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Michel Bouvier

Université de Montréal, Biochemistry (Biochemie), Faculty Member

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Professor of Biochemistry and Molecular Medicine, General Director of Institut for Research in Immunology and Cancer, Canada Research (IRIC) Chair in Signal Transduction and Molecular Pharmacology.
Supervisors: Jacques De Champlain and Robert J. Lefkowitz

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Anna M . Czarnecka

Princeton University

Mohamed Abdel-Daim

Suez Canal University

University of Padjadjaran (UNPAD)

Suhnrita Chaudhuri

University of Illinois at Chicago

Professor Dr. Loutfy H . Madkour

Al-Baha University, Kingdom of Saudi Arabia

Hokkaido University

Michael Szardenings

Fraunhofer

Hany Abdelhafiz

University of Colorado Denver

Guido Santos Rosales

FAU Erlangen-Nuernberg

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Papers by Michel Bouvier

The G protein-coupled receptor TBXA2R activates ERMs to promote motility, invasion, and metastasis of triple-negative breast cancer cells

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.

Role of SUMOylation in Full Antiestrogenicity

by Mohamed El Ezzy and Michel Bouvier

Molecular and Cellular Biology, 2012

The selective estrogen receptor downregulator (SERD) fulvestrant can be used as second-line treat... more The selective estrogen receptor downregulator (SERD) fulvestrant can be used as second-line treatment for patients relapsing after treatment with tamoxifen, a selective estrogen receptor modulator (SERM). Unlike tamoxifen, SERDs are devoid of partial agonist activity. While the full antiestrogenicity of SERDs may result in part from their capacity to downregulate levels of estrogen receptor alpha (ERα) through proteasome-mediated degradation, SERDs are also fully antiestrogenic in the absence of increased receptor turnover in HepG2 cells. Here we report that SERDs induce the rapid and strong SUMOylation of ERα in ERα-positive and -negative cell lines, including HepG2 cells. Four sites of SUMOylation were identified by mass spectrometry analysis. In derivatives of the SERD ICI164,384, SUMOylation was dependent on the length of the side chain and correlated with full antiestrogenicity. Preventing SUMOylation by the overexpression of a SUMO-specific protease (SENP) deSUMOylase partiall...

Monitoring ligand-dependent assembly of receptor ternary complexes in live cells by BRETFect

by Mohamed El Ezzy and Michel Bouvier

Proceedings of the National Academy of Sciences of the United States of America, Mar 27, 2018

There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of... more There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of ternary complexes in live cells. Here we describe bioluminescence resonance energy transfer with fluorescence enhancement by combined transfer (BRETFect), a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate. Its unique donor-intermediate-acceptor relay system is designed so that the acceptor can receive energy either directly from the donor or indirectly via the intermediate in a combined transfer, taking advantage of the entire luciferase emission spectrum. BRETFect was used to study the ligand-dependent cofactor interaction properties of the estrogen receptors ERα and ERβ, which form homo- or heterodimers whose distinctive regulatory properties are difficult to dissect using traditional methods. BRETFect uncovered...

The AF-2 cofactor binding region is key for the selective SUMOylation of estrogen receptor alpha by antiestrogens

by Mohamed El Ezzy and Michel Bouvier

Journal of Biological Chemistry

Pharmacological fingerprint of antipsychotic drugs at the serotonin 5-HT2A receptor

Research Square (Research Square), Jun 1, 2023

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 unidenti ed 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-speci c apparent dissociation constants determined from functional analyses revealed distinct coupling-modulating capacities of the tested antipsychotics at the different 5-HT-activated pathways. Computational analyses of the pharmacological and structural ngerprints supports a mechanistically based clustering that recapitulate the clinical classi cation (typical/ rst 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 identi cation of better and safer neuropsychiatric drugs and allows formulating hypotheses on the links between speci c signaling cascades and in the clinical outcomes of the existing drugs.

Pharmacological fingerprint of antipsychotic drugs at the serotonin 5-HT2A receptor

The intricate involvement of the serotonin 5-HT2A receptor (5-HT2AR) both in schizophrenia and in... more The intricate involvement of the serotonin 5-HT2A receptor (5-HT2AR) 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-HT2AR 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 activ...

