MAGAZINE teurs. Nous utilisons actuellement différents modèles animaux afin d'apporter la preuve ... more MAGAZINE teurs. Nous utilisons actuellement différents modèles animaux afin d'apporter la preuve du concept que la reptine est effectivement une cible attractive. ‡ A little less respite for hepatocellular carcinoma? RÉFÉRENCES 1. Blanc JF, Lalanne C, Plomion C, et al. Proteomic analysis of differentially expressed proteins in hepatocellular carcinoma developed in patients with chronic viral hepatitis C.
bioRxiv (Cold Spring Harbor Laboratory), Mar 29, 2023
Metastasis, the process by which cancer cells colonize distant organs, relies on the ability of t... more Metastasis, the process by which cancer cells colonize distant organs, relies on the ability of these cells to migrate and invade the surrounding tissues. The ezrin, radixin, and moesin family (ERM) of proteins are critical regulators of cell morphology transformations required for cancer cell movement and invasion. Yet, how ERMs are activated during metastasis remains poorly understood. Here, we identified the thromboxane A2 receptor (TBXA2R), a G protein-coupled receptor, as a critical activator of ERMs that promotes motility, invasion, and metastasis of triple-negative breast cancer (TNBC) cells. We found that ERM activation downstream of TBXA2R signaling depends on the Gα q/11 and Gα 12/13 subfamilies, the small GTPase RhoA, and its Ser/Thr kinase effector SLK. We also showed that TBXA2R signaling increases TNBC cell motility and invasion in vitro and metastasis in vivo, depending on ERMs. These findings unveil a novel mechanism by which a member of the largest class of receptors activates key metastatic determinants to promote TNBC metastasis, which could have important implications for developing novel therapeutic strategies.
Background: G Protein-Coupled Receptors (GPCRs) are a large and diverse family of membrane protei... more Background: G Protein-Coupled Receptors (GPCRs) are a large and diverse family of membrane proteins whose members participate in the regulation of most cellular and physiological processes and therefore represent key pharmacological targets. Although several bioinformatics resources support research on GPCRs, most of these have been designed based on the traditional assumption that monomeric GPCRs constitute the functional receptor unit. The increase in the frequency and number of reports about GPCR dimerization/oligomerization and the implication of oligomerization in receptor function makes necessary the ability to store and access information about GPCR dimers/oligomers electronically. We present here the requirements and ontology (the information scheme to describe oligomers and associated concepts and their relationships) for an information system that can manage the elements of information needed to describe comprehensively the phenomena of both homo-and hetero-oligomerization of GPCRs. The comprehensive information management
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2022
H. performed all binding and functional assays and analyses for adrenergic and dopamine receptors... more H. performed all binding and functional assays and analyses for adrenergic and dopamine receptors with input from D.W. J.X. determined the '9087-α 2A AR-G oA and '4622-α 2A AR-G oA structures and made α 2A AR mutations assisted by G.C. and Z.L., with supervision from Y.D. J.M.B. performed and analyzed the in vivo pharmacology experiments assisted by V.C., supervised and coanalyzed by A.I.B. C.A. tested select compounds in the panel of G protein and β-arrestin subtypes and receptor internalization with supervision from M.B. S.G. modeled compound '7075 and PS75. X.-P.H. tested compounds in GPCRome and hERG assays. M.F.S. performed contact area calculation. P.S. synthesized bespoke compounds and performed pK a and analytical testing with supervision of P.G. D.W. performed ELISA experiments. C.M.W. tested compounds for μOR activity. C.K. performed permeability calculations with supervision of M.P.J. Y.S.M. supervised compound synthesis of Enamine compounds purchased from the ZINC15 database and 12 billion catalog, assisted by N.A.T. J.J.I. built the ZINC15 ultralarge libraries.
