Different EGF-induced receptor dimer conformations for signaling and internalization.

The structural basis of the activation and internalization of EGF receptors (EGFR) is still a matter of debate despite the importance of this target in cancer treatment. Whether agonists induce dimer formation or act on preformed dimers remains discussed. Here, we provide direct evidence that EGF-induced EGFR dimer formation as best illustrated by the very large increase in FRET between snap-tagged EGFR subunits induced by agonists. We confirm that Erlotinib-related TK (tyrosine kinase) inhibitors also induce dimer formation despite the inactive state of the binding domain. Surprisingly, TK inhibitors do not inhibit EGF-induced EGFR internalization despite their ability to fully block EGFR signaling. Only Erlotinib-related TK inhibitors promoting asymmetric dimers could slow down this process while the lapatinib-related ones have almost no effect. These results reveal that the conformation of the intracellular TK dimer, rather than the known EGFR signaling, is critical for EGFR internalization. These results also illustrate clear differences in the mode of action of TK inhibitors on the EGFR and open novel possibilities to control EGFR signaling for cancer treatment.

Comparative analysis of lanthanide excited state quenching by electronic energy and electron transfer processes.

The relative sensitivities of structurally related Eu(III) complexes to quenching by electron and energy transfer processes have been compared. In two sets of 9-coordinate complexes based on 1,4,7-triazacyclononane, the Eu emission lifetime decreased as the number of conjugated sensitising groups and the number of unbound ligand N atoms increased, consistent with photoinduced electron transfer to the excited Eu(III) ion that is suppressed by N-protonation. Quenching of the Eu 5D0 excited state may also occur by electronic energy transfer, and the quenching of a variety of 9-coordinate complexes by a cyanine dye with optimal spectral overlap occurs by an efficient FRET process, defined by a Förster radius (R0) value of 68 Å and characterised by second rate constants in the order of 109 M-1 s-1; these values were insensitive to changes in the ligand structure and to the overall complex hydrophilicity. Quenching of the Eu and Tb excited states by energy transfer to Mn(II) and Cu(II) aqua ions occurred over much shorter distances, with rate constants of around 106 M-1 s-1, owing to the much lower spectral overlap integral. The calculated R0 values were estimated to be between 2.5 to 4 Å in the former case, suggesting the presence of a Dexter energy transfer mechanism that requires much closer contact, consistent with the enhanced sensitivity of the rate of quenching to the degree of steric shielding of the lanthanide ion provided by the ligand.

Synthesis and Evaluation of Europium Complexes that Switch on Luminescence in Lysosomes of Living Cells.

A set of four luminescent EuIII complexes bearing an extended aryl-alkynylpyridine chromophore has been studied, showing very different pH-dependent behaviour in their absorption and emission spectral response. For two complexes with pKa values of 6.45 and 6.20 in protein-containing solution, the emission lifetime increases very significantly following protonation. By varying the gate time during signal acquisition, the 'switch-on' intensity ratio could be optimised, and enhancement factors of between 250 to 1330 were measured between pH 8 and 4. The best-behaved probe showed no significant emission dependence on the concentration of endogenous cations, reductants, and serum albumin. It was examined in live-cell imaging studies to monitor time-dependent lysosomal acidification, for which the increase in observed image brightness due to acidification was a factor of 50 in NIH-3T3 cells.

Design of novel tripyridinophane-based Eu(III) complexes as efficient luminescent labels for bioassay applications.