Structure-based discovery of nonopioid analgesics acting through the α 2A -adrenergic receptor

Science

Because nonopioid analgesics are much sought after, we computationally docked more than 301 milli... more Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α 2A -adrenergic receptor (α 2A AR), seeking new α 2A AR agonists chemotypes that lack the sedation conferred by known α 2A AR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential G i and G o signaling. Experimental structures of α 2A AR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit ‘9087 [mean effective concentration (EC 50 ) of 52 nanomolar] and two analogs, ‘7075 and PS75 (EC 50 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine.

Visualization of real-time receptor endocytosis in dopamine neurons enabled by NTSR1-Venus knock-in mice

Frontiers in Cellular Neuroscience, Nov 29, 2022

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.

Post-endocytotic Deubiquitination and Degradation of the Metabotropic γ-Aminobutyric Acid Receptor by the Ubiquitin-specific Protease 14

Journal of Biological Chemistry, 2016

Mechanisms controlling the metabotropic ␥-aminobutyric acid receptor (GABA B) cell surface stabil... more Mechanisms controlling the metabotropic ␥-aminobutyric acid receptor (GABA B) cell surface stability are still poorly understood. In contrast with many other G protein-coupled receptors (GPCR), it is not subject to agonist-promoted internalization, but is constitutively internalized and rapidly downregulated. In search of novel interacting proteins regulating receptor fate, we report that the ubiquitin-specific protease 14 (USP14) interacts with the GABA B(1b) subunit's second intracellular loop. Probing the receptor for ubiquitination using bioluminescence resonance energy transfer (BRET), we detected a constitutive and phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination of the receptor at the cell surface. PMA also increased internalization and accelerated receptor degradation. Overexpression of USP14 decreased ubiquitination while treatment with a small molecule inhibitor of the deubiquitinase (IU1) increased receptor ubiquitination. Treatment with the internalization inhibitor Dynasore blunted both USP14 and IU1 effects on the receptor ubiquitination state, suggesting a post-endocytic site of action. Overexpression of USP14 also led to an accelerated degradation of GABA B in a catalytically independent fashion. We thus propose a model whereby cell surface ubiquitination precedes endocytosis, after which USP14 acts as an ubiquitin-binding protein that targets the ubiquitinated receptor to lysosomal degradation and promotes its deubiquitination.

GLP-1R signaling neighborhoods associate with the susceptibility to adverse drug reactions of incretin mimetics

Nature Communications

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...

Dissecting the allosteric networks governing agonist efficacy and potency in G protein-coupled receptors

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.

Les années 2010

médecine/sciences, 2015

les années 2010 m/s fête ses 30 ans en mars 2015 Programme 9 h Introduction-Christian Bréchot (di... more les années 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

Les années 2000

médecine/sciences, 2015

Post-endocytotic Deubiquitination and Degradation of the Metabotropic γ-Aminobutyric Acid Receptor by the Ubiquitin-specific Protease 14

Journal of Biological Chemistry, 2016

Mechanisms controlling the metabotropic ␥-aminobutyric acid receptor (GABA B) cell surface stabil... more Mechanisms controlling the metabotropic ␥-aminobutyric acid receptor (GABA B) cell surface stability are still poorly understood. In contrast with many other G protein-coupled receptors (GPCR), it is not subject to agonist-promoted internalization, but is constitutively internalized and rapidly downregulated. In search of novel interacting proteins regulating receptor fate, we report that the ubiquitin-specific protease 14 (USP14) interacts with the GABA B(1b) subunit's second intracellular loop. Probing the receptor for ubiquitination using bioluminescence resonance energy transfer (BRET), we detected a constitutive and phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination of the receptor at the cell surface. PMA also increased internalization and accelerated receptor degradation. Overexpression of USP14 decreased ubiquitination while treatment with a small molecule inhibitor of the deubiquitinase (IU1) increased receptor ubiquitination. Treatment with the internalization inhibitor Dynasore blunted both USP14 and IU1 effects on the receptor ubiquitination state, suggesting a post-endocytic site of action. Overexpression of USP14 also led to an accelerated degradation of GABA B in a catalytically independent fashion. We thus propose a model whereby cell surface ubiquitination precedes endocytosis, after which USP14 acts as an ubiquitin-binding protein that targets the ubiquitinated receptor to lysosomal degradation and promotes its deubiquitination.