Dopamine (DA) neurons are primarily concentrated in substantia nigra (SN) and ventral tegmental a... more Dopamine (DA) neurons are primarily concentrated in substantia nigra (SN) and ventral tegmental area (VTA). A subset of these neurons expresses the neurotensin receptor NTSR1 and its putative ligand neurotensin (Nts). NTSR1, a G protein-coupled receptor (GPCR), which classically activates Gαq/calcium signaling, is a potential route for modulating DA activity. Drug development efforts have been hampered by the receptor's complex pharmacology and a lack of understanding about its endogenous location and signaling responses. Therefore, we have generated NTSR1-Venus knockin (KI) mice to study NTSR1 receptors in their physiological context. In primary hippocampal neurons, we show that these animals express functional receptors that respond to agonists by increasing intracellular calcium release and trafficking to endosomes. Moreover, systemic agonist administration attenuates locomotion in KIs as it does in control animals. Mapping receptor protein expression at regional and cellular levels, located NTSR1-Venus on the soma and dendrites of dopaminergic SN/VTA neurons. Direct monitoring of receptor endocytosis, as a proxy for activation, enabled profiling of NTSR1 agonists in neurons, as well as acute SN/VTA containing brain slices. Taken together, NTSR1-Venus animals express traceable receptors that will improve understanding of NTSR1 and DA activities and more broadly how GPCRs act in vivo.
The intricate involvement of the serotonin 5-HT 2A receptor (5-HT 2A R) both in schizophrenia and... more The intricate involvement of the serotonin 5-HT 2A receptor (5-HT 2A R) both in schizophrenia and in the activity of antipsychotic drugs is widely acknowledged. The currently marketed antipsychotic drugs, although effective in managing the symptoms of schizophrenia to a certain extent, are not without their repertoire of serious side effects. There is a need for better therapeutics to treat schizophrenia for which understanding the mechanism of action of the current antipsychotic drugs is imperative. With bioluminescence resonance energy transfer (BRET) assays, we trace the signaling signature of six antipsychotic drugs belonging to three generations at the 5-HT 2A R for the entire spectrum of signaling pathways activated by serotonin (5-HT). The antipsychotic drugs display previously unidentified pathway preference at the level of the individual Gα subunits and β-arrestins. In particular, risperidone, clozapine, olanzapine and haloperidol showed G protein-selective inverse agonist activity. In addition, G proteinselective partial agonism was found for aripiprazole and cariprazine. Pathway-specific apparent dissociation constants determined from functional analyses revealed distinct coupling-modulating capacities of the tested antipsychotics at the different 5-HTactivated pathways. Computational analyses of the pharmacological and structural fingerprints support a mechanistically based clustering that recapitulate the clinical classification (typical/first generation, atypical/second generation, third generation) of the antipsychotic drugs. The study provides a new framework to functionally classify antipsychotics that should represent a useful tool for the identification of better and safer neuropsychiatric drugs and allows formulating hypotheses on the links between specific signaling cascades and in the clinical outcomes of the existing drugs.
G protein-coupled receptors are important drug targets that engage and activate signaling transdu... more G protein-coupled receptors are important drug targets that engage and activate signaling transducers in multiple cellular compartments. Delineating therapeutic signaling from signaling associated with adverse events is an important step towards rational drug design. The glucagon-like peptide-1 receptor (GLP-1R) is a validated target for the treatment of diabetes and obesity, but drugs that target this receptor are a frequent cause of adverse events. Using recently developed biosensors, we explored the ability of GLP-1R to activate 15 pathways in 4 cellular compartments and demonstrate that modifications aimed at improving the therapeutic potential of GLP-1R agonists greatly influence compound efficacy, potency, and safety in a pathway- and compartment-selective manner. These findings, together with comparative structure analysis, time-lapse microscopy, and phosphoproteomics, reveal unique signaling signatures for GLP-1R agonists at the level of receptor conformation, functional sel...
G protein-coupled receptors (GPCRs) translate binding of extracellular ligands into intracellular... more G protein-coupled receptors (GPCRs) translate binding of extracellular ligands into intracellular responses through conformational changes. Ligand properties are described by the maximum response (efficacy) and the agonist concentration at half-maximal response (potency). Integrating structural changes with pharmacological properties remains challenging and has not yet been performed at the resolution of individual amino acids. We use epinephrine and β2-adrenergic receptor as a model to integrate residue-level pharmacology data with intramolecular residue contact data describing receptor activation. This unveils the allosteric networks driving ligand efficacy and potency. We provide detailed insights into how structural rearrangements are linked to fundamental pharmacological properties at single-residue level in a receptor-ligand system. Our approach can be used to determine such pharmacological networks for any receptor-ligand complex.
Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbatio... more Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.
Communication across membranes controls critical cellular processes and is achieved by receptors ... more Communication across membranes controls critical cellular processes and is achieved by receptors translating extracellular signals into selective cytoplasmic responses. While receptor tertiary structures can now be readily characterized, receptor associations into quaternary structures are very challenging to study and their implications in signal transduction remain poorly understood. Here, we report a computational approach for predicting membrane receptor self-associations, and designing receptor oligomers with various quaternary structures and signaling properties. Using this approach, we designed chemokine receptor CXCR4 dimers with reprogrammed stabilities, conformations, and abilities to activate distinct intracellular signaling proteins. In agreement with our predictions, the designed CXCR4s dimerized through distinct conformations and displayed different quaternary structural changes upon activation. Consistent with the active state models, all engineered CXCR4 oligomers ac...
In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recr... more In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor ...
The ability of individual G protein-coupled receptors (GPCR) to engage multiple signaling pathway... more The ability of individual G protein-coupled receptors (GPCR) to engage multiple signaling pathways opens opportunities for the development of better drugs. This requires new knowledge and tools to determine the G protein subtypes and arrestins engaged by a given receptor. Here, we used a new BRET-based effector membrane translocation assay (EMTA) that monitors activation of each Gα protein through the recruitment of selective G protein effectors and βarrestins to the plasma membrane. Profiling of 100 therapeutically relevant GPCR revealed a great diversity of coupling profiles with some receptors displaying exquisite selectivity, whereas others promiscuitely engage all four G protein families. Comparison with existing datasets points to commonalities but also to critical differences between studies. Combining a biosensor subset allowed detecting activity of nearly all GPCR thus providing a new tool for safety screens and systems pharmacology. Overall, this work describes unique resources for studying GPCR function and drug discovery.
Two-thirds of human hormones and one-third of clinical drugs act on membrane receptors that coupl... more Two-thirds of human hormones and one-third of clinical drugs act on membrane receptors that couple to G proteins to achieve appropriate functional responses. While G protein transducers from literature are annotated in the Guide to Pharmacology database, two recent large-scale datasets now expand the receptor-G protein ‘couplome’. However, these three datasets differ in scope and reported G protein couplings giving different coverage and conclusions on GPCR-G protein signaling. Here, we report a common coupling map uncovering novel couplings supported by both large-scale studies, the selectivity/promiscuity of GPCRs and G proteins, and how the co-coupling and co-expression of G proteins compare to the families from phylogenetic relationships. The coupling map and insights on GPCR-G protein selectivity will catalyze advances in receptor research and cellular signaling towards the exploitation of G protein signaling bias in design of safer drugs.
Activation of the G protein-coupled receptors by agonists may result in the activation of one or ... more Activation of the G protein-coupled receptors by agonists may result in the activation of one or more G proteins, and in the recruitment of arrestins. The balance of activation of different pathways can be influenced by the ligand. Using BRET-based biosensors, we showed that the vasopressin V2 receptor activates or at least engages many different G proteins across all G protein subfamilies in response to its native agonist arginine vasopressin (AVP). This includes members of the Gi/o and G12/13 families that have not been previously reported. These signalling pathways are also activated by the synthetic peptide desmopressin and natural homologs of AVP, namely oxytocin and the non-mammalian hormone vasotocin. They demonstrated varying degrees of functional selectivity relative to AVP, as quantified using the operational model for quantifying ligand bias. Additionally, we modelled G protein activation as a Michaelis-Menten reaction. This approach provided a complementary way to quanti...
ABSTRACTMalaria causes millions of deaths worldwide and is considered a huge public health proble... more ABSTRACTMalaria causes millions of deaths worldwide and is considered a huge public health problem for underdeveloped countries. The most severe cases of malaria present complications of the host circulatory system, which may cause clogging and rupture of blood vessels, leading to death or important sequelae. Because of the previously suggested role of thrombin and platelet aggregation in Plasmodium falciparum biology, we hypothesized that one of the GPCR-like proteins identified in the genome of the parasite, P. falciparum serpentine receptor 12 (PfSR12), could be a thrombin-activated GPCR. To test this hypothesis we used a series of Bioluminescence and Bioluminescence Resonance Energy Transfer (BRET)-based biosensors to investigate the signaling activity of PfSR12. Using an Obelin based biosensor, thrombin promoted a PfSR12-dependent cytosolic Ca2+ rise in HEK293 cells. This Ca2+ mobilization was accompanied by DAG formation and PKC activation as detected using DAG and PKC BRET-ba...
