Bioluminescence Resonance Energy Transfer Technology Research Articles

NanoBRET in C. elegans illuminates functional receptor interactions in real time

BackgroundProtein-protein interactions form the basis of every organism and thus, investigating their dynamics, intracellular protein localization, trafficking and interactions of distinct proteins such as receptors and their ligand-binding are of general interest. Bioluminescence resonance energy transfer (BRET) is a powerful tool to investigate these aspects in vitro. Since in vitro approaches mostly neglect the more complex in vivo situation, we established BRET as an in vivo tool for studying protein interactions in the nematode C. elegans.ResultsWe generated worms expressing NanoBRET sensors and elucidated the interaction of two ligand-G protein-coupled receptor (GPCR) pairs, the neuropeptide receptor NPR-11 and the Adhesion GPCR LAT-1. Furthermore, we adapted the enhanced bystander BRET technology to measure subcellular protein localization. Using this approach, we traced ligand-induced internalization of NPR-11 in vivo.ConclusionsOur results indicate that in vivo NanoBRET is a tool to investigate specific protein interactions and localization in a physiological setting in real time in the living organism C. elegans.

Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform.

Large-scale genomics studies have generated vast resources for in-depth understanding of vital biological and pathological processes. A rising challenge is to leverage such enormous information to rapidly decipher the intricate protein-protein interactions (PPIs) for functional characterization and therapeutic interventions. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform with both high sensitivity and robustness in a mammalian cell environment remains to be established. Here we describe the development and integration of a highly sensitive NanoLuc luciferase-based bioluminescence resonance energy transfer technology, termed BRET(n), which enables ultra-high-throughput (uHTS) PPI detection in live cells with streamlined co-expression of biosensors in a miniaturized format. We further demonstrate the application of BRET(n) in uHTS format in chemical biology research, including the discovery of chemical probes that disrupt PRAS40 dimerization and pathway connectivity profiling among core members of the Hippo signaling pathway. Such hippo pathway profiling not only confirmed previously reported PPIs, but also revealed two novel interactions, suggesting new mechanisms for regulation of Hippo signaling. Our BRET(n) biosensor platform with uHTS capability is expected to accelerate systematic PPI network mapping and PPI modulator-based drug discovery.

Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2.

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

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.

Spinophilin as a novel regulator of M 3 muscarinic receptor‐mediated insulin release in vitro and in vivo

Spinophilin (SPL), a multidomain scaffolding protein known to modulate the activity of different G-protein-coupled receptors, regulates various central nervous system (CNS) functions. However, little is known about the role of SPL expressed in peripheral cell types including pancreatic β cells. In this study, we examined the ability of SPL to modulate the activity of β-cell M(3) muscarinic acetylcholine receptors (M3Rs), which play an important role in facilitating insulin release and maintaining normal blood glucose levels. We demonstrated, by using both in vitro and in vivo approaches (mouse insulinoma cells and SPL-deficient mice), that SPL is a potent negative regulator of M3R-mediated signaling and insulin release. Additional biochemical and biophysical studies, including the use of bioluminescence resonance energy transfer technology, suggested that SPL is able to recruit regulator of G-protein signaling 4 (RGS4) to the M3R signaling complex in an agonist-dependent fashion. Since RGS4 is a member of the RGS family of proteins that act to reduce the lifetime of activated G proteins, these findings support the concept that the inhibitory effects of SPL on M3R activity are mediated by RGS4. These data suggest that SPL or other G-protein-coupled receptor-associated proteins may serve as novel targets for drug therapy aimed at improving β-cell function for the treatment of type 2 diabetes.

Application of BRET technology in interaction of G protein-coupled receptor with β-arrestins: recent progress

G protein-coupled receptors constitute an important super family of cell surface proteins.β-arrestin 1and 2 are widely expressed intracellular adaptor and scaffolding proteins playing important roles in G protein-coupled receptor desensitization,internalization,intracellular trafficking,and G protein-independent signaling.Recent development in bioluminescence resonance energy transfer(BRET)technology makes it possible to monitor the GPCR-β-arrestin complexes in live cells in a real-time manner;and investigators can further elucidate the ever-expanding roles ofβ-arrestins in mediating GPCR function,which will help to develop the new generation of drugs targeting GPCRs.