In this work, the development of highly luminescent europium(III) complexes in water solution is reported, including their syntheses, analyses of their photophysical properties and applications in bioassays. Three Eu(III) complexes are derived from new ligands based on a tripyridinophane platform. There are four distinct sections in the structure of these ligands: an 18-membered polyaminocarboxylic macrocycle to bind efficiently lanthanide ions in aqueous solutions, three chromophoric subunits (4-(phenylethynyl)pyridine moieties) to effectively sensitize the emission of the metal, two peripheral moieties to solubilise the complex in aqueous media (sulfonate, sulfobetaine or glucose groups) and a free NH2 group available for grafting or bioconjugation. In our synthetic procedure, a pivotal macrocyclic platform is obtained with a high yield in the crucial macrocyclization step due to a metal template ion effect (74% yield). In Tris aqueous buffer (pH 7.4), the Eu(III) complexes show a maximum excitation wavelength at 320 nm, a suitable overall quantum yield (14%), a relatively long lifetime (0.80 ms) and a one-photon brightness in the range of 10 000 M-1 cm-1. Importantly, these photophysical properties are retained at dilute concentrations, even in the presence of a very large excess of potentially competing species, such as EDTA or Mg2+ ions. Furthermore, we report the bioconjugation of a Eu(III) complex labelled by an N-hydroxysuccinimide ester reactive group with an antibody (anti-glutathione-S-transferase) and the successful application of the corresponding antibody conjugate in the detection of GST-biotin in a fluoroimmunoassay. These new complexes provide a solution for high sensitivity in Homogeneous Time-Resolved Fluorescence (HTRF®) bioassays.

Tripyridinophane Platform Containing Three Acetate Pendant Arms: An Attractive Structural Entry for the Development of Neutral Eu(III) and Tb(III) Complexes in Aqueous Solution.

We report a detailed characterization of Eu3+ and Tb3+ complexes derived from a tripyridinophane macrocycle bearing three acetate side arms (H3tpptac). Tpptac3- displays an overall basicity (∑ log KiH) of 24.5, provides the formation of mononuclear ML species, and shows a good binding affinity for Ln3+ (log KLnL = 17.5-18.7). These complexes are also thermodynamically stable at physiological pH (pEu = 18.6, pTb = 18.0). It should be noted that the pGd value of Gd-tpptac (18.4) is only slightly lower than that of commercially available MRI contrast agents such as Gd-dota (pGd = 19.2). Moreover, a very good selectivity for these ions over the endogenous cations (log KCuL = 14.4, log KZnL = 12.9, and log KCaL = 9.3) is observed. The X-ray structure of the terbium complex shows the metal coordinated by the nine N6O3 donor set of the ligand and one inner-sphere water molecule. DFT calculations result in two Eu-tpptac structures with similar bond energies (ΔE = 0.145 eV): one structure in which the water is coordinated to the metal ion and one structure in which the water molecule is farther away from the ion, bound to the ligand with an OH-π bond. By detailed luminescence experiments, we demonstrate that the europium complex in aqueous solution presents a hydration equilibrium between nine-coordinate, dehydrated [Eu-tpptac]0 and ten-coordinate, monohydrated [Eu-tpptac(H2O)]0 species. A similar trend is observed for the terbium complex. Despite the presence of this hydration equilibrium, the H3tpptac ligand sensitizes Eu3+ and Tb3+ luminescence efficiently in buffered water at physiological pH. Particularly, the terbium complex displays a long excited-state lifetime of 2.24 ms and an overall quantum yield of 33% with a brightness of 3600 M-1 cm-1. Such features of Ln3+ complexes of H3tpptac indicate that this platform appears to be particularly appealing for the further development of luminescent lanthanide labels.

pH dependence of water anomaly temperature investigated by Eu(III) cryptate luminescence.

Although water has been extensively studied, not all of its unique properties have been fully understood. There is still controversy about the temperature at which hydrogen bonds are broken or weakened, producing the anomalous temperature dependence of many water properties. Different temperatures between 23 and 48 °C have been reported, but no study has scrutinized the reasons for this discrepancy. We suggest the determining role of pH in the alteration of the water anomaly temperature. We employed a luminescent europium trisbipyridine cryptate, which is highly sensitive to changes in the arrangement of water molecules and whose luminescence intensity and lifetime are not significantly influenced by variations over a broad pH range. Our results revealed an increase of the crossover temperature from circa 35 °C at pH 3.5 to circa 45 °C at pH 7 to 9, which explains the discrepancies of previous studies. The pH dependence of water anomaly temperature is an important property for a better understanding of water and water-based systems and applications.