2010 m/s fête ses 30 ans en mars 2015 Programme 9 h Introduction -Christian Bréchot (directeur gé... more 2010 m/s fête ses 30 ans en mars 2015 Programme 9 h Introduction -Christian Bréchot (directeur général de l'Institut P asteur), Yves Lévy (président-directeur g én ér a l d e l 'Inserm ), Rémi Quirion (scientifique e n c h e f d u Québec), Axel Kahn (président h o n o r a ir e d e l 'université P a r is Descartes) 9 h 30-11 h Session 1 -Comment lit-on une revue scientifique aujourd'hui ? • Le cerveau des écrans du savoir : Olivier Houdé (directeur d u l a b o r a t o ir e d e psy c h o l o g ie d u d é v e l o ppe m e n t -université P a r is D e sc a r t e s) • La science biomédicale en français : Anne-Marie Moulin (présid e n t e d u c o m it é c o n su l t a t if d e d é o n t o l o g ie e t d 'éth iq u e d e l 'IR D ) • Le point de vue d'un doyen : Gérard Friedlander (d o y e n d e l a f a c u l t é d e m é d e c in e d e l 'u n iv e r sit é P a r is D e sc a r t e s) • La vision d'un industriel : Antoine Bril (d ir e c t e u r R & D d e l 'In s t it u t d e r e c h e r c h e s in t e r n a t io n a l e s S e r v ie r ) 11 h 30-13 h Session 2 -1 face int (comment une revue rend-elle compte des avancées technologiques ?) • Im agerie temps réel : Jean-Louis Martin (professeur d e b io l o g ie à l 'É c o l e P o l y t e c h n iq u e ) • Génom ique et société : Bertrand Jordan (a n c ie n d ir e c t e u r d e M a r se il l e -Nic e G é n o po l e ) • Biothérapies : Nathalie Cartier (d ir e c t r ic e d u g r o u pe Bio t h é r a pie In s e r m U745) 14 h-15 h 30 Session 3 -1 i face au temps des complexités (comment une revue rend-elle compte des nouvelles complexités ?) • Complexité/Big data : Jacques Haiech (professeur d e b io t e c h n o l o g ie , u n iv e r sit é d e S t r a sb o u r g ) • Microbiote : Bernard Henrissat (d ir e c t e u r d 'éq u ipe a u l a b o r a t o ir e a r c h it e c t u r e e t fo n c t io n d e s m a c r o m o l é c u l e s b io l o g iq u e s, C N R S e t u n iv e r s it é Aix -Marseille) • DOHaD/Épigénétique : Claudine Junten (pro fesseure de génétique, U M R Inra 1198, Jouy-en-J osas) • Médicament : LaurenceLafanechère (d ir e c t r ic e é q u ipe In se r m /UJF-In s t it u t Albert B o n n io t -La Tro n c h e ) 16 h-17 h 30 Session 4 -Publier dans aujourd'hui et demain (pourquoi publier encore dans (ffi demain ? Le rôle pédagogique de la revue ■ et les Mooc) • CatherineJessus (d ir e c t r ic e d e l 'IN S B d u C N R S ) • Michel Bouvier (d ir e c t e u r g é n é r a l , In s t it u t d e r e c h e r c h e I Rie-u n iv e r s it é d e M o n t r é a l ) • Jacques Biot (président d e l 'É c o l e P o l y t e c h n iq u e ) 17 h 30-18 h Conclusions -Dialogue Philippe Lazar (ancien directeur général de l'Inserm)/ M onique Canto-Sperber (ancienne d ir e c t r ic e d e l 'É c o l e N o r m a l e Supérieure) 18 h 30 Cocktail
G protein-coupled receptor (GPCR) oligomerization is a growing concept that has emerged from seve... more 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 heterodimers 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 immu-noprecipitated 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.