Pharmacological Characterization of Histone Deacetylase Inhibitor and Tumor Cell-Growth Inhibition Properties of New Benzofuranone Compounds

Epigenetic modifications, such as DNA methylation or histone deacetylation, are early events in cell tumorigenesis. The consequences of these modifications are repression of gene transcription and, notably, of tumor suppressor gene transcription. New therapeutic strategies aim to 'normalize' the epigenetic status of cancer cells. Histone deacetylase inhibitors (HDACi) have shown promising effects against proliferation and resistance to apoptosis of a large number of cancer cells. Vorinostat (SAHA), a hydroxamate HDACi, has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of refractory cutaneous T-cell lymphoma (CTCL). However, HDACi are poorly specific, present toxicities and many have very low half-lives in the plasma. Thus, the development of new compounds is necessary in order to increase the potential of HDACi in cancer treatment. We designed an assay, based on bioluminescence resonance energy transfer (BRET) technology, to screen and characterize HDACi activity in living cells. Using our specific and reproducible BRET assay, we characterized the pharmacological properties of benzofuranone HDACi compounds for the induction of histone acetylation and performed a comparison with the properties of suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA). We defined a benzofuranone HDACi compound that induced histone acetylation at nanomolar concentrations and showed an increased duration of histone acetylation. These properties correlated with the pharmacological properties of this HDACi for the growth inhibition of cancer cells. We, thus, demonstrated the applicability of BRET technology for the screening and characterization of new HDACi compounds in living cells, and identified an interesting benzofuranone HDACi.

The New Era of Bioluminescence Resonance Energy Transfer Technology

Bioluminescence resonance energy transfer (BRET) assay is a comparatively new cell-based assay technology that is assuming more prominent roles in the field of studying protein-protein interactions, protein dimerization and signal transduction. In the last few years BRET related research has gained significant momentum in terms of adding versatility in the assay format as well as a variety of new applications where it has been suitably used. Beyond the scope of quantitative measurement of protein-protein interactions and protein dimerization, molecular imaging applications based on BRET assays have broaden its scope as a great tool for high-throughput screening (HTS) of pharmacologically important compounds. This article will highlight the landmarks in BRET research, with those which have significant contributions towards making it an attractive single format assay that shuttles between in vitro and in vivo measurements.

Mapping the VDAC1 Oligomerization Contact Sites and N-Terminus Translocation

Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. Accumulating evidence implicates the voltage-dependent anion channel (VDAC) as functioning in mitochondria-mediated apoptosis involving cytochrome c release, leading to caspases activation and apoptosis. The mechanisms regulating cytochrome c release and the molecular architecture of the cytochrome c conducting channel remain unknown. Previously, we demonstrated that apoptosis induction was accompanied by VDAC oligomerization, as revealed by cross-linking and directly monitored in living cells using Bioluminescence Resonance Energy Transfer technology. Moreover, apoptosis inhibitors inhibited VDAC oligomerization and a correlation between the levels of VDAC oligomerization and apoptosis was observed. Here, we combined site-directed mutagenesis with chemical cross-linking to reveal the contact sites between VDAC1 molecules in dimers and higher oligomers. Replacing hydrophobic amino acids with charged amino acids in b-strands 1,2 and 19, but not 14, interfered with VDAC1 oligomerization and apoptosis induction. Cysteine cross-linking results, from introducing cysteine at a defined position in cysteine-less VDAC1 and applying the cysteine-specific cross-linker, BMOE, supported the close vicinity of b-strands 1,2 and 19 in VDAC1 dimer. Moreover, the results suggest that VDAC1 exists as a dimer that undergoes conformational changes upon apoptosis induction to assemble into a higher oligomeric state. Additionally we demonstrated that the N-terminal region of VDAC1 lies inside the pore, but could also move and interact with the N-terminus from a second molecule to form a dimer. Our results suggest that the glycine rich sequence 21-GYGFG-25 is involved in the N-terminus translocation from the internal pore to the channel face. These results provide structural insight into cellular VDAC1's oligomeric state and its N-terminal region location and translocation.