Les années 2000

médecine/sciences, 2015

ADDITIONS AND CORRECTIONS-Quantitative assessment of b1-and b2-adrenergic rereceptor homo-and heterodimerization by bioluminescence resonance energy transfer. Vol. 277 (2002) 44925-44931

Reply: beyond receptor dimerization.: beyond receptor dimerization

β2-Adrenergic receptor down-regulation

by Michel Bouvier and Stefano Marullo

Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of rec... more Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as downregulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosomeplasma membrane recycling pathway to the endosomelysosome degradation pathway. This model is based on previous studies of epidermal growth factor (EGF) receptor degradation and implies that receptors need to be endocytosed to be down-regulated.

β-Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors

by Michel Bouvier and Pascale Charest

Proceedings of the …, 2003

It is becoming increasingly clear that signaling via G protein-coupled receptors is a diverse phe... more

VRQ397 (CRAVKY): a novel noncompetitive V2 receptor antagonist

by Michel Bouvier, Pierre Hardy, and F. Hamdan

AJP: Regulatory, Integrative and Comparative Physiology, 2009

Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibri... more Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibrium and, to a lesser extent, on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, arginine vasopressin (AVP), and hence are competitive ligands. Peptides that reproduce specific sequences of a given receptor have lately been reported to interfere with its action, and if such molecules arise from regions remote from the binding site they would be anticipated to exhibit noncompetitive antagonism, but this has yet to be shown for V2R. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, cravky (labeled VRQ397), was characterized. VRQ397 was potent (IC(50) = 0.69 +/- 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function, specifically desmopressin-L-desamino-8-arginine-vasopressin (DDAVP)-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events. A dose-response profile revealed a noncompetitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). Specificity of VRQ397 was further confirmed by its inability to bind to homologous V1 and oxytocin receptors and its inefficacy to alter responses to stimulation of these receptors. VRQ397 exhibited pharmacological permissiveness on V2R-induced signals, as it inhibited DDAVP-induced PGI(2) generation but not that of cAMP or recruitment of beta-arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. We hereby describe the discovery of a first potent noncompetitive antagonist of V2R, which exhibits functional selectivity, in line with properties of a negative allosteric modulator.

The G protein-coupled receptor TBXA2R activates ERMs to promote motility, invasion, and metastasis of triple-negative breast cancer cells

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.

Role of SUMOylation in Full Antiestrogenicity

by Mohamed El Ezzy and Michel Bouvier

Molecular and Cellular Biology, 2012

The selective estrogen receptor downregulator (SERD) fulvestrant can be used as second-line treat... more The selective estrogen receptor downregulator (SERD) fulvestrant can be used as second-line treatment for patients relapsing after treatment with tamoxifen, a selective estrogen receptor modulator (SERM). Unlike tamoxifen, SERDs are devoid of partial agonist activity. While the full antiestrogenicity of SERDs may result in part from their capacity to downregulate levels of estrogen receptor alpha (ERα) through proteasome-mediated degradation, SERDs are also fully antiestrogenic in the absence of increased receptor turnover in HepG2 cells. Here we report that SERDs induce the rapid and strong SUMOylation of ERα in ERα-positive and -negative cell lines, including HepG2 cells. Four sites of SUMOylation were identified by mass spectrometry analysis. In derivatives of the SERD ICI164,384, SUMOylation was dependent on the length of the side chain and correlated with full antiestrogenicity. Preventing SUMOylation by the overexpression of a SUMO-specific protease (SENP) deSUMOylase partiall...

Monitoring ligand-dependent assembly of receptor ternary complexes in live cells by BRETFect

by Mohamed El Ezzy and Michel Bouvier

Proceedings of the National Academy of Sciences of the United States of America, Mar 27, 2018

There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of... more There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of ternary complexes in live cells. Here we describe bioluminescence resonance energy transfer with fluorescence enhancement by combined transfer (BRETFect), a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate. Its unique donor-intermediate-acceptor relay system is designed so that the acceptor can receive energy either directly from the donor or indirectly via the intermediate in a combined transfer, taking advantage of the entire luciferase emission spectrum. BRETFect was used to study the ligand-dependent cofactor interaction properties of the estrogen receptors ERα and ERβ, which form homo- or heterodimers whose distinctive regulatory properties are difficult to dissect using traditional methods. BRETFect uncovered...