MAGAZINE teurs. Nous utilisons actuellement différents modèles animaux afin d'apporter la preuve ... more MAGAZINE teurs. Nous utilisons actuellement différents modèles animaux afin d'apporter la preuve du concept que la reptine est effectivement une cible attractive. ‡ A little less respite for hepatocellular carcinoma? RÉFÉRENCES 1. Blanc JF, Lalanne C, Plomion C, et al. Proteomic analysis of differentially expressed proteins in hepatocellular carcinoma developed in patients with chronic viral hepatitis C.
bioRxiv (Cold Spring Harbor Laboratory), Mar 29, 2023
Metastasis, the process by which cancer cells colonize distant organs, relies on the ability of t... more Metastasis, the process by which cancer cells colonize distant organs, relies on the ability of these cells to migrate and invade the surrounding tissues. The ezrin, radixin, and moesin family (ERM) of proteins are critical regulators of cell morphology transformations required for cancer cell movement and invasion. Yet, how ERMs are activated during metastasis remains poorly understood. Here, we identified the thromboxane A2 receptor (TBXA2R), a G protein-coupled receptor, as a critical activator of ERMs that promotes motility, invasion, and metastasis of triple-negative breast cancer (TNBC) cells. We found that ERM activation downstream of TBXA2R signaling depends on the Gα q/11 and Gα 12/13 subfamilies, the small GTPase RhoA, and its Ser/Thr kinase effector SLK. We also showed that TBXA2R signaling increases TNBC cell motility and invasion in vitro and metastasis in vivo, depending on ERMs. These findings unveil a novel mechanism by which a member of the largest class of receptors activates key metastatic determinants to promote TNBC metastasis, which could have important implications for developing novel therapeutic strategies.
Background: G Protein-Coupled Receptors (GPCRs) are a large and diverse family of membrane protei... more Background: G Protein-Coupled Receptors (GPCRs) are a large and diverse family of membrane proteins whose members participate in the regulation of most cellular and physiological processes and therefore represent key pharmacological targets. Although several bioinformatics resources support research on GPCRs, most of these have been designed based on the traditional assumption that monomeric GPCRs constitute the functional receptor unit. The increase in the frequency and number of reports about GPCR dimerization/oligomerization and the implication of oligomerization in receptor function makes necessary the ability to store and access information about GPCR dimers/oligomers electronically. We present here the requirements and ontology (the information scheme to describe oligomers and associated concepts and their relationships) for an information system that can manage the elements of information needed to describe comprehensively the phenomena of both homo-and hetero-oligomerization of GPCRs. The comprehensive information management
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2022
H. performed all binding and functional assays and analyses for adrenergic and dopamine receptors... more H. performed all binding and functional assays and analyses for adrenergic and dopamine receptors with input from D.W. J.X. determined the '9087-α 2A AR-G oA and '4622-α 2A AR-G oA structures and made α 2A AR mutations assisted by G.C. and Z.L., with supervision from Y.D. J.M.B. performed and analyzed the in vivo pharmacology experiments assisted by V.C., supervised and coanalyzed by A.I.B. C.A. tested select compounds in the panel of G protein and β-arrestin subtypes and receptor internalization with supervision from M.B. S.G. modeled compound '7075 and PS75. X.-P.H. tested compounds in GPCRome and hERG assays. M.F.S. performed contact area calculation. P.S. synthesized bespoke compounds and performed pK a and analytical testing with supervision of P.G. D.W. performed ELISA experiments. C.M.W. tested compounds for μOR activity. C.K. performed permeability calculations with supervision of M.P.J. Y.S.M. supervised compound synthesis of Enamine compounds purchased from the ZINC15 database and 12 billion catalog, assisted by N.A.T. J.J.I. built the ZINC15 ultralarge libraries.