Oligomerization of the Mitochondrial Protein Voltage-Dependent Anion Channel Is Coupled to the Induction of Apoptosis

Accumulating evidence implicates that the voltage-dependent anion channel (VDAC) functions in mitochondrion-mediated apoptosis and as a critical player in the release of apoptogenic proteins, such as cytochrome c, triggering caspase activation and apoptosis. The mechanisms regulating cytochrome c release and the molecular architecture of the cytochrome c-conducting channel remain unknown. Here the relationship between VDAC oligomerization and the induction of apoptosis was examined. We demonstrated that apoptosis induction by various stimuli was accompanied by highly increased VDAC oligomerization, as revealed by cross-linking and directly monitored in living cells using bioluminescence resonance energy transfer technology. VDAC oligomerization was induced in all cell types and with all apoptosis inducers used, including staurosporine, curcumin, As(2)O(3), etoposide, cisplatin, selenite, tumor necrosis factor alpha (TNF-α), H(2)O(2), and UV irradiation, all acting through different mechanisms yet all involving mitochondria. Moreover, correlation between the levels of VDAC oligomerization and apoptosis was observed. Furthermore, the apoptosis inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited VDAC oligomerization. Finally, a caspase inhibitor had no effect on VDAC oligomerization and cytochrome c release. We propose that VDAC oligomerization is involved in mitochondrion-mediated apoptosis and may represent a general mechanism common to numerous apoptogens acting via different initiating cascades. Thus, targeting the oligomeric status of VDAC, and hence apoptosis, offers a therapeutic strategy for combating cancers and neurodegenerative diseases.

Different Effects of the Different Natural CC Chemokine Receptor 2b Ligands on β-Arrestin Recruitment, GαiSignaling, and Receptor Internalization

The chemokine receptor CCR2, which has been implicated in a variety of inflammatory, autoimmune, and cardiovascular conditions, binds several natural chemokine ligands. Here, we assessed the recruitment of β-arrestin to CCR2 in response to these ligands using bioluminescence resonance energy transfer technology. Compared with CCL2, which was considered as a full agonist, other CCR2 ligands were partial agonists with reduced efficacy and potency. Agonist potencies were not a function of their affinity for CCR2. Efficacy of arrestin recruitment matched that of agonist-induced CCR2 internalization. Although the potency and efficacy rank orders of the ligands in arrestin recruitment were similar to those observed for Gα(i1) activation, arrestin recruitment was at least in part resistant to Gα(i/o)-inactivating pertussis toxin, suggesting partial independence from Gα(i/o). The degree of pertussis toxin resistance of arrestin recruitment was different between the chemokines. Moreover, qualitative differences between the arrestin responses to the different ligands were identified in the stability of the response: although CCL7-induced arrestin recruitment had a half-life of less than 15 min, CCL8 and CCL13 induced stable CCR2-arrestin interactions. Finally, the ligands stabilized different conformations of the CCR2 homodimer. Our results support the validity of models for receptor-ligand interactions in which different ligands stabilize different receptor conformations also for endogenous receptor ligands, with corresponding implications for drug development targeting CCR2.

Enhanced BRET technology for the monitoring of agonist-induced and agonist-independent interactions between GPCRs and β-arrestins

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.

Recent advances in bioluminescence resonance energy transfer technologies to study GPCR heteromerization

The field of G protein-coupled receptor (GPCR) research has undergone a transformation in recent years due to the notion of heteromerization. In order to progress our understanding of the functional implications of this phenomenon, as well as its applicability across the diversity of GPCR subtypes, we need to continually look to improve the technologies we use to evaluate protein-protein interactions in as near a physiological setting as possible. The bioluminescence resonance energy transfer (BRET) technology has been intimately associated with the study of GPCR-GPCR interactions for the past ten years, and over this period, both the tools and the methods of analysis have continually evolved. In this review, we highlight recent advances in the BRET technology and focus particularly on the drive to establish the specificity of GPCR heteromers.