HTS-compatible FRET-based conformational sensors clarify membrane receptor activation.

Cell surface receptors represent a vast majority of drug targets. Efforts have been conducted to develop biosensors reporting their conformational changes in live cells for pharmacological and functional studies. Although Förster resonance energy transfer (FRET) appears to be an ideal approach, its use is limited by the low signal-to-noise ratio. Here we report a toolbox composed of a combination of labeling technologies, specific fluorophores compatible with time-resolved FRET and a novel method to quantify signals. This approach enables the development of receptor biosensors with a large signal-to-noise ratio. We illustrate the usefulness of this toolbox through the development of biosensors for various G-protein-coupled receptors and receptor tyrosine kinases. These receptors include mGlu, GABAB, LH, PTH, EGF and insulin receptors among others. These biosensors can be used for high-throughput studies and also revealed new information on the activation process of these receptors in their cellular environment.

Single-Nanoparticle Cell Barcoding by Tunable FRET from Lanthanides to Quantum Dots.

Fluorescence barcoding based on nanoparticles provides many advantages for multiparameter imaging. However, creating different concentration-independent codes without mixing various nanoparticles and by using single-wavelength excitation and emission for multiplexed cellular imaging is extremely challenging. Herein, we report the development of quantum dots (QDs) with two different SiO2 shell thicknesses (6 and 12 nm) that are coated with two different lanthanide complexes (Tb and Eu). FRET from the Tb or Eu donors to the QD acceptors resulted in four distinct photoluminescence (PL) decays, which were encoded by simple time-gated (TG) PL intensity detection in three individual temporal detection windows. The well-defined single-nanoparticle codes were used for live cell imaging and a one-measurement distinction of four different cells in a single field of view. This single-color barcoding strategy opens new opportunities for multiplexed labeling and tracking of cells.

Multicolor time-resolved Förster resonance energy transfer microscopy reveals the impact of GPCR oligomerization on internalization processes.

Identifying the interacting partners and the dynamics of the molecular networks constitutes the key point in understanding cellular processes. Different methods often based on energy transfer strategies have been developed to examine the molecular dynamics of protein complexes. However, these methods suffer a couple of drawbacks: a single complex can be studied at a time, and its localization and tracking cannot generally be investigated. Here, we report a multicolor time-resolved Förster resonance energy transfer microscopy method that allows the identification of up to 3 different complexes simultaneously, their localization in cells, and their tracking after activation. Using this technique, we studied GPCR oligomerization and internalization in human embryonic kidney 293 cells. We definitively show that receptors can internalize as oligomers and that receptor coexpression deeply impacts oligomer internalization processes.

Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors.

In multimeric cell-surface receptors, the conformational changes of the extracellular ligand-binding domains (ECDs) associated with receptor activation remain largely unknown. This is the case for the dimeric metabotropic glutamate receptors even though a number of ECD structures have been solved. Here, using an innovative approach based on cell-surface labeling and FRET, we demonstrate that a reorientation of the ECDs is associated with receptor and G-protein activation. Our approach helps identify partial agonists and highlights allosteric interactions between the effector and binding domains. Any approach expected to stabilize the active conformation of the effector domain increased the agonist potency in stabilizing the active ECDs conformation. These data provide key information on the structural dynamics and drug action at metabotropic glutamate receptors and validate an approach for tackling such analysis on other receptors.

Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

Comparative analysis of conjugated alkynyl chromophore-triazacyclononane ligands for sensitized emission of europium and terbium.