Dopamine (DA) neurons are primarily concentrated in substantia nigra (SN) and ventral tegmental a... more Dopamine (DA) neurons are primarily concentrated in substantia nigra (SN) and ventral tegmental area (VTA). A subset of these neurons expresses the neurotensin receptor NTSR1 and its putative ligand neurotensin (Nts). NTSR1, a G protein-coupled receptor (GPCR), which classically activates Gαq/calcium signaling, is a potential route for modulating DA activity. Drug development efforts have been hampered by the receptor's complex pharmacology and a lack of understanding about its endogenous location and signaling responses. Therefore, we have generated NTSR1-Venus knockin (KI) mice to study NTSR1 receptors in their physiological context. In primary hippocampal neurons, we show that these animals express functional receptors that respond to agonists by increasing intracellular calcium release and trafficking to endosomes. Moreover, systemic agonist administration attenuates locomotion in KIs as it does in control animals. Mapping receptor protein expression at regional and cellular levels, located NTSR1-Venus on the soma and dendrites of dopaminergic SN/VTA neurons. Direct monitoring of receptor endocytosis, as a proxy for activation, enabled profiling of NTSR1 agonists in neurons, as well as acute SN/VTA containing brain slices. Taken together, NTSR1-Venus animals express traceable receptors that will improve understanding of NTSR1 and DA activities and more broadly how GPCRs act in vivo.
The intricate involvement of the serotonin 5-HT 2A receptor (5-HT 2A R) both in schizophrenia and... more The intricate involvement of the serotonin 5-HT 2A receptor (5-HT 2A R) both in schizophrenia and in the activity of antipsychotic drugs is widely acknowledged. The currently marketed antipsychotic drugs, although effective in managing the symptoms of schizophrenia to a certain extent, are not without their repertoire of serious side effects. There is a need for better therapeutics to treat schizophrenia for which understanding the mechanism of action of the current antipsychotic drugs is imperative. With bioluminescence resonance energy transfer (BRET) assays, we trace the signaling signature of six antipsychotic drugs belonging to three generations at the 5-HT 2A R for the entire spectrum of signaling pathways activated by serotonin (5-HT). The antipsychotic drugs display previously unidentified pathway preference at the level of the individual Gα subunits and β-arrestins. In particular, risperidone, clozapine, olanzapine and haloperidol showed G protein-selective inverse agonist activity. In addition, G proteinselective partial agonism was found for aripiprazole and cariprazine. Pathway-specific apparent dissociation constants determined from functional analyses revealed distinct coupling-modulating capacities of the tested antipsychotics at the different 5-HTactivated pathways. Computational analyses of the pharmacological and structural fingerprints support a mechanistically based clustering that recapitulate the clinical classification (typical/first generation, atypical/second generation, third generation) of the antipsychotic drugs. The study provides a new framework to functionally classify antipsychotics that should represent a useful tool for the identification of better and safer neuropsychiatric drugs and allows formulating hypotheses on the links between specific signaling cascades and in the clinical outcomes of the existing drugs.
G protein-coupled receptors are important drug targets that engage and activate signaling transdu... more G protein-coupled receptors are important drug targets that engage and activate signaling transducers in multiple cellular compartments. Delineating therapeutic signaling from signaling associated with adverse events is an important step towards rational drug design. The glucagon-like peptide-1 receptor (GLP-1R) is a validated target for the treatment of diabetes and obesity, but drugs that target this receptor are a frequent cause of adverse events. Using recently developed biosensors, we explored the ability of GLP-1R to activate 15 pathways in 4 cellular compartments and demonstrate that modifications aimed at improving the therapeutic potential of GLP-1R agonists greatly influence compound efficacy, potency, and safety in a pathway- and compartment-selective manner. These findings, together with comparative structure analysis, time-lapse microscopy, and phosphoproteomics, reveal unique signaling signatures for GLP-1R agonists at the level of receptor conformation, functional sel...
G protein-coupled receptors (GPCRs) translate binding of extracellular ligands into intracellular... more G protein-coupled receptors (GPCRs) translate binding of extracellular ligands into intracellular responses through conformational changes. Ligand properties are described by the maximum response (efficacy) and the agonist concentration at half-maximal response (potency). Integrating structural changes with pharmacological properties remains challenging and has not yet been performed at the resolution of individual amino acids. We use epinephrine and β2-adrenergic receptor as a model to integrate residue-level pharmacology data with intramolecular residue contact data describing receptor activation. This unveils the allosteric networks driving ligand efficacy and potency. We provide detailed insights into how structural rearrangements are linked to fundamental pharmacological properties at single-residue level in a receptor-ligand system. Our approach can be used to determine such pharmacological networks for any receptor-ligand complex.
Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbatio... more Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.
Communication across membranes controls critical cellular processes and is achieved by receptors ... more Communication across membranes controls critical cellular processes and is achieved by receptors translating extracellular signals into selective cytoplasmic responses. While receptor tertiary structures can now be readily characterized, receptor associations into quaternary structures are very challenging to study and their implications in signal transduction remain poorly understood. Here, we report a computational approach for predicting membrane receptor self-associations, and designing receptor oligomers with various quaternary structures and signaling properties. Using this approach, we designed chemokine receptor CXCR4 dimers with reprogrammed stabilities, conformations, and abilities to activate distinct intracellular signaling proteins. In agreement with our predictions, the designed CXCR4s dimerized through distinct conformations and displayed different quaternary structural changes upon activation. Consistent with the active state models, all engineered CXCR4 oligomers ac...
In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recr... more In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor ...
The ability of individual G protein-coupled receptors (GPCR) to engage multiple signaling pathway... more The ability of individual G protein-coupled receptors (GPCR) to engage multiple signaling pathways opens opportunities for the development of better drugs. This requires new knowledge and tools to determine the G protein subtypes and arrestins engaged by a given receptor. Here, we used a new BRET-based effector membrane translocation assay (EMTA) that monitors activation of each Gα protein through the recruitment of selective G protein effectors and βarrestins to the plasma membrane. Profiling of 100 therapeutically relevant GPCR revealed a great diversity of coupling profiles with some receptors displaying exquisite selectivity, whereas others promiscuitely engage all four G protein families. Comparison with existing datasets points to commonalities but also to critical differences between studies. Combining a biosensor subset allowed detecting activity of nearly all GPCR thus providing a new tool for safety screens and systems pharmacology. Overall, this work describes unique resources for studying GPCR function and drug discovery.
Two-thirds of human hormones and one-third of clinical drugs act on membrane receptors that coupl... more Two-thirds of human hormones and one-third of clinical drugs act on membrane receptors that couple to G proteins to achieve appropriate functional responses. While G protein transducers from literature are annotated in the Guide to Pharmacology database, two recent large-scale datasets now expand the receptor-G protein ‘couplome’. However, these three datasets differ in scope and reported G protein couplings giving different coverage and conclusions on GPCR-G protein signaling. Here, we report a common coupling map uncovering novel couplings supported by both large-scale studies, the selectivity/promiscuity of GPCRs and G proteins, and how the co-coupling and co-expression of G proteins compare to the families from phylogenetic relationships. The coupling map and insights on GPCR-G protein selectivity will catalyze advances in receptor research and cellular signaling towards the exploitation of G protein signaling bias in design of safer drugs.
Activation of the G protein-coupled receptors by agonists may result in the activation of one or ... more Activation of the G protein-coupled receptors by agonists may result in the activation of one or more G proteins, and in the recruitment of arrestins. The balance of activation of different pathways can be influenced by the ligand. Using BRET-based biosensors, we showed that the vasopressin V2 receptor activates or at least engages many different G proteins across all G protein subfamilies in response to its native agonist arginine vasopressin (AVP). This includes members of the Gi/o and G12/13 families that have not been previously reported. These signalling pathways are also activated by the synthetic peptide desmopressin and natural homologs of AVP, namely oxytocin and the non-mammalian hormone vasotocin. They demonstrated varying degrees of functional selectivity relative to AVP, as quantified using the operational model for quantifying ligand bias. Additionally, we modelled G protein activation as a Michaelis-Menten reaction. This approach provided a complementary way to quanti...
ABSTRACTMalaria causes millions of deaths worldwide and is considered a huge public health proble... more ABSTRACTMalaria causes millions of deaths worldwide and is considered a huge public health problem for underdeveloped countries. The most severe cases of malaria present complications of the host circulatory system, which may cause clogging and rupture of blood vessels, leading to death or important sequelae. Because of the previously suggested role of thrombin and platelet aggregation in Plasmodium falciparum biology, we hypothesized that one of the GPCR-like proteins identified in the genome of the parasite, P. falciparum serpentine receptor 12 (PfSR12), could be a thrombin-activated GPCR. To test this hypothesis we used a series of Bioluminescence and Bioluminescence Resonance Energy Transfer (BRET)-based biosensors to investigate the signaling activity of PfSR12. Using an Obelin based biosensor, thrombin promoted a PfSR12-dependent cytosolic Ca2+ rise in HEK293 cells. This Ca2+ mobilization was accompanied by DAG formation and PKC activation as detected using DAG and PKC BRET-ba...
