Allosteric Modulators of GABAB Receptors: Mechanism of Action and Therapeutic Perspective

γ-aminobutyric acid (GABA) plays important roles in the central nervous system, acting as a neurotransmitter on both ionotropic ligand-gated Cl--channels, and metabotropic G-protein coupled receptors (GPCRs). These two types of receptors called GABAA (and C) and GABAB are the targets of major therapeutic drugs such as the anxiolytic benzodiazepines, and antispastic drug baclofen (lioresal®), respectively. Although the multiplicity of GABAA receptors offer a number of possibilities to discover new and more selective drugs, the molecular characterization of the GABAB receptor revealed a unique, though complex, heterodimeric GPCR. High throughput screening strategies carried out in pharmaceutical industries, helped identifying new compounds positively modulating the activity of the GABAB receptor. These molecules, almost devoid of apparent activity when applied alone, greatly enhance both the potency and efficacy of GABAB agonists. As such, in contrast to baclofen that constantly activates the receptor everywhere in the brain, these positive allosteric modulators induce a large increase in GABAB-mediated responses only WHERE and WHEN physiologically needed. Such compounds are then well adapted to help GABA to activate its GABAB receptors, like benzodiazepines favor GABAA receptor activation. In this review, the way of action of these molecules will be presented in light of our actual knowledge of the activation mechanism of the GABAB receptor. We will then show that, as expected, these molecules have more pronounced in vivo responses and less side effects than pure agonists, offering new potential therapeutic applications for this new class of GABAB ligands

Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser 280 by Hallucinogenic versus Nonhallucinogenic Agonists

International audienceThe serotonin 5-HT 2A receptor is a primary target of psy-chedelic hallucinogens such as lysergic acid diethyl-amine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT 2A receptor ago-nists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hy-drophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT 2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT 2A ago-nist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser 280) located in the third intracellular loop of the 5-HT 2A receptor, a region important for its desensitiza-tion. The specific phosphorylation of Ser 280 by hallucino-gens was further validated by quantitative mass spec-trometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific anti-body. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT 2A receptors at Ser 280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser 280 to as-partic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucino-gens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT 2A receptor in response to hallucinogenic versus nonhallucinogenic ago-nists, which underlies their distinct capacity to desensi-tize the receptor. Molecular & Cellular Proteomics 13: 10.1074/mcp.M113.036558, 1273-1285, 2014. Among the G Protein-Coupled Receptors (GPCRs) 1 activated by serotonin (5-hydroxytryptamine, 5-HT), the 5-HT 2A receptor continues to attract particular attention in view of its broad physiological role and implication in the actions of numerous psychotropic agents (1, 2). It is a primary target of widely used atypical antipsychotics such as clozapine, risperi-done, and olanzapine, which act as antagonists or inverse agonists (1, 3). The activation of 5-HT 2A receptors expressed in the prefrontal cortex has also been implicated in the psy-cho-mimetic effects of psychedelic hallucinogens, such as lysergic acid diethylamide (LSD), mescaline, and psilocybin, which are often used to model positive symptoms of schizo-phrenia (4-8). However, these psychoactive effects are not reproduced by structurally-related agonists, such as ergota-mine and the anti-Parkinson agent lisuride, despite the fact that they exhibit comparable affinities and efficacies at 5-HT 2A receptors (7, 9). This paradox was partially resolved by the demonstration that hallucinogens induce a specific transcrip-From the ‡CNRS

Beyond Autonomy: Coersion and Morality in Clinical Relationships

International audienc

Appeal No. 0678: Perto Drilling Corp., Inc. v. Division of Mineral Resources Management

Chief\u27s Order 99-15

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

Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by positive allosteric modulators.: Asymmetric Functioning of a GPCR Dimer

International audienceThe recent discovery of positive allosteric modulators (PAMs) for G-protein-coupled receptors open new possibilities to control a number of physiological and pathological processes. Understanding the mechanism of action of such compounds will provide new information on the activation process of these important receptors. Within the last 10 years, a number of studies indicate that G-protein-coupled receptors can form dimers, but the functional significance of this phenomenon remains elusive. Here we used the metabotropic glutamate receptors as a model, because these receptors, for which PAMs have been identified, are constitutive dimers. We used the quality control system of the GABA(B) receptor to generate metabotropic glutamate receptor dimers in which a single subunit binds a PAM. We show that one PAM/dimer is sufficient to enhance receptor activity. Such a potentiation can still be observed if the subunit unable to bind the PAM is also made unable to activate G-proteins. However, the PAM acts as a non-competitive antagonist when it binds in the subunit that cannot activate G-proteins. These data are consistent with a single heptahelical domain reaching the active state per dimer during receptor activation

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

Peer reviewe