A series of europium and terbium complexes based on a functionalized triazacyclononane carboxylate or phosphinate macrocyclic ligand is described. The influence of the anionic group, that is, carboxylate, methylphosphinate, or phenylphosphinate, on the photophysical properties was studied and rationalized on the basis of DFT calculated structures. The nature, number, and position of electron-donating or electron-withdrawing aryl substituents were varied systematically within the same phenylethynyl scaffold in order to optimize the brightness of the corresponding europium complexes and investigate their two-photon absorption properties. Finally, the europium complexes were examined in cell-imaging applications, and selected terbium complexes were studied as potential oxygen sensors.

Luminescent lanthanide cryptates: from the bench to the bedside.

The design and application of luminescent lanthanide cryptates for sensing biological interactions is highlighted through the review of the work performed in our laboratory and with academic collaborations. The path from the initial applications probing biochemical interaction in vitro to "state-of-the-art" cellular assays toward clinical applications using homogeneous time-resolved fluorescence technology is described. An overview of the luminescent lanthanide macrocyclic compounds developed at Cisbio in the recent past is given with an emphasis on specific constraints required by specific applications. Recent assays for drug-discovery and diagnostic purposes using both antibody-based and suicide-enzyme-based technology are illustrated. New perspectives in the field of molecular medicine and time-resolved microscopy are discussed.

Bright, highly water-soluble triazacyclononane europium complexes to detect ligand binding with time-resolved FRET microscopy.

Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water-soluble, and negatively charged sulfonic- or carboxylic acid derivatives of para-substituted aryl-alkynyl triazacyclononane complexes are described. Introduction of the charged solubilizing moieties suppresses cellular uptake or adsorption to living cells making them applicable for labeling and performing assays on membrane receptors. These europium complexes are applied to monitor fluorescent ligand binding on cell-surface proteins with time-resolved Förster resonance energy transfer (TR-FRET) assays in plate-based format and using TR-FRET microscopy.

A new approach to analyze cell surface protein complexes reveals specific heterodimeric metabotropic glutamate receptors.

G-protein-coupled receptors (GPCRs) can form heteromeric complexes. Herein, we describe a new approach to test the heteromerization of 2 receptors, or 2 receptor subunits, and to study the stoichiometry of the resulting complexes. As a proof-of-concept study, we investigated whether metabotropic glutamate receptors (mGluRs), in addition to being well-known homodimers, can form heteromers. To that aim, we combine the benefits of time-resolved fluorescence resonance energy transfer (trFRET) with the specific, cell-surface labeling of SNAP- and CLIP-tagged rat mGluR subunits, expressed in a mammalian cell line. First, we show that mGlu2 and mGlu4 subunits (but not mGlu2 and mGlu1) can heteromerize. Moreover, our trFRET data are consistent with mGluR subunits forming strict homodimeric receptors on single expression, and a combination of strict heterodimeric and strict homodimeric receptors on coexpression. Second, a comprehensive analysis reveals that from the 21 possible pairs of 2 mGluR subunits out of 7 subtypes (mGlu1 to 8, but not 6), only 11 are able to form heterodimers. These findings were further validated by biochemical and functional complementation studies. In addition to describing a new method to analyze cell-surface receptor complexes, our data reveal a new level of complexity within the mGluR family.

Developing chemical genetic approaches to explore G protein-coupled receptor function: validation of the use of a receptor activated solely by synthetic ligand (RASSL).

