A comparative analysis of resonance energy transfer methods for Alzheimer related protein-protein interactions in living cells

Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are extensively used to analyze protein interactions occurring in living cells. Although these two techniques are broadly applied in cellular biology, comparative analysis of their strengths and limitations is lacking. To this end, we analyzed a small network of proteins involved in the amyloidogenic processing of the Alzheimer β-amyloid precursor using FRET based cytometry, BRET, and fluorescence lifetime imaging microscopy (FLIM). Using all three methods, we were able to detect the interactions of the amyloid precursor protein with APBB1, APBB2, and APP itself. And we found an unreported interacting pair, APP-APH1A. In addition, we show that these four interacting pairs exhibit a strong FRET correlation with the acceptor/donor expression ratios. Overall the FRET based cytometry was the most sensitive and reliable approach to screen for new interacting proteins. Therefore, we applied FRET based cytometry to study competitive binding of two proteins, APBB1 and APBB2, with the same APP target.     

Constraints on GPCR Heterodimerization Revealed by the Type-4 Induced-Association BRET Assay

G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically important family of cell-surface receptors encoded by the human genome. In many instances, the distinct signaling behavior of certain GPCRs has been explained in terms of the formation of heteromers with, for example, distinct signaling properties and allosteric cross-regulation. Confirmation of this has, however, been limited by the paucity of reliable methods for probing heteromeric GPCR interactions in situ. The most widely used assays for GPCR stoichiometry, based on resonance energy transfer, are unsuited to reporting heteromeric interactions. Here, we describe a targeted bioluminescence resonance energy transfer (BRET) assay, called type-4 BRET, which detects both homo- and heteromeric interactions using induced multimerization of protomers within such complexes, at constant expression. Using type-4 BRET assays, we investigate heterodimerization among known GPCR homodimers: the CXC chemokine receptor 4 and sphingosine-1-phosphate receptors. We observe that CXC chemokine receptor 4 and sphingosine-1-phosphate receptors can form heterodimers with GPCRs from their immediate subfamilies but not with more distantly related receptors. We also show that heterodimerization appears to disrupt homodimeric interactions, suggesting the sharing of interfaces. Broadly, these observations indicate that heterodimerization results from the divergence of homodimeric receptors and will therefore likely be restricted to closely related homodimeric GPCRs.     

Analysis of in vitro SUMOylation using bioluminescence resonance energy transfer (BRET)

We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase (Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions.     

Biochemical and biophysical characterization of serotonin 5-HT2C receptor homodimers on the plasma membrane of living cells

While many studies have provided evidence of homodimerization and heterodimerization of G-protein-coupled receptors (GPCRs), few studies have used fluorescence resonance energy transfer (FRET) combined with confocal microscopy to visualize receptor dimerization on the plasma membrane, and there have been no reports demonstrating the expression of serotonin receptor dimers/oligomers on the plasma membrane of living cells. In the study presented here, biochemical and biophysical techniques were used to determine if 5-HT(2C) receptors exist as homodimers on the plasma membrane of living cells. Immunoprecipitation followed by Western blotting revealed the presence of immunoreactive bands the predicted size of 5-HT(2C) receptor monomers and homodimers that were detergent and cross-linker sensitive. Bioluminescence resonance energy transfer (BRET) was assessed in HEK293 cells expressing 5-HT(2C) receptors labeled with Renilla luciferase and yellow fluorescent protein. BRET levels were not altered by pretreatment with serotonin. Confocal microscopy provided direct visualization of FRET on the plasma membrane of live cells expressing 5-HT(2C) receptors labeled with cyan (donor) and yellow (acceptor) fluorescent proteins. FRET, assessed by acceptor photobleaching, was dependent on the donor/acceptor ratio and independent of acceptor expression levels, indicating that FRET resulted from receptor clustering and not from overexpression of randomly distributed receptors, providing evidence for GPCR dimers/oligomers in a clustered distribution on the plasma membrane. The results of this study suggest that 5-HT(2C) receptors exist as constitutive homodimers on the plasma membrane of living cells. In addition, a confocal-based FRET method for monitoring receptor dimerization directly on the plasma membrane of living cells is described.     

