A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells

New technologies based on luminescence have been essential to monitor the organization, signaling, trafficking or ligand binding of G Protein-Coupled Receptors (GPCRs), but they rely on the overexpression of genetically modified receptors. As more and more studies indicate the importance of studying native receptors in their natural environment, it is essential to develop approaches allowing the specific labeling of native receptors. Here we report an innovative ligand directed approach to specifically label residues of native GPCRs upon ligand binding. To this end, we developed a ligand-directed toolbox based on a novel approach that uses molecular modules to build fluorescent ligand-directed probes that can label an archetypical aminergic GPCR (D1R). Our probes can be readily prepared before the labeling reaction from two molecular modules: an activated electrophilic linker which includes a fluorescent dye and a GPCR ligand that may include nucleophilic groups. Thanks to a fast and specific click reaction, the nucleophilic ligand can barely react with the activated linker before it is bound to the native target GPCR and the labeling reaction occurs. Subsequently, the ligand unbinds the GPCR pocket, leaving the receptor fluorescently labeled and fully functional. This novel labeling approach allowed us to label both D1 receptor in transfected cells and native receptors in neuronal cell lines. This approach will pave the way to develop new reagents and assays to monitor endogenous GPCRs distribution, trafficking, activity or binding properties in their native environment.Funding Agence Nationale de la Recherche ANR-17-CE11-0046 Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe CTQ2017-89222-R Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe PCI2018-093047 Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF - A way of making Europe PID2020-120499RB-I00 Catalan government 2017 SGR 1604 the European Union's Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement No. 801342 (TecniospringINDUSTRY) and the Government of Catalonia's Agency for Business Competitive-ness (ACCIÓ). TECSPR19-1-0062Peer reviewe

A modular ligand-directed approach to label endogenous aminergic GPCRs in live cells

In the last two decades, new technologies based on luminescence have been developed to monitor the organization, signalling or ligand binding of G Protein-Coupled Receptors. These technologies rely on the overexpression of genetically modified (and/or fluorescently tagged) receptors of interest. However, there is an increasing interest in developping approaches to conjugate chemical labels to specific residues of native GPCRs, despite of the low reactivity and the high abundance of such residues.Peer reviewe

A modular ligand-directed approach to label endogenous aminergic GPCRs in live cells

Peer reviewe

A MODULAR LIGAND-DIRECTED APPROACH TO LABEL ENDOGENOUS AMINERGIC GPCRS IN LIVE CELLS

In the last two decades, new technologies based on luminescence have been developed to monitor the organization, signalling or ligand binding of G Protein-Coupled Receptors. These technologies rely on the overexpression of genetically modified (and/or fluorescently tagged) receptors of interest. However, there is an increasing interest in developping approaches to conjugate chemical labels to specific residues of native GPCRs, despite of the low reactivity and the high abundance of such residues. Ligand directed approaches, may offer a solution to this problem. Such approaches consist in molecular probes that include a selective ligand moiety and a reactive moiety. Upon ligand binding, the labelling is directed to a nucleophilic amino acid in the vicinity of the binding pocket. However, this requires the use of non-nucleophilic ligand moieties, which is particularly difficult since many GPCR ligands contain amines or other nucleophilic functional groups. In the present work, we developed an innovative ligand-directed toolbox based on a novel approach. The later uses molecular modules to build fluorescent ligand-directed probes to label an archetypical aminergic GPCR (D1R). Our molecular probes can be readily prepared before the labelling reaction from two molecular modules: an activated electrophilic linker which includes a fluorescent dye and a GPCR ligand that may include nucleophilic groups. Thanks to a fast and specific chemical reaction, the nucleophilic ligand can barely react with the activated linker before it is bound to the native target GPCR and the labelling reaction occurs. Subsequently, the ligand will unbind the GPCR pocket, leaving the receptor fluorescently labelled and fully functional. This novel labelling approach allowed us to label endogenous D1 receptor both in transfected cells and primary cultures of neurons and will pave the way to develop new reagents and assays to monitor endogenous GPCRs distribution and activity in their native environment.Peer reviewe

A modular ligand-directed approach to label endogenous aminergic GPCRs in live cells

In the last two decades, new technologies based on luminescence have been developed to monitor the organization, signalling or ligand binding of G Protein-Coupled Receptors. These technologies rely on the overexpression of genetically modified (and/or fluorescently tagged) receptors of interest. However, there is an increasing interest in developping approaches to conjugate chemical labels to specific residues of native GPCRs, despite of the low reactivity and the high abundance of such residues.Peer reviewe

A modular ligand-directed approach to label endogenous aminergic GPCRs in live cells

Peer reviewe

Scaffold remodeling in space and time controls synaptic transmission

International audienc

Le récepteur métabotropique du glutamate mGlu7 (voie de signalisation et fonction dans les neurones)

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Développement de capteurs d'interaction protéique pour la modélisation de lacroissance du muscle.