Molecular evolution and chemical genetics have been applied to generate functional pairings of mutated G protein-coupled receptors (GPCRs) and nonendogenous ligands. These mutant receptors, referred to as receptors activated solely by synthetic ligands (RASSLs) or designer receptors exclusively activated by designer drugs (DREADDs), have huge potential to define physiological roles of GPCRs and to validate receptors in animal models as therapeutic targets to treat human disease. However, appreciation of ligand bias and functional selectivity of different ligands at the same receptor suggests that RASSLs may signal differently than wild-type receptors activated by endogenous agonists. We assessed this by generating forms of wild-type human M(3) muscarinic receptor and a RASSL variant that responds selectively to clozapine N-oxide. Although the RASSL receptor had reduced affinity for muscarinic antagonists, including atropine, stimulation with clozapine N-oxide produced effects very similar to those generated by acetylcholine at the wild-type M(3)-receptor. Such effects included the relative movement of the third intracellular loop and C-terminal tail of intramolecular fluorescence resonance energy transfer sensors and the ability of the wild type and evolved mutant to regulate extracellular signal-regulated kinase 1/2 phosphorylation. Each form interacted similarly with ß-arrestin 2 and was internalized from the cell surface in response to the appropriate ligand. Furthermore, the pattern of phosphorylation of specific serine residues within the evolved receptor in response to clozapine N-oxide was very similar to that produced by acetylcholine at the wild type. Such results provide confidence that, at least for the M(3) muscarinic receptor, results obtained after transgenic expression of this RASSL are likely to mirror the actions of acetylcholine at the wild type receptor.

Modulating the photophysical properties of azamacrocyclic europium complexes with charge-transfer antenna chromophores.

Two europium complexes with bis(bipyridine) azamacrocyclic ligands featuring pendant arms with or without π-conjugated donor groups are synthesized and fully characterized by theoretical calculations and NMR spectroscopy. Their photophysical properties, including two-photon absorption, are investigated in water and in various organic solvents. The nonfunctionalized ligand gives highly water-stable europium complexes featuring bright luminescence properties but poor two-photon absorption cross sections. On the other hand, the europium complex with an extended conjugated antenna ligand presents a two-photon absorption cross section of 45 GM at 720 nm but is poorly luminescent in water. A detailed solvent-dependent photophysical study indicates that this luminescence quenching is not due to the direct coordination of O-H vibrators to the metal center but to the increase of nonradiative processes in a protic solvent induced by an internal isomerization equilibrium.

Targeted pH switched europium complexes monitoring receptor internalisation in living cells.

We report the design and evaluation of pH responsive luminescent europium(iii) probes that allow conjugation to targeting vectors to monitor receptor internalisation in cells. The approach adopted here can be used to tag proteins selectively and to monitor uptake into more acidic organelles, thereby enhancing the performance of time-resolved internalisation assays that require pH monitoring in real time.

Oligomerization of a G protein-coupled receptor in neurons controlled by its structural dynamics.

G protein coupled receptors (GPCRs) play essential roles in intercellular communication. Although reported two decades ago, the assembly of GPCRs into dimer and larger oligomers in their native environment is still a matter of intense debate. Here, using number and brightness analysis of fluorescently labeled receptors in cultured hippocampal neurons, we confirm that the metabotropic glutamate receptor type 2 (mGlu2) is a homodimer at expression levels in the physiological range, while heterodimeric GABAB receptors form larger complexes. Surprisingly, we observed the formation of larger mGlu2 oligomers upon both activation and inhibition of the receptor. Stabilizing the receptor in its inactive conformation using biochemical constraints also led to the observation of oligomers. Following our recent observation that mGlu receptors are in constant and rapid equilibrium between several states under basal conditions, we propose that this structural heterogeneity limits receptor oligomerization. Such assemblies are expected to stabilize either the active or the inactive state of the receptor.

FRET-Based Sensors Unravel Activation and Allosteric Modulation of the GABAB Receptor.

The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABAB, which is a target for various therapeutic applications. It is an obligatory heterodimer made of GB1 and GB2 that can be regulated by positive allosteric modulators (PAMs). The molecular mechanism of activation of the GABAB receptor remains poorly understood. Here, we have developed FRET-based conformational GABAB sensors compatible with high-throughput screening. We identified conformational changes occurring within the extracellular and transmembrane domains upon receptor activation, which are smaller than those observed in the related metabotropic glutamate receptors. These sensors also allow discrimination between agonists of different efficacies and between PAMs that have different modes of action, which has not always been possible using conventional functional assays. Our study brings important new information on the activation mechanism of the GABAB receptor and should facilitate the screening and identification of new chemicals targeting this receptor.