Applications of BRET to study dynamic G-protein coupled receptor interactions in living cells

Summary Protein-protein interactions are fundamental processes for many biological systems including those involving the superfamily of G-protein coupled receptors (GPCRs). When addressing key questions concerning the regulation of GPCR-protein complexes and their functional significance, the development and refinement of non-invasive techniques to study these interactions will be of great value. One such technique, bioluminescence resonance energy transfer (BRET), is a recently described biophysical method that represents a powerful tool with which to measure protein-protein interactions in live cells, in real time. This minireview highlights the impact that evolving techniques such as BRET have had on the study of dynamic protein interactions involving GPCRs. In particular, the application of BRET to the study of protein interactions involving the receptors for hypothalamic peptide hormones, thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone (GnRH), will be discussed. Using these receptors, BRET has successfully been used to demonstrate formation of both agonist-dependent and independent GPCR-GPCR complexes (oligomerization) and the agonist-dependent interaction of GPCRs with their intracellular adaptor protein partners, the arrestins. In summary, BRET is a highly snnsitive method that will not only aid in advancing our understanding of GPCR signalling and trafficking bout coud also potentially lead to the development of novel therapeutics that target these GPCR-protein complexes.     

Dimerization in the absence of higher-order oligomerization of the G protein-coupled secretin receptor

Oligomerization of G protein-coupled receptors has been proposed to affect receptor function and regulation; however, little is known about the molecular nature of such complexes. We previously utilized bioluminescence resonance energy transfer (BRET) to demonstrate that the prototypic Family B secretin receptor can form oligomers. We now explore the order of oligomerization present utilizing unique bimolecular fluorescence complementation and energy transfer techniques. The non-fluorescent carboxyl-terminal and amino-terminal halves of yellow fluorescent protein (YFP) were fused to the carboxyl terminus of the secretin receptor. These constructs bound secretin normally and signaled in response to secretin like wild type receptor. When co-expressed on COS cells, these constructs physically interacted to yield typical YFP fluorescence in biosynthetic compartments and at the plasma membrane, reflecting receptor homo-dimerization. However, the addition of another potential partner in form of Rlu- or CFP-tagged secretin receptor yielded no significant BRET or FRET signal, respectively, under conditions in which intact YFP-tagged secretin receptor yielded such a signal. Absence of higher-order receptor oligomers was further confirmed using saturation BRET techniques. Absence of significant resonance transfer to the secretin receptor homo-dimer was true for carboxyl-terminally-tagged secretin receptor, as well as for receptor incorporating the transfer partner into each of the three distinct intracellular loop domains. These results suggest that the secretin receptor can exist only as a structurally-specific homo-dimer, without being present as higher-order oligomers.     

Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis

G protein-coupled receptor (GPCR) oligomerization is a growing concept that has emerged from several studies suggesting that GPCRs can form both homo- and heterodimers. Using both coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) approaches, we established that the vasopressin V1a, V2, and the oxytocin receptors exist as homo- and hetero-dimers in transfected human embryonic kidney 293T cells. Each receptor protomer had a similar propensity to form homo- and heterodimers, indicating that their relative expression levels may determine the homo-/heterodimer ratio. The finding that immature forms of the receptor can be immunoprecipitated as homo- and heterodimers and the detection by BRET of such oligomer in endoplasmic reticulum-enriched fractions suggest that the oligomerization processes take place early during biosynthesis. Treatment with agonists or antagonists did not modify the BRET among any of the vasopressin and oxytocin receptor pairs studied, indicating that the dimerization state of the receptors is not regulated by ligand binding once they have reached the cell surface. Taken together, these results strongly support the notion that GPCR dimerization is a constitutive process.     

