Physical responses of bacterial chemoreceptors

Chemoreceptors of the bacterium Escherichia coli are thought to form trimers of homodimers that undergo conformational changes upon ligand binding and thereby signal a cytoplasmic kinase. We monitored the physical responses of trimers in living cells lacking other chemotaxis proteins by fluorescently tagging receptors and measuring changes in fluorescence anisotropy. These changes were traced to changes in energy transfer between fluorophores on different dimers of a trimer: attractants move these fluorophores farther apart, and repellents move them closer together. These measurements allowed us to define the responses of bare receptor oligomers to ligand binding and compare them to the corresponding response in kinase activity. Receptor responses could be fit by a simple "two-state" model in which receptor dimers are in either active or inactive conformations, from which energy bias and dissociation constants could be estimated. Comparison with responses in kinase-activity indicated that higher-order interactions are dominant in receptor clusters.     

Secretin receptor oligomers form intracellularly during maturation through receptor core domains

Oligomerization of numerous G protein-coupled receptors has been documented, including the prototypic family B secretin receptor. The clinical significance of oligomerization of this receptor became clear with the recent observation that a misspliced form present in pancreatic cancer could associate with the wild-type receptor and act as a dominant negative inhibitor of its normal growth inhibitory function. Our goal was to explore the molecular mechanism of this interaction using bioluminescence (BRET) and fluorescence (FRET) resonance energy transfer and fluorescence microscopy with a variety of receptor constructs tagged with luciferase or cyan or yellow fluorescent proteins. BRET signals comparable to those obtained from cells coexpressing differentially tagged wild-type receptors were observed for similarly tagged secretin receptors in which all or part of the amino-terminal domain was deleted. As expected, neither of these constructs bound secretin, and only the partially truncated construct sorted to the plasma membrane. Receptors lacking the majority of the carboxyl-terminal domain, including that important for phosphorylation-mediated desensitization, also produced BRET signals above background. These findings suggested that the receptor's membrane-spanning core is responsible for secretin receptor oligomerization. Interestingly, alanine substitutions for a -GxxxG- helix interaction motif in transmembrane segment 7 created nonfunctional receptors that were capable of forming oligomers. Furthermore, treatment of receptor-expressing cells with brefeldin A did not eliminate the BRET signals, and morphologic FRET experiments confirmed the expected subcellular localizations of receptor oligomers. We conclude that secretin receptor oligomerization occurs through -GxxxG- motif-independent interactions of transmembrane segments during the maturation of nascent molecules.     

Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor1

Background information. The idea that GPCRs (G‐protein‐coupled receptors) may exist as homo‐ or hetero‐oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM₁ (human M₁ muscarinic acetylcholine receptor subtype). Results. We analysed the hM₁ oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native‐PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO‐M1 cells (Chinese‐hamster ovary cells expressing muscarinic M₁ receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M₁ receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M₁ receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor. Conclusions. Our results suggest that MT7 binds to a dimeric form of hM₁ receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre‐existing muscarinic receptor homodimers.     

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.     

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.     

Opsin oligomerization in a heterologous cell system

Using bioluminescence resonance energy transfer (BRET) we studied opsin oligomerization in heterologous expression systems and quantitatively assessed its oligomerization state. BRET2 saturation and competition experiments were performed with live COS-7 cells expressing Rluc-and GFP2-tagged receptor constructs. BRET2 saturation curves obtained were hyperbolic, and the calculated oligomerization state (N = 1 for dimers) suggested that opsin (N = 1.34 +/- 0.25) forms higher oligomers. Very high BRET2 values obtained for the opsin homo-dimer pair indicated a large energy transfer efficiency (E) and for cases where E > 0.1 a modified saturation curve was proposed. The existence of homo-dimer complexes was additionally supported by competition assay results and was also observed in HEK-293 cells. Furthermore, evidence was provided for homo-and hetero-dimerization of family A (beta2-adrenergic) and B (gastric inhibitory polypeptide, GIP) receptors. In summary, these experiments demonstrate homo-and hetero-dimerization for opsin, beta 2-adrenergic, and GIP receptors.     

Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB1 cannabinoid receptors

Human OX₁ orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB₁ cannabinoid receptors. The latter has been suggested to be important for orexin receptor responses and trafficking. In this study, we wanted to assess the ability of the other combinations of receptors to also form similar complexes. Vectors for expression of human OX₁, OX₂ and CB₁ receptors, C-terminally fused with either Renilla luciferase or GFP² green fluorescent protein variant, were generated. The constructs were transiently expressed in Chinese hamster ovary cells, and constitutive dimerization between the receptors was assessed by bioluminescence energy transfer (BRET). Orexin receptor subtypes readily formed homo- and hetero(di)mers, as suggested by significant BRET signals. CB₁ receptors formed homodimers, and they also heterodimerized with both orexin receptors. Interestingly, BRET efficiency was higher for homodimers than for almost all heterodimers. This is likely to be due to the geometry of the interaction; the putatively symmetric dimers may place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP² to CB₁ produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX₁–OX₂ interaction. In conclusion, orexin receptors have a significant propensity to make homo- and heterodi-/oligomeric complexes. However, it is unclear whether this affects their signaling. As orexin receptors efficiently signal via endocannabinoid production to CB₁ receptors, dimerization could be an effective way of forming signal complexes with optimal cannabinoid concentrations available for cannabinoid receptors.     

Preparation and characterization of Alexa Fluor 594-labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor

We have prepared and characterized a new fluorescent derivative of murine epidermal growth factor (EGF), Alexa Fluor 594-labeled EGF (A-EGF), for fluorescence studies of EGF-EGF receptor interactions. We describe the synthesis of this derivative and its physical and biological characterization. The significant overlap between the excitation and the emission spectra of A-EGF makes this probe well suited to fluorescence resonance energy homo-transfer. Using time-resolved fluorescence to examine the oligomeric state of the EGF receptor, we have observed resonance energy homo-transfer of A-EGF bound to EGF receptors in cells, but not of A-EGF bound to EGF receptors in membrane vesicles. Our results, interpreted in the context of recent crystallographic studies of the ligand-binding domains of EGF receptors, suggest that observed fluorescence resonance energy transfer does not result from transfer within receptor dimers, but rather results from transfer within higher-order oligomers. Furthermore, our results support a structural model for oligomerization of EGF receptors in which dimers are positioned head-to-head with respect to the ligand-binding site, consistent with the head-to-head interactions observed between adjacent receptor dimers by X-ray crystallography.     

Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair

Spectral variants of the green fluorescent protein (GFP) have been extensively used as reporters to image molecular interactions in living cells by fluorescence resonance energy transfer (FRET). However, those GFP variants which are the most efficient donor acceptor pairs for FRET measurements show a high degree of spectral overlap which has hampered in the past their use in FRET applications. Here we use spectral imaging and subsequent un-mixing to quantitatively separate highly overlapping donor and acceptor emissions in FRET measurements. We demonstrate the method in fixed and living cells using a novel GFP based FRET pair (GFP2-YFP (yellow)), which has an increased FRET efficiency compared to the most commonly used FRET pair consisting of cyan fluorescent protein and YFP. Moreover, GFP2 has its excitation maximum at 396 nm at which the YFP acceptor is excited only below the detection level and thus this FRET pair is ideal for applications involving sensitized emission.     

Identification of regions of leukotriene C4 synthase which direct the enzyme to its nuclear envelope localization

Leukotrienes (LTs) are fatty acid derivatives formed by oxygenation of arachidonic acid via the 5-lipoxygenase (5-LO) pathway. Upon activation of inflammatory cells 5-LO is translocated to the nuclear envelope (NE) where it converts arachidonic acid to the unstable epoxide LTA4. LTA4 is further converted to LTC4 by conjugation with glutathione, a reaction catalyzed by the integral membrane protein LTC4 synthase (LTC4S), which is localized on the NE and endoplasmic reticulum (ER). We now report the mapping of regions of LTC4S that are important for its subcellular localization. Multiple constructs encoding fusion proteins of green fluorescent protein (GFP) as the N-terminal part and various truncated variants of human LTC4S as C-terminal part were prepared and transfected into HEK 293/T or COS-7 cells. Constructs encoding hydrophobic region 1 of LTC4S (amino acids 6-27) did not give distinct membrane localized fluorescence. In contrast hydrophobic region 2 (amino acids 60-89) gave a localization pattern similar to that of full length LTC4S. Hydrophobic region 3 (amino acids 114-135) directed GFP to a localization indistinguishable from that of full length LTC4S. A minimal directing sequence, amino acids 117-132, was identified by further truncation. The involvement of the hydrophobic regions in the homo-oligomerization of LTC4S was investigated using bioluminescence resonance energy transfer (BRET) analysis in living cells. BRET data showed that hydrophobic regions 1 and 3 each allowed oligomerization to occur. These regions most likely form transmembrane helices, suggesting that homo-oligomerization of LTC4S is due to helix-helix interactions in the membrane.     