2010 m/s fête ses 30 ans en mars 2015 Programme 9 h Introduction -Christian Bréchot (directeur gé... more 2010 m/s fête ses 30 ans en mars 2015 Programme 9 h Introduction -Christian Bréchot (directeur général de l'Institut P asteur), Yves Lévy (président-directeur g én ér a l d e l 'Inserm ), Rémi Quirion (scientifique e n c h e f d u Québec), Axel Kahn (président h o n o r a ir e d e l 'université P a r is Descartes) 9 h 30-11 h Session 1 -Comment lit-on une revue scientifique aujourd'hui ? • Le cerveau des écrans du savoir : Olivier Houdé (directeur d u l a b o r a t o ir e d e psy c h o l o g ie d u d é v e l o ppe m e n t -université P a r is D e sc a r t e s) • La science biomédicale en français : Anne-Marie Moulin (présid e n t e d u c o m it é c o n su l t a t if d e d é o n t o l o g ie e t d 'éth iq u e d e l 'IR D ) • Le point de vue d'un doyen : Gérard Friedlander (d o y e n d e l a f a c u l t é d e m é d e c in e d e l 'u n iv e r sit é P a r is D e sc a r t e s) • La vision d'un industriel : Antoine Bril (d ir e c t e u r R & D d e l 'In s t it u t d e r e c h e r c h e s in t e r n a t io n a l e s S e r v ie r ) 11 h 30-13 h Session 2 -1 face int (comment une revue rend-elle compte des avancées technologiques ?) • Im agerie temps réel : Jean-Louis Martin (professeur d e b io l o g ie à l 'É c o l e P o l y t e c h n iq u e ) • Génom ique et société : Bertrand Jordan (a n c ie n d ir e c t e u r d e M a r se il l e -Nic e G é n o po l e ) • Biothérapies : Nathalie Cartier (d ir e c t r ic e d u g r o u pe Bio t h é r a pie In s e r m U745) 14 h-15 h 30 Session 3 -1 i face au temps des complexités (comment une revue rend-elle compte des nouvelles complexités ?) • Complexité/Big data : Jacques Haiech (professeur d e b io t e c h n o l o g ie , u n iv e r sit é d e S t r a sb o u r g ) • Microbiote : Bernard Henrissat (d ir e c t e u r d 'éq u ipe a u l a b o r a t o ir e a r c h it e c t u r e e t fo n c t io n d e s m a c r o m o l é c u l e s b io l o g iq u e s, C N R S e t u n iv e r s it é Aix -Marseille) • DOHaD/Épigénétique : Claudine Junten (pro fesseure de génétique, U M R Inra 1198, Jouy-en-J osas) • Médicament : LaurenceLafanechère (d ir e c t r ic e é q u ipe In se r m /UJF-In s t it u t Albert B o n n io t -La Tro n c h e ) 16 h-17 h 30 Session 4 -Publier dans aujourd'hui et demain (pourquoi publier encore dans (ffi demain ? Le rôle pédagogique de la revue ■ et les Mooc) • CatherineJessus (d ir e c t r ic e d e l 'IN S B d u C N R S ) • Michel Bouvier (d ir e c t e u r g é n é r a l , In s t it u t d e r e c h e r c h e I Rie-u n iv e r s it é d e M o n t r é a l ) • Jacques Biot (président d e l 'É c o l e P o l y t e c h n iq u e ) 17 h 30-18 h Conclusions -Dialogue Philippe Lazar (ancien directeur général de l'Inserm)/ M onique Canto-Sperber (ancienne d ir e c t r ic e d e l 'É c o l e N o r m a l e Supérieure) 18 h 30 Cocktail
G protein-coupled receptor (GPCR) oligomerization is a growing concept that has emerged from seve... more 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 heterodimers 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 immu-noprecipitated 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.
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Papers by Michel Bouvier