Third-Party Bioluminescence Resonance Energy Transfer Indicates Constitutive Association of Membrane Proteins: Application to Class A G-Protein-Coupled Receptors and G-Proteins

Many of the molecules that mediate G-protein signaling are thought to constitutively associate with each other in variably stable signaling complexes. Much of the evidence for signaling complexes has come from Förster resonance energy transfer and bioluminescence resonance energy transfer (BRET) studies. However, detection of constitutive protein association with these methods is hampered by nonspecific energy transfer that occurs when donor and acceptor molecules are in close proximity by chance. We show that chemically-induced recruitment of local third-party BRET donors or acceptors reliably separates nonspecific and specific BRET. We use this method to reexamine the constitutive association of class A G-protein-coupled receptors (GPCRs) with other GPCRs and with heterotrimeric G-proteins. We find that β2 adrenoreceptors constitutively associate with each other and with several other class A GPCRs. In contrast, GPCRs and G-proteins are unlikely to exist in stable constitutive preassembled complexes.     

Adenosine A2A-dopamine D2 receptor-receptor heteromerization: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer

There is evidence for strong functional antagonistic interactions between adenosine A2A receptors (A2ARs) and dopamine D2 receptors (D2Rs). Although a close physical interaction between both receptors has recently been shown using co-immunoprecipitation and co-localization assays, the existence of a A2AR-D2R protein-protein interaction still had to be demonstrated in intact living cells. In the present work, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques were used to confirm the occurrence of A2AR-D2R interactions in co-transfected cells. The degree of A2AR-D2R heteromerization, measured by BRET, did not vary after receptor activation with selective agonists, alone or in combination. BRET competition experiments were performed using a chimeric D2R-D1R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D2R were replaced by those of the dopamine D1 receptor (D1R). Although the wild type D2R was able to decrease the BRET signal, the chimera failed to achieve any effect. This suggests that the helix 5-I3-helix 6-E3 portion of D2R holds the site(s) for interaction with A2AR. Modeling of A2AR and D2R using a modified rhodopsin template followed by molecular dynamics and docking simulations gave essentially two different possible modes of interaction between D2R and A2AR. In the most probable one, helix 5 and/or helix 6 and the N-terminal portion of I3 from D2R approached helix 4 and the C-terminal portion of the C-tail from the A2AR, respectively.     

Detecting and imaging protein-protein interactions during G protein-mediated signal transduction in vivo and In situ by using fluorescence-based techniques

An important goal in cell biology has been to observe dynamic interactions between protein molecules within a living cell as they execute the reactions of a particular biochemical pathway. An important step toward achieving this goal has been the development of noninvasive fluorescence-based detection and imaging techniques for determining whether and when specific biomolecules in a cell become associated with one another. Furthermore, these techniques, which take advantage of phenomena known as bioluminescence- and fluorescence resonance energy transfer (BRET and FRET, respectively) as well as biomolecular fluorescence complementation (BiFC), can provide information about where and when protein-protein interactions occur in the cell. Increasingly BRET, FRET, and BiFC are being used to probe interactions between components involved in G protein-mediated signal transduction. Heptahelical (7TM) receptors, heterotrimeric guanine nucleotide binding proteins (G proteins) and their proximal downstream effectors constitute the core components of these ubiquitous signaling pathways. Signal transduction is initiated by the binding of agonist to heptahelical (7TM) receptors that in turn activate their cognate G proteins. The activated G protein subsequently regulates the activity of specific effectors. 7TM receptors, G proteins, and effectors are all membrane-associated proteins, and for decades two opposing hypotheses have vied for acceptance. The predominant hypothesis has been that these proteins move about independently of one another in membranes and that signal trandduction occurs when they encounter each other as the result of random collisions. The contending hypothesis is that signaling is propagated by organized complexes of these proteins. Until recently, the data supporting these hypotheses came from studying signaling proteins in solution, in isolated membranes, or in fixed cells. Although the former hypothesis has been favored, recent studies using BRET and FRET have generally supported the latter hypothesis as being the most likely scenario operating in living cells. In addition to the core components, there are many other proteins involved in G protein signaling, and BRET and FRET studies have been used to investigate their interactions as well. This review describes various BRET, FRET, and BiFC techniques, how they have been or can be applied to the study of G protein signaling, what caveats are involved in interpreting the results, and what has been learned about G protein signaling from the published studies.     

Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells

Green bioluminescence in Renilla species is generated by a approximately 100% efficient RET (resonance energy transfer) process that is caused by the direct association of a blue-emitting luciferase [Rluc (Renilla luciferase)] and an RGFP (Renilla green fluorescent protein). Despite the high efficiency, such a system has never been evaluated as a potential reporter of protein-protein interactions. To address the question, we compared and analysed in mammalian cells the bioluminescence of Rluc and RGFP co-expressed as free native proteins, or as fused single-chain polypeptides and tethered partners of self-assembling coiled coils. Here, we show that: (i) no spontaneous interactions generating detectable BRET (bioluminescence RET) signals occur between the free native proteins; (ii) high-efficiency BRET similar to that observed in Renilla occurs in both fusion proteins and self-interacting chimaeras, but only if the N-terminal of RGFP is free; (iii) the high-efficiency BRET interaction is associated with a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogues. Here, we propose a new functional complementation assay based on the detection of the high-efficiency BRET signal that is generated when the reporters Rluc and RGFP are brought into close proximity by a pair of interacting proteins to which they are linked. To demonstrate its performance, we implemented the assay to measure the interaction between GPCRs (G-protein-coupled receptors) and beta-arrestins. We show that complementation-induced BRET allows detection of the GPCR-beta-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response.     

gp130 dimerization in the absence of ligand: preformed cytokine receptor complexes

It is established that cytokine receptors signal after ligand binding as homo- or hetero-dimers in heteromeric complexes, but it is unclear, when dimerization occurs. To investigate gp130 dimerization, we performed co-precipitation experiments with the endogenous cytokine receptors gp130 and leukemia inhibitory factor receptor (LIF-R) and with gp130 variants carrying two different C-terminal peptide tags. Furthermore, fluorescence resonance energy transfer (FRET) was employed to detect dimerization of two fluorescent-tagged gp130 variants. Confocal laser scanning microscopy was used for FRET detection in live cells. gp130 and LIF-R could be coprecipitated in the absence of ligand. The interaction, however, was intensified by the addition of LIF. Similar results were obtained with the gp130 variants and confirmed by FRET analysis in live cells. The present study clearly demonstrates the existence of preformed but inactive gp130/LIF-R hetero- and gp130/gp130 homo-dimers. The addition of ligand enhanced the respective dimer formation and was required for signal transduction.     

Melanocortin receptors form constitutive homo- and heterodimers

A significant number of G protein-coupled receptors are shown to form homo- or heterodimers/oligomers, and oligomerization of GPCRs may be a quite general phenomenon. We have here explored the possibility that the two closely related human melanocortin receptor 1 (MC(1)R) and melanocortin receptor 3 (MC(3)R) form dimers. Using bioluminescence resonance energy transfer (BRET(2)) we demonstrate that MC(1) and MC(3)Rs form homo- and heterodimers, when expressed in Cos-7 cells. Treatment with agonist, partial agonist or antagonists did not modify the BRET(2) signal for any of the receptor pairs studied, suggesting that the dimerization is not regulated by ligand binding. Rather our results indicate that melanocortin receptors exist as constitutively pre-formed dimers.     

Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein

We report here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)–fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyze yellow-green (560 nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. Our results demonstrate for the first time that an efficient sequential BRET–FRET energy transfer process based on firefly luciferase bioluminescence can be employed to assay physiologically important protease activities.     