The AF-2 cofactor binding region is key for the selective SUMOylation of estrogen receptor alpha by antiestrogens

by Mohamed El Ezzy and Michel Bouvier

Journal of Biological Chemistry

Pharmacological fingerprint of antipsychotic drugs at the serotonin 5-HT2A receptor

Research Square (Research Square), Jun 1, 2023

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 unidenti ed 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-speci c apparent dissociation constants determined from functional analyses revealed distinct coupling-modulating capacities of the tested antipsychotics at the different 5-HT-activated pathways. Computational analyses of the pharmacological and structural ngerprints supports a mechanistically based clustering that recapitulate the clinical classi cation (typical/ rst 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 identi cation of better and safer neuropsychiatric drugs and allows formulating hypotheses on the links between speci c signaling cascades and in the clinical outcomes of the existing drugs.

Pharmacological fingerprint of antipsychotic drugs at the serotonin 5-HT2A receptor

The intricate involvement of the serotonin 5-HT2A receptor (5-HT2AR) both in schizophrenia and in... more The intricate involvement of the serotonin 5-HT2A receptor (5-HT2AR) 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-HT2AR 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 activ...

Structure-based discovery of nonopioid analgesics acting through the α 2A -adrenergic receptor

Science

Because nonopioid analgesics are much sought after, we computationally docked more than 301 milli... more Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α 2A -adrenergic receptor (α 2A AR), seeking new α 2A AR agonists chemotypes that lack the sedation conferred by known α 2A AR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential G i and G o signaling. Experimental structures of α 2A AR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit ‘9087 [mean effective concentration (EC 50 ) of 52 nanomolar] and two analogs, ‘7075 and PS75 (EC 50 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine.

Visualization of real-time receptor endocytosis in dopamine neurons enabled by NTSR1-Venus knock-in mice

Frontiers in Cellular Neuroscience, Nov 29, 2022

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.

Post-endocytotic Deubiquitination and Degradation of the Metabotropic γ-Aminobutyric Acid Receptor by the Ubiquitin-specific Protease 14

Journal of Biological Chemistry, 2016

Mechanisms controlling the metabotropic ␥-aminobutyric acid receptor (GABA B) cell surface stabil... more Mechanisms controlling the metabotropic ␥-aminobutyric acid receptor (GABA B) cell surface stability are still poorly understood. In contrast with many other G protein-coupled receptors (GPCR), it is not subject to agonist-promoted internalization, but is constitutively internalized and rapidly downregulated. In search of novel interacting proteins regulating receptor fate, we report that the ubiquitin-specific protease 14 (USP14) interacts with the GABA B(1b) subunit's second intracellular loop. Probing the receptor for ubiquitination using bioluminescence resonance energy transfer (BRET), we detected a constitutive and phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination of the receptor at the cell surface. PMA also increased internalization and accelerated receptor degradation. Overexpression of USP14 decreased ubiquitination while treatment with a small molecule inhibitor of the deubiquitinase (IU1) increased receptor ubiquitination. Treatment with the internalization inhibitor Dynasore blunted both USP14 and IU1 effects on the receptor ubiquitination state, suggesting a post-endocytic site of action. Overexpression of USP14 also led to an accelerated degradation of GABA B in a catalytically independent fashion. We thus propose a model whereby cell surface ubiquitination precedes endocytosis, after which USP14 acts as an ubiquitin-binding protein that targets the ubiquitinated receptor to lysosomal degradation and promotes its deubiquitination.

GLP-1R signaling neighborhoods associate with the susceptibility to adverse drug reactions of incretin mimetics

Nature Communications

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...

Dissecting the allosteric networks governing agonist efficacy and potency in G protein-coupled receptors

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.

Les années 2010

médecine/sciences, 2015

les années 2010 m/s fête ses 30 ans en mars 2015 Programme 9 h Introduction-Christian Bréchot (di... more les années 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

Les années 2000

médecine/sciences, 2015

Post-endocytotic Deubiquitination and Degradation of the Metabotropic γ-Aminobutyric Acid Receptor by the Ubiquitin-specific Protease 14

Journal of Biological Chemistry, 2016

Mechanisms controlling the metabotropic ␥-aminobutyric acid receptor (GABA B) cell surface stabil... more Mechanisms controlling the metabotropic ␥-aminobutyric acid receptor (GABA B) cell surface stability are still poorly understood. In contrast with many other G protein-coupled receptors (GPCR), it is not subject to agonist-promoted internalization, but is constitutively internalized and rapidly downregulated. In search of novel interacting proteins regulating receptor fate, we report that the ubiquitin-specific protease 14 (USP14) interacts with the GABA B(1b) subunit's second intracellular loop. Probing the receptor for ubiquitination using bioluminescence resonance energy transfer (BRET), we detected a constitutive and phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination of the receptor at the cell surface. PMA also increased internalization and accelerated receptor degradation. Overexpression of USP14 decreased ubiquitination while treatment with a small molecule inhibitor of the deubiquitinase (IU1) increased receptor ubiquitination. Treatment with the internalization inhibitor Dynasore blunted both USP14 and IU1 effects on the receptor ubiquitination state, suggesting a post-endocytic site of action. Overexpression of USP14 also led to an accelerated degradation of GABA B in a catalytically independent fashion. We thus propose a model whereby cell surface ubiquitination precedes endocytosis, after which USP14 acts as an ubiquitin-binding protein that targets the ubiquitinated receptor to lysosomal degradation and promotes its deubiquitination.

Les années 2000

médecine/sciences, 2015

ADDITIONS AND CORRECTIONS-Quantitative assessment of b1-and b2-adrenergic rereceptor homo-and heterodimerization by bioluminescence resonance energy transfer. Vol. 277 (2002) 44925-44931

Reply: beyond receptor dimerization.: beyond receptor dimerization

β2-Adrenergic receptor down-regulation

by Michel Bouvier and Stefano Marullo

Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of rec... more Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as downregulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosomeplasma membrane recycling pathway to the endosomelysosome degradation pathway. This model is based on previous studies of epidermal growth factor (EGF) receptor degradation and implies that receptors need to be endocytosed to be down-regulated.

β-Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors

by Michel Bouvier and Pascale Charest

Proceedings of the …, 2003

It is becoming increasingly clear that signaling via G protein-coupled receptors is a diverse phe... more

VRQ397 (CRAVKY): a novel noncompetitive V2 receptor antagonist

by Michel Bouvier, Pierre Hardy, and F. Hamdan

AJP: Regulatory, Integrative and Comparative Physiology, 2009

Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibri... more Vasopressin type 2 receptor (V2R) exhibits mostly important properties for hydroosmotic equilibrium and, to a lesser extent, on vasomotricity. Drugs currently acting on this receptor are analogs of the natural neuropeptide, arginine vasopressin (AVP), and hence are competitive ligands. Peptides that reproduce specific sequences of a given receptor have lately been reported to interfere with its action, and if such molecules arise from regions remote from the binding site they would be anticipated to exhibit noncompetitive antagonism, but this has yet to be shown for V2R. Six peptides reproducing juxtamembranous regions of V2R were designed and screened; the most effective peptide, cravky (labeled VRQ397), was characterized. VRQ397 was potent (IC(50) = 0.69 +/- 0.25 nM) and fully effective in inhibiting V2R-dependent physiological function, specifically desmopressin-L-desamino-8-arginine-vasopressin (DDAVP)-induced cremasteric vasorelaxation; this physiological functional assay was utilized to avoid overlooking interference of specific signaling events. A dose-response profile revealed a noncompetitive property of VRQ397; correspondingly, VRQ397 bound specifically to V2R-expressing cells could not displace its natural ligand, AVP, but modulated AVP binding kinetics (dissociation rate). Specificity of VRQ397 was further confirmed by its inability to bind to homologous V1 and oxytocin receptors and its inefficacy to alter responses to stimulation of these receptors. VRQ397 exhibited pharmacological permissiveness on V2R-induced signals, as it inhibited DDAVP-induced PGI(2) generation but not that of cAMP or recruitment of beta-arrestin2. Consistent with in vitro and ex vivo effects as a V2R antagonist, VRQ397 displayed anticipated in vivo aquaretic efficacy. We hereby describe the discovery of a first potent noncompetitive antagonist of V2R, which exhibits functional selectivity, in line with properties of a negative allosteric modulator.