An endogenous green fluorescent protein–photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer

Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria. It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.     

Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells

A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100 Å of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.     

Mapping the architecture of secretin receptors with intramolecular fluorescence resonance energy transfer using acousto-optic tunable filter-based spectral imaging

The molecular structure and agonist-induced conformational changes of class II G protein-coupled receptors are poorly understood. In this work, we developed and characterized a series of dual cyan fluorescent protein (CFP)-tagged and yellow fluorescent protein (YFP)-tagged secretin receptor constructs for use in various functional and fluorescence analyses of receptor structural variants. CFP insertions within the first or second intracellular loop domains of this receptor were tolerated poorly or partially, respectively, in receptors tagged with a carboxyl-terminal yellow fluorescent protein that itself had no effect on secretin binding or cAMP production. A similar CFP insertion into the third intracellular loop resulted in a plasma membrane-localized receptor that bound secretin and signaled normally. This fully active third-loop variant exhibited a significant decrease in fluorescence resonance energy transfer signals that were recorded with an acousto-optic tunable filter microscope after exposure to secretin agonist but not to a receptor antagonist. These data demonstrate changes in the relative positions of intracellular structures that support a model for secretin receptor activation.     

Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase

Proteolytic processing plays crucial roles in physiological and pathophysiological cellular functions such as peptide generation, cell cycle, and apoptosis. We developed a novel biophysical bioluminescence resonance energy transfer (BRET) system between a secreted Vargula luciferase (Vluc) and an enhanced yellow fluorescent protein (EYFP) for visualization of cell biological processes. The bioluminescence spectrum of the fusion protein (Vluc-EYFP) is bimodal (lambdamax = 460 nm (Vluc) and 525nm (EYFP)), indicating that the excited-state energy of Vluc transfers to EYFP (in short, BRET). The BRET signal can be measured in the culture medium and pursue quantitative production of two neuropeptides, nocistatin (NST) and nociceptin/orphanin FQ (N/OFQ) in living cells. NST and N/OFQ are located in tandem on the same precursor, but NST exhibits antagonistic action against N/OFQ-induced central functions. Insertion of a portion of the NST-N/OFQ precursor (Glu-Gln-Lys-Gln-Leu-Gln-Lys-Arg-Phe-Gly-Gly-Phe-Tyr-Gly) in Vluc-EYFP makes the fusion protein cleavable at Lys-Arg in NG108-15 cells, and proprotein convertase 1 enhances this digestion. The change in BRET signals quantifies the processing of the fusion protein. Our novel intramolecular BRET system using a secreted luciferase is useful for investigating peptide processing in living cells.     

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.     

FRET analysis reveals a critical conformational change within the Na,K-ATPase alpha1 subunit N-terminus during GPCR-dependent endocytosis

Dopamine is a major regulator of sodium reabsorption in proximal tubule epithelia. It induces the endocytosis of plasma membrane Na,K-ATPase molecules, and this results in a reduced capacity of the cells to transport sodium. Dopamine induces the phosphorylation of Ser-18 in the alpha1-subunit of Na,K-ATPase. Fluorescence resonance energy transfer analysis of cells expressing YFP-alpha1 and beta1-CFP reveals that treatment of the cells with dopamine increases energy transfer between CFP and YFP. This is consistent with a protein conformational change that results in the N-terminal end of alpha1 moving closer to the internal face of the plasma membrane.     

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.     

The physical and functional thermal sensitivity of bacterial chemoreceptors

The bacterium Escherichia coli exhibits chemotactic behavior at temperatures ranging from approximately 20 °C to at least 42 °C. This behavior is controlled by clusters of transmembrane chemoreceptors made from trimers of dimers that are linked together by cross-binding to cytoplasmic components. By detecting fluorescence energy transfer between various components of this system, we studied the underlying molecular behavior of these receptors in vivo and throughout their operating temperature range. We reveal a sharp modulation in the conformation of unclustered and clustered receptor trimers and, consequently, in kinase activity output. These modulations occurred at a characteristic temperature that depended on clustering and were lower for receptors at lower adaptational states. However, in the presence of dynamic adaptation, the response of kinase activity to a stimulus was sustained up to 45 °C, but sensitivity notably decreased. Thus, this molecular system exhibits a clear thermal sensitivity that emerges at the level of receptor trimers, but both receptor clustering and adaptation support the overall robust operation of the system at elevated temperatures.