Oligomerisation of C. elegans Olfactory Receptors,ODR-10 and STR-112, in Yeast

It is widely accepted that vertebrate G-Protein Coupled Receptors (GPCRs) associate with each other as homo- or hetero-dimers or higher-order oligomers. The C. elegans genome encodes hundreds of olfactory GPCRs, which may be expressed in fewer than a dozen chemosensory neurons, suggesting an opportunity for oligomerisation. Here we show, using three independent lines of evidence: co-immunoprecipitation, bioluminescence resonance energy transfer and a yeast two-hybrid assay that nematode olfactory receptors (ORs) oligomerise when heterologously expressed in yeast. Specifically, the nematode receptor ODR-10 is able to homo-oligomerise and can also form heteromers with the related nematode receptor STR-112. ODR-10 also oligomerised with the rat I7 OR but did not oligomerise with the human somatostatin receptor 5, a neuropeptide receptor. In this study, the question of functional relevance was not addressed and remains to be investigated.     

Detection of heteromerization of more than two proteins by sequential BRET-FRET

Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.     

Use of fluorescence resonance energy transfer to analyze oligomerization of G-protein-coupled receptors expressed in yeast

Oligomerization or dimerization of G-protein-coupled receptors (GPCRs) has emerged as an important theme in signal transduction. This concept has recently gained widespread interest due to the application of direct and noninvasive biophysical techniques such as fluorescence resonance energy transfer (FRET), which have shown unequivocally that several types of GPCR can form dimers or oligomers in living cells. Current challenges are to determine which GPCRs can self-associate and/or interact with other GPCRs, to define the molecular principles that govern these specific interactions, and to establish which aspects of GPCR function require oligomerization. Although these questions ultimately must be addressed by using GPCRs expressed endogenously in their native cell types, analysis of GPCR oligomerization in heterologous expression systems will be useful to survey which GPCRs can interact, to conduct structure-function studies, and to identify peptides or small molecules that disrupt GPCR oligomerization and function. Here, we describe methods employing scanning fluorometry to detect FRET between GPCRs tagged with enhanced cyan and yellow fluorescent proteins (CFP and YFP) in living yeast cells. This approach provides a powerful means to analyze oligomerization of a variety of GPCRs that can be expressed in yeast, such as adrenergic, adenosine, C5a, muscarinic acetylcholine, vasopressin, opioid, and somatostatin receptors.     

Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions

A substantial range of protein-protein interactions can be readily monitored in real time using bioluminescence resonance energy transfer (BRET). The procedure involves heterologous coexpression of fusion proteins, which link proteins of interest to a bioluminescent donor enzyme or acceptor fluorophore. Energy transfer between these proteins is then detected. This protocol encompasses BRET1, BRET2 and the recently described eBRET, including selection of the donor, acceptor and substrate combination, fusion construct generation and validation, cell culture, fluorescence and luminescence detection, BRET detection and data analysis. The protocol is particularly suited to studying protein-protein interactions in live cells (adherent or in suspension), but cell extracts and purified proteins can also be used. Furthermore, although the procedure is illustrated with references to mammalian cell culture conditions, this protocol can be readily used for bacterial or plant studies. Once fusion proteins are generated and validated, the procedure typically takes 48-72 h depending on cell culture requirements.     

Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer

We describe a noncompetitive homogeneous bioluminescent immunoassay based on the antigen-dependent reassociation of antibody variable domains (open sandwich bioluminescent immunoassay, OS-BLIA). The reassociation of two chimeric proteins, an antibody heavy-chain fragment (V(H))-Renilla luciferase (Rluc) and an antibody light-chain fragment (V(L))-enhanced yellow fluorescent protein (EYFP), was monitored by a bioluminescence resonance energy transfer (BRET) between the two. Upon simple mixing of the reagents with the sample, an antigen-dependent increase in BRET was observed with a measurable concentration range of 0.1 to approximately 10 microg/ml antigen hen egg lysozyme. Compared with our comparable assays based on fluorescence resonance energy transfer (FRET), a 10-fold improvement in the sensitivity was attained, probably due to a reduction in reagent concentration.