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.

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 NH group available for grafting or bioconjugation.

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.

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

A set of four luminescent Eu 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 pK values of 6.45 and 6.

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 Eu and Tb complexes derived from a tripyridinophane macrocycle bearing three acetate side arms (Htpptac). Tpptac displays an overall basicity (∑ log ) of 24.5, provides the formation of mononuclear ML species, and shows a good binding affinity for Ln (log = 17.

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.

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 SiO shell thicknesses (6 and 12 nm) that are coated with two different lanthanide complexes (Tb and Eu).

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 (mGlu) is a homodimer at expression levels in the physiological range, while heterodimeric GABA receptors form larger complexes.

Pharmacological evidence for a metabotropic glutamate receptor heterodimer in neuronal cells.

Metabotropic glutamate receptors (mGluRs) are mandatory dimers playing important roles in regulating CNS function. Although assumed to form exclusive homodimers, 16 possible heterodimeric mGluRs have been proposed but their existence in native cells remains elusive. Here, we set up two assays to specifically identify the pharmacological properties of rat mGlu heterodimers composed of mGlu2 and 4 subunits.

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

The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABA, 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 GABA receptor remains poorly understood.

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.

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living 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.

Time-resolved FRET strategy to screen GPCR ligand library.

Screening chemical libraries to find specific drugs for G protein-coupled receptors is still of major interest. Indeed, because of their major roles in all physiological functions, G protein-coupled receptors remain major targets for drug development programs. Currently, interest in GPCRs as drug targets has been boosted by the discovery of biased ligands, thus allowing the development of drugs not only specific for one target but also for the specific signaling cascade expected to have the therapeutic effect.

EuroTracker® dyes: design, synthesis, structure and photophysical properties of very bright europium complexes and their use in bioassays and cellular optical imaging.

The development of the brightest luminescent europium(iii) complexes is traced, including analysis of the C3-symmetric core complex based on a functionalized triazacyclononane and identification of the most suitable strongly absorbing chromophore. Strategies for the synthesis of the complexes, including enantiopure analogues, are outlined and opportunities for applications in time-resolved microscopy and spectral imaging emphasised. Practicable examples are introduced, including selective organelle staining for cellular optical imaging at 65 nm resolution and the development of new bioassays using time-resolved FRET methods.

Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy.

Efficient cell-to-cell communication relies on the accurate signalling of cell surface receptors. Understanding the molecular bases of their activation requires the characterization of the dynamic equilibrium between active and resting states. Here, we monitor, using single-molecule Förster resonance energy transfer, the kinetics of the reorientation of the extracellular ligand-binding domain of the metabotropic glutamate receptor (mGluR), a class C G-protein-coupled receptor.

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.

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.

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.

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.

Time-resolved Förster resonance energy transfer-based technologies to investigate G protein-coupled receptor machinery: high-throughput screening assays and future development.

High-throughput screening requires easy-to-monitor, rapid, robust, reliable, and miniaturized methods to test thousands of compounds on a target in a short period, in order to find active drugs. Only a few methods have been proved to fulfill all these requirements. New screening approaches based on fluorescence and especially on the principle of resonance energy transfer are being developed to study one of the main targets in the pharmaceutical industry, namely, the G protein-coupled receptors (GPCRs).

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.

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.