The BRET technology and its application to screening assays

The bioluminescence resonance energy transfer (BRET) method is based on resonance energy transfer between a light-emitting enzyme and a fluorescent acceptor. Since its first description in 1999, several versions of BRET have been described using different substrates and energy donor/acceptor couples. Today, BRET is considered as one of the most versatile techniques for studying the dynamics of protein-protein interactions in living cells. Various studies have applied BRET-based assays to screen new receptor ligands and inhibitors of disease-related-proteases. Inhibitors of protein-protein interactions are likely to become a new major class of therapeutic drugs, and BRET technology is expected to play an important role in the identification of such compounds. This review describes the original BRET-based methodology, more recent variants, and potential applications to drug screening.

In this issue

AbstractBioluminescent screeningBacart et al.: Biotechnol. J. 2008, 3, 311–324The bioluminescence resonance energy transfer (BRET) method is based on resonance energy transfer between a light‐emitting enzyme and a fluorescent acceptor. Since its first description in 1999, several versions of BRET have been presented using different substrates and energy donor/acceptor couples. Today, BRET is considered one of the most versatile techniques for studying the dynamics of protein‐protein interactions in living cells. Various studies have applied BRET‐based assays to screen new receptor ligands and inhibitors of disease‐related‐proteases. Inhibitors of proteinprotein interactions are likely to become a new major class of therapeutic drugs, and BRET technology is expected to play an important role in the identification of such compounds. This review describes the original BRET‐based methodology, more recent variants, and potential applications to drug screening.Switchgrass transformationMazarei et al.: Biotechnol. J. 2008, 3, 354–359Transient assay systems using protoplasts have been utilized in several plant species and are a powerful tool for rapid functional gene analysis and biochemical manipulations. A protoplast system has not been used in switchgrass (Panicum virgatum L.), even though it is a bioenergy crop that has received considerable attention. Here researchers from Knoxville (TN, USA) and Palestine report the first protocol to isolate large numbers of viable protoplasts from both leaves and roots of two switchgrass genotypes. They show that protoplast transformation with either a 35S or a ubiquitin promoter resulted in an increase in GUS activity compared to the untransformed controls. Their results indicate an efficient protoplast isolation and a transient assay system that can be used to facilitate gene expression studies in switchgrass.Stem cells on chipsJu et al.: Biotechnol. J. 2008, 3, 383–391Directional induction and differentiation of mesenchymal stem cells (MSCs) is very important to clinical therapy, but the mechanisms that govern differentiation are not well understood. Traditional plate culture cannot precisely control cellular behavior because cells take up substances while secreting cytokines and wastes. Here, Chinese researchers used a microfluidic device to culture MSCs inside a microchamber. Hepatic differentiation medium was perfused to evaluate the ability of MSCs to differentiate toward hepatic cells on the chip. Parallel differentiation on 96‐well plates was used to provide a detailed comparison of the differences between the two culturing methods. Their results indicate that a microfluidic platform might be a potential tool for cost‐effective and automated cell culture, and has potential applications in reliable cell‐based screens and assays.

The GAF Domain of the cGMP-Binding, cGMP-Specific Phosphodiesterase (PDE5) Is a Sensor and a Sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFa F163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET (2) assays. BRET (2) ratios of the wild type GAFa fusion protein, but not GAFa F163A, increased in the presence of cGMP but not cAMP. Higher basal BRET (2) ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFa F163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET (2) technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET (2) sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.

Monitoring Interactions between Receptor Tyrosine Kinases and Their Downstream Effector Proteins in Living Cells Using Bioluminescence Resonance Energy Transfer

A limited number of whole-cell assays allow monitoring of receptor tyrosine kinase (RTK) activity in a signaling pathway-specific manner. We present the general use of the bioluminescence resonance energy transfer (BRET) technology to quantitatively study the pharmacology and signaling properties of the receptor tyrosine kinase (RTK) superfamily. RTK BRET-2 assays monitor, in living cells, the specific interaction between RTKs and their effector proteins, which control the activation of specific downstream signaling pathways. A total of 22 BRET assays have been established for nine RTKs derived from four subfamilies [erythroblastic leukemia viral (v-erb-b) oncogene homolog (ErbB), platelet-derived growth factor (PDGF), neurotrophic tyrosine kinase receptor (TRK), vascular endothelial growth factor (VEGF)] monitoring the interactions with five effectors (Grb2, p85, Stat5a, Shc46, PLCgamma1). These interactions are dependent on the RTK kinase activity and autophosphorylation of specific tyrosine residues in the carboxyl terminus. RTK BRET assays are highly sensitive for quantifying ligand-independent (constitutive), agonist-induced, or antagonist-inhibited RTK activity levels. We studied the signaling properties of the PDGF receptor, alpha polypeptide (PDGFRA) isoforms (V561D; D842V and delta842-845) carrying activating mutations identified in gastrointestinal stromal tumors (GIST). All three PDGFRA isoforms are fully constitutively activated, insensitive to the growth factor PDGF-BB, but show differential sensitivity of their constitutive activity to be inhibited by the inhibitor imatinib (Gleevec). Epidermal growth factor receptor (EGFR) BRET structure-function studies identify the tyrosine residues 1068, 1114, and 1148 as the main residues mediating the interaction of EGFR with the adapter protein Grb2. The BRET technology provides an assay platform to study signaling pathway-specific RTK structure-function and will facilitate drug discovery efforts for the identification of novel RTK modulators.

Monitoring interactions between G-protein-coupled receptors and β-arrestins

beta-Arrestins 1 and 2 are ubiquitously expressed intracellular adaptor and scaffolding proteins that play important roles in GPCR (G-protein-coupled receptor) desensitization, internalization, intracellular trafficking and G-protein-independent signalling. Recent developments in BRET (bioluminescence resonance energy transfer) technology enable novel insights to be gained from real-time monitoring of GPCR-beta-arrestin complexes in live cells for prolonged periods. In concert with confocal microscopy, assays for studying internalization and recycling kinetics such as ELISAs, and techniques for measuring downstream signalling pathways such as those involving MAPKs (mitogen-activated protein kinases), investigators can now use a range of experimental tools to elucidate the ever-expanding roles of beta-arrestins in mediating GPCR function.

Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein–protein interactions in live cells

Bioluminescence resonance energy transfer (BRET) is an increasingly popular technique for studying protein–protein interactions in live cells. It is particularly suitable for real-time monitoring of such interactions, however, the timescale over which assays can be carried out is currently relatively short (minutes) due to substrate instability. We present a new derivation of the BRET technology, termed ‘extended BRET’ (eBRET), which now enables protein–protein interactions to be monitored in real-time for many hours. This capability has significant benefits for investigating cellular function over extended timescales, as we have illustrated using the agonist-induced G-protein coupled receptor/β-arrestin interaction. The potential for studying the modulation of such interactions by agonists, antagonists, inhibitors, dominant negative mutants and co-expressed accessory proteins is substantial. Furthermore, the advantages of eBRET have important implications for the development of high-throughput BRET screening systems, an ever-expanding area of interest for the pharmaceutical industry.

Detection of human immunodeficiency virus type 1 Nef and CD4 physical interaction in living human cells by using bioluminescence resonance energy transfer.

CD4 down-regulation by human immunodeficiency virus type 1 (HIV-1) Nef protein is a key function for virus virulence. This activity may be mediated by a direct Nef-CD4 interaction. We investigated the formation, in situ, of such a complex between proteins using bioluminescence resonance energy transfer technology and co-immunoprecipitations. Our data clearly demonstrate that Nef and CD4 interact in intact human cells. Moreover, our results clearly indicate that the dileucine motif of the CD4 cytoplasmic domain, critical for the Nef-induced CD4 down-regulation, is not implicated in the Nef/CD4 complex formation in the cellular context.