CONTEXTE et ENJEUX :Ce projet a pour but l’élaboration et la validation d’un système de mesure en temps réel de l'étatmétabolique (physiologique et pathologique) des cellules musculaires, le but ultime étant de letransférer in vivo au muscle squelettique murin. Notre stratégie est d’analyser l’état d’activation desvoies signalétiques clefs pour la survie et la destinée des cellules musculaires en étudiant l'interactionde certains de leurs composants moléculaires par le développement de capteurs BRET(Bioluminescence Resonnance Energy Transfert). Une fois validés ces capteurs permettront dedéterminer in cellulo l'influence de stress environnementaux et de différents génotypes sur l'étatmétabolique du muscle en développement. Deux modèles de capteurs « prédictifs » du musclesquelettique ont été développés; 1) le capteur 4EBP1/EIF4E qui mesure l’activation de la voieIGF-1/AKT/mTOR : Cette voie constitue la voie majeure de régulation de la croissance et de la surviedu muscle squelettique. La détermination de son état d'activation repose sur l’analyse del’interaction/dissociation entre les protéines 4EBP1/EIF4E, verrou ultime de l’initiation de la traductiondes mARNs (et donc de la synthèse protéique). 2) Le capteur mitofusines pour évaluer l'intégrité desmitochondries musculaires : La biogenèse et le métabolisme mitochondrial reposent sur un équilibreentre fusion et fission de ces organites qui conditionne in fine la survie cellulaire. L'hétérodimerisationdes protéines mitofusines Mfn1 et Mfn2 localisées à la surface externe des mitochondries contrôle cecycle de fusion/fission.RESULTATS1) Les différents plasmides nécessaires au projet ont été construits et les expériences de BRET ontdémontré la faisabilité de l’approche pour détecter l’interaction en temps réel dans des cellulesvivantes d’une part entre les Mitofusines d’autre part entre 4EBP1 et EIF4E.2) La fonctionnalité du complexe 4EBP1/EIF4E a été vérifiée in vitro en démontrant l’interaction desprotéines exogènes avec la coiffe 5’ des mRNAs (par immunoprécipitation sur billes de methyl-7GTP-Sepharose).3) Comme attendu nous avons observé par une approche biochimique que l’interaction entre 4EBP1 etEIF4E mesurée par BRET dépend du niveau de phosphorylation de 4EBP1 (et donc de l’activation dela voie mTORC1). En effet l’introduction de mutations mimant les formes phosphorylées de 4EBP1entraîne une diminution de l’affinité entre 4EBP1 et EIF4E. Réciproquement, des mutants de 4EBP1résistants à la phosphorylation par TORC1 ont une affinité accrue pour la protéine EIF4E.4) La surexpression d’un inhibiteur de la voie mTORC1 (Redd1) augmente l’affinité entre 4EBP1 etEIF4E suggérant que le capteur 4EBP1/EIF4E est bien sensible à l’état d’activation de la voiemTORC1.5) En collaboration avec Julie Perroy (IGF CNRS Montpellier) nous avons déterminé par imagerie lalocalisation in cellulo du BRET mesuré et nous observons qu’une part de l’interaction entre lesmitofusines est bien localisée au niveau de la surface mitochondriale et d’autre part que celle ducomplexe 4EBP1/EIF4E s’effectue principalement au niveau du compartiment nucléaire

The Complex Formed by Group I Metabotropic Glutamate Receptor (mGluR) and Homer1a Plays a Central Role in Metaplasticity and Homeostatic Synaptic Scaling

International audienceG-protein-coupled receptors can be constitutively activated following physical interaction with intracellular proteins. The first example described was the constitutive activation of Group I metabotropic glutamate receptors (mGluR: mGluR1,5) following their interaction with Homer1a, an activity-inducible early-termination variant of the scaffolding protein Homer that lacks dimerization capacity (Ango et al., 2001). Homer1a disrupts the links, maintained by the long form of Homer (cross-linking Homers), between mGluR1,5 and the Shank-GKAP-PSD-95-ionotropic glutamate receptor network. Two characteristics of the constitutive activation of the Group I mGluR-Homer1a complex are particularly interesting: (1) it affects a large number of synapses in which Homer1a is upregulated following enhanced, long-lasting neuronal activity; and (2) it mainly depends on Homer1a protein turnover. The constitutively active Group I mGluR-Homer1a complex is involved in the two main forms of non-Hebbian neuronal plasticity: "metaplasticity" and "homeostatic synaptic scaling," which are implicated in a large series of physiological and pathologic processes. Those include non-Hebbian plasticity observed in visual system, synapses modulated by addictive drugs (rewarded synapses), chronically overactivated synaptic networks, normal sleep, and sleep deprivation