Sensing of amino acids in a dopaminergic circuitry promotes rejection of an incomplete diet in Drosophila.

The brain is the central organizer of food intake, matching the quality and quantity of the food sources with organismal needs. To ensure appropriate amino acid balance, many species reject a diet lacking one or several essential amino acids (EAAs) and seek out a better food source. Here, we show that, in Drosophila larvae, this behavior relies on innate sensing of amino acids in dopaminergic (DA) neurons of the brain. We demonstrate that the amino acid sensor GCN2 acts upstream of GABA signaling in DA neurