arrestin is believed to undergo an important conformational change in the structure that is necessary for its signal transduction. The bioluminescence resonance energy transfer (BRET) technique involves the fusion of donor (luciferase) and acceptor (fluorescent) molecules to the interested proteins. Co-expression of these fusion proteins enables direct detection of their interactions in living cells. Here we describe the use of our established BRET technique to track the interaction of βAR with both GRK and β-arrestin. The assay described here allows the measurement of the BRET signal for detecting the interaction of β2AR with GRK2 and the conformational change of β-arrestin2 following βAR stimulation.

Effects of radiofrequency field exposure on proteotoxic-induced and heat-induced HSF1 response in live cells using the bioluminescence resonance energy transfer technique.

As of today, only acute effects of RF fields have been confirmed to represent a potential health hazard and they are attributed to non-specific heating (≥ 1°C) under high-level exposure. Yet, the possibility that environmental RF impact living matter in the absence of temperature elevation needs further investigation. Since HSF1 is both a thermosensor and the master regulator of heat-shock stress response in eukaryotes, it remains to assess HSF1 activation in live cells under exposure to low-level RF signals. We thus measured basal, temperature-induced, and chemically induced HSF1 trimerization, a mandatory step on the cascade of HSF1 activation, under RF exposure to continuous wave (CW), Global System for Mobile (GSM), and Wi-Fi-modulated 1800MHz signals, using a bioluminescence resonance energy transfer technique (BRET) probe. Our results show that, as expected, HSF1 is heat-activated by acute exposure of transiently transfected HEK293T cells to a CW RF field at a specific absorption rate of 24W/kg for 30min. However, we found no evidence of HSF1 activation under the same RF exposure condition when the cell culture medium temperature was fixed. We also found no experimental evidence that, at a fixed temperature, chronic RF exposure for 24h at a SAR of 1.5 and 6W/kg altered the potency or the maximal capability of the proteasome inhibitor MG132 to activate HSF1, whatever signal used. We only found that RF exposure to CW signals (1.5 and 6W/kg) and GSM signals (1.5W/kg) for 24h marginally decreased basal HSF1 activity.

Regulators of G-protein signaling, RGS2 and RGS4, inhibit protease-activated receptor 4-mediated signaling by forming a complex with the receptor and G\u03b1 in live cells

BackgroundProtease-activated receptor 4 (PAR4) is a seven transmembrane G-protein coupled receptor (GPCR) activated by endogenous proteases, such as thrombin. PAR4 is involved in various pathophysiologies including cancer, inflammation, pain, and thrombosis. Although regulators of G-protein signaling (RGS) are known to modulate GPCR/Gα-mediated pathways, their specific effects on PAR4 are not fully understood at present. We previously reported that RGS proteins attenuate PAR1- and PAR2-mediated signaling through interactions with these receptors in conjunction with distinct Gα subunits.MethodsWe employed a bioluminescence resonance energy transfer technique and confocal microscopy to examine potential interactions among PAR4, RGS, and Gα subunits. The inhibitory effects of RGS proteins on PAR4-mediated downstream signaling and cancer progression were additionally investigated by using several assays including ERK phosphorylation, calcium mobilization, RhoA activity, cancer cell proliferation, and related gene expression.ResultsIn live cells, RGS2 interacts with PAR4 in the presence of Gαq while RGS4 binding to PAR4 occurs in the presence of Gαq and Gα12/13. Co-expression of PAR4 and Gαq induced a shift in the subcellular localization of RGS2 and RGS4 from the cytoplasm to plasma membrane. Combined PAR4 and Gα12/13 expression additionally promoted translocation of RGS4 from the cytoplasm to the membrane. Both RGS2 and RGS4 abolished PAR4-activated ERK phosphorylation, calcium mobilization and RhoA activity, as well as PAR4-mediated colon cancer cell proliferation and related gene expression.ConclusionsRGS2 and RGS4 forms ternary complex with PAR4 in Gα-dependent manner and inhibits its downstream signaling. Our findings support a novel physiological function of RGS2 and RGS4 as inhibitors of PAR4-mediated signaling through selective PAR4/RGS/Gα coupling.EjpkaY-cVsUCvUePDRpq7pVideo

Effects of radiofrequency fields on RAS and ERK kinases activity in live cells using the bioluminescence resonance energy transfer technique

Purpose: The present study was conducted to re-evaluate the effect of low-level 1800 MHz RF signals on RAS/MAPK activation in live cells.Material and methods: Using Bioluminescence Resonance Energy Transfer technique (BRET), we assessed the effect of Continuous wave (CW) and Global System for Mobile (GSM)-modulated 1800 MHz signals (up to 2 W/kg) on ERK and RAS kinases' activity in live HuH7 cells.Results: We found that radiofrequency field (RF) exposure for 24 h altered neither basal level of RAS and ERK activation nor the potency of phorbol-12-myristate-13-acetate (PMA) to activate RAS and ERK kinases. However, we found that exposure to GSM-modulated 1800 MHz signals at 2 W/kg decreased the PMA maximal efficacy to activate both RAS and ERK kinases' activity. Exposure with CW 1800 MHz signal at 2 W/kg only decreased maximal efficacy of PMA to activate ERK but not RAS. No effects of RF exposure at 0.5 W/kg was observed on maximal efficacy of PMA to activate either RAS or ERK whatever the signal used.Conclusions: Our results indicate that RF exposure decreases the efficiency of the cascade of events, which, from the binding of PMA to its receptor(s), leads to the activation of RAS and ERK kinases.

β-Arrestin-2 BRET Biosensors Detect Different β-Arrestin-2 Conformations in Interaction with GPCRs.

β-Arrestins are critical regulators of G protein-coupled receptors (GPCRs) that desensitize G protein signaling, promote receptor internalization, and initiate signaling on their own. Recent structural findings indicate that β-arrestins adopt different conformations upon interaction with agonist-activated GPCRs. Here, we established a β-arrestin-2 conformational bioluminescence resonance energy transfer (BRET) sensor composed of the bright Nanoluc BRET donor and the red-shifted CyOFP1 BRET acceptor. The sensor monitors early intramolecular conformational changes of β-arrestin-2 in complex with a wide panel of different class A and class B GPCRs upon agonist activation and with orphan GPCRs known to spontaneously recruit β-arrestin-2. The introduction of the R170E mutant in the β-arrestin-2 sensor allowed the detection of a partially active β-arrestin-2 conformation, which is not dependent on receptor phosphorylation, while the deletion of the β-arrestin-2 finger-loop region detected the "tail-conformation" corresponding to the interaction of β-arrestin with the carboxyl-terminal domain of GPCRs. The new sensors combine the advantages of the BRET technique in terms of sensitivity, robustness, and suitability for real-time measurements with a high responsiveness toward early conformational changes to help to elucidate the different conformational states of β-arrestins associated with GPCR activation in living cells.

Multimeric Purinoceptor Detection by Bioluminescence Resonance Energy Transfer.

Assays based on bioluminescence resonance energy transfer (BRET) provide a sensitive and simple method to study protein-protein interactions in live cells. Here we describe a protocol using BRET technique to investigate potential interactions between P2X subunits. This approach combined with bimolecular fluorescence complementation (BiFC) can also be employed to determine the stoichiometry of heteromeric P2X receptors.

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.

Assembly of DNA-Templated Bioluminescent Modules for Amplified Detection of Protein Biomarkers.

By virtue of its self-illuminating mechanism, the bioluminescence resonance energy transfer (BRET) technique has recently emerged as a promising platform for point-of-care (POC) diagnostics. However, due to the difficulty of incorporating generic affinity elements, such as aptamers and antibodies, current BRET-based methods are still not applicable to most clinically important biomarkers. Furthermore, the inability of these methods to amplify BRET signals leads to limited sensitivity in some applications. Here, we present a modular strategy for amplified BRET detection of protein biomarkers in human peripheral blood samples. In this strategy, a DNA-templated bioluminescent module was constructed by simultaneously binding luciferase and green fluorescent protein to one DNA template in a site-specific manner. The proposed modules showed high energy transfer efficiency and could be assembled into long self-illuminating polymers. Owing to this modular design, aptamers and antibodies were rationally incorporated, enabling specific assembly of multiple bioluminescent modules on one target. This strategy realized amplified BRET assays for human α-thrombin and prostate specific antigen (PSA) with the detection limit in the picomolar range using either a spectrophotometer or a smartphone. The modularity of our strategy allowed detection of different biomarkers by simple exchange of affinity elements. Furthermore, the self-illumination and isothermal amplification performance of this strategy make it an attractive tool for POC diagnostics.

The regulators of G protein signaling RGS16 and RGS18 inhibit protease-activated receptor 2/Gi/o signaling through distinct interactions with Gα in live cells.

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor (GPCR) activated by endogenous proteases, in particular, trypsin. Although regulators of G protein signaling (RGS) are known to inhibit GPCR/Gα-mediated signaling, their specific effects on PAR2 are poorly understood at present. Here, we use a bioluminescence resonance energy transfer technique to investigate whether RGS16 and RGS18 bind PAR2 in live cells to regulate PAR2/Gαi/o -mediated signaling. Notably, we find that RGS16 binds to PAR2 in the presence of Gαi while RGS18 does not interact with PAR2, regardless of the presence of Gα. Both RGS16 and RGS18 inhibit PAR2/Gαi/o -mediated signaling. To our knowledge, the current study is the first to highlight the effects of RGS proteins on PAR2-mediated signaling.

Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins.

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

Activation of the TRPV1 Thermoreceptor Induced by Modulated or Unmodulated 1800 MHz Radiofrequency Field Exposure

The existence of effects of radiofrequency field exposure at environmental levels on living tissues and organisms remains controversial, in particular regarding potential "nonthermal" effects produced in the absence of temperature elevation. Therefore, we investigated whether TRPV1, one of the most studied thermosensitive channels, can be activated by the heat produced by radiofrequency fields and by some specific nonthermal interaction with the fields. We have recently shown that TRPV1 activation can be assessed in real-time on live cells using the bioluminescence resonance energy transfer technique. Taking advantage of this innovative assay, we monitored TRPV1 thermal and chemical modes of activation under radiofrequency exposure at 1800 MHz using different signals (CW, GSM, UMTS, LTE, Wi-Fi and WiMAX) at specific absorption rates between 8 and 32 W/kg. We showed that, as expected, TRPV1 channels were activated by the heat produced by radiofrequency field exposure of transiently-transfected HEK293T cells, but found no evidence of TRPV1 activation in the absence of temperature elevation under radiofrequency field exposure. There was no evidence either that, at fixed temperature, radiofrequency exposure altered the maximal efficacy of the agonist Capsaicin to activate TRPV1.

Hu antigen R (HuR) multimerization contributes to glioma disease progression

Among primary brain cancers, gliomas are the most deadly and most refractory to current treatment modalities. Previous reports overwhelmingly support the role of the RNA-binding protein Hu antigen R (HuR) as a positive regulator of glioma disease progression. HuR expression is consistently elevated in tumor tissues, and a cytoplasmic localization appears essential for HuR-dependent oncogenic transformation. Here, we report HuR aggregation (multimerization) in glioma and the analysis of this tumor-specific HuR protein multimerization in clinical brain tumor samples. Using a split luciferase assay, a bioluminescence resonance energy transfer technique, and site-directed mutagenesis, we examined the domains involved in HuR multimerization. Results obtained with the combination of the split HuR luciferase assay with the bioluminescence resonance energy transfer technique suggested that multiple (at least three) HuR molecules come together during HuR multimerization in glioma cells. Using these data, we developed a model of HuR multimerization in glioma cells. We also demonstrate that exposing glioma cells to the HuR inhibitor tanshinone group compound 15,16-dihydrotanshinone-I or to the newly identified compound 5 disrupts HuR multimerization modules and reduces tumor cell survival and proliferation. In summary, our findings provide new insights into HuR multimerization in glioma and highlight possible pharmacological approaches for targeting HuR domains involved in cancer cell-specific multimerization.

MP17-08 THE IDENTIFICATION OF ESTROGEN RECEPTOR GENE NETWORKS IN BENIGN PROSTATIC HYPERPLASIA

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology1 Apr 2017MP17-08 THE IDENTIFICATION OF ESTROGEN RECEPTOR GENE NETWORKS IN BENIGN PROSTATIC HYPERPLASIA Teresa Liu, Jalissa Wynder, Taryn James, Jill Macoska, and William Ricke Teresa LiuTeresa Liu More articles by this author , Jalissa WynderJalissa Wynder More articles by this author , Taryn JamesTaryn James More articles by this author , Jill MacoskaJill Macoska More articles by this author , and William RickeWilliam Ricke More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.597AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Benign prostatic hyperplasia (BPH) is a disease that develops in aging men as the ratio of 17β-estradiol (E2) to testosterone (T) increases. Little is known about the role of estrogens and estrogen-regulated genes in BPH. Estrogens bind and activate estrogen receptors (ER), ERα and ERβ, to form homo- and heterodimers. These ERs play opposing roles within the prostate; ERα is pro-proliferative and ERβ is anti-proliferative. While ER activation and gene networks have been extensively studied in other organs, little is known in the prostate. The objective of this is study was to identify genes regulated by E2 and selective estrogen receptor modulators (SERMs). METHODS RNA-seq was performed on BPH1 cells treated with E2 (binds ERα and ERβ), SERMs that bind ERβ (3β-diol, DPN, and WAY20070) and ERα (PPT) to identify genes specifically regulated by either ERα or ERβ. Sequences were mapped and assembled using TopHat, HTSeq, and edgeR. Genes reported were greater than 2-fold change over vehicle control with p-value < 0.05. CRISPR/Cas9 was used to knockout ERα and/or ERβ in BPH-1 cells to further validate ER-specific gene regulation as well as ER dimerization using Bioluminescence resonance energy transfer techniques. RESULTS RNA-seq analysis of BPH1 cells treated with ER agonists revealed both overlapping and distinct gene expression profiles for each compound. By examining the overlap of genes induced or repressed by both the natural and synthetic compounds specific for each receptor, we have identified 144 ERα-specific, 222 ERβ-specific, and 42 ERα/ERβ-heterodimer genes. Luciferase assays in the CRISPR knockout cells confirm the receptor specificity of the synthetic compounds used in this study. Additionally, dimerization studies confirm the contribution of ERα and/or ERβ to the regulation of gene expression. CONCLUSIONS Using E2 and SERMs, we were able to identify and validate ERα- and ERβ-regulated genes in benign prostate cells. Further studies assessing these estrogen-regulated genes will provide a basis for biomarkers in the study of BPH/LUTD. Furthermore, given the opposing roles of ERα and ERβ, these genes will allow us to further assess the function of ER in disease promotion in patients and animal models as well as monitor the efficacy of therapeutic SERMs in the treatment of BPH/LUTS. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e214 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Teresa Liu More articles by this author Jalissa Wynder More articles by this author Taryn James More articles by this author Jill Macoska More articles by this author William Ricke More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Nanoluciferase signal brightness using furimazine substrates opens bioluminescence resonance energy transfer to widefield microscopy.

Fluorescence and bioluminescence resonance energy transfer (FRET, BRET) techniques are powerful tools for studying protein-protein interactions in cellular assays. In contrast to fluorescent proteins, chemiluminescent proteins do not require excitation light, known to trigger autofluorescence, phototoxicity, and photobleaching. Regrettably, low signal intensity of luciferase systems restricts their usage as they require specialized microscopes equipped with ultra low-light imaging cameras. In this study, we report that bioluminescence quantification in living cells using a standard widefield automated microscope dedicated to screening and high content analysis is possible with the newer luciferase systems, Nanoluciferase (Nluc). With such equipment, we showed that robust intramolecular BRET can be measured using a combination of Nluc and yellow fluorescent protein (YFP). Using the human Superoxide Dismutase 1 (SOD1) dimer model, we next validated that intermolecular BRET could be quantified at a single cell level. The enhanced signal brightness of Nluc enabling BRET imaging to widefield microscopy shows strong potential to open up single cell protein-protein interactions studies to a wider audience. © 2016 International Society for Advancement of Cytometry.

Regulator of G protein signaling 8 inhibits protease-activated receptor 1/Gi/o signaling by forming a distinct G protein-dependent complex in live cells

Activation of seven-transmembrane-domain-possessing G protein-coupled receptors (GPCRs) by extracellular stimuli elicits intracellular responses. One class of GPCRs–protease-activated receptors (PARs)—is activated by endogenous proteases, such as thrombin and trypsin. Members of the regulator of G protein signaling (RGS) family stimulate GTP hydrolysis of G protein alpha (Gα) subunits, thereby inhibiting GPCR/Gα-mediated signaling. We previously reported that RGS2 and RGS4 inhibit PAR1/Gα-mediated signaling by interacting with PAR1 in a Gα-dependent manner. Here, employing the bioluminescence resonance energy transfer (BRET) technique, we identified RGS8 as a novel PAR1-interacting protein. Very little BRET activity was observed between PAR1-Venus (PAR1-Ven) and RGS8-Luciferase (RGS8-Luc) in the absence of Gα. However, in the presence of Gαo, BRET activity was specifically and significantly increased. This interaction was confirmed by biochemical and immunofluorescence assays. Notably, RGS8 inhibited PAR1/Gαi/o-mediated adenylyl cyclase and ERK activation, and prevented Gαo-induced neurite outgrowth and activation of Necdin protein, a downstream target of Gαo. Our findings suggest a novel function of RGS8 and reveal cellular mechanisms by which RGS8 mediates PAR1 inhibition.

Analysis of Arrestin Recruitment to Chemokine Receptors by Bioluminescence Resonance Energy Transfer.

Chemokine receptors recruit the multifunctional scaffolding protein beta arrestin in response to binding of their chemokine ligands. Given that arrestin recruitment represents a signaling axis that is in part independent from G-protein signaling, it has become a hallmark of G protein-coupled receptor functional selectivity. Therefore, quantification of arrestin recruitment has become a requirement for the delineation of chemokine and drug candidate activity along different signaling axes. Bioluminescence resonance energy transfer (BRET) techniques provide methodology for such quantification that can reveal differences between nonredundant chemokines binding the same receptor, and that can be upscaled for high-throughput testing. We here provide protocols for the careful setup of BRET-based arrestin recruitment assays, and examples for the application of such systems in dose-response or time-course experiments. Suggestions are given for troubleshooting, optimizing test systems, and the interpretation of results obtained with BRET-based assays, which indeed yield an intricate blend of quantitative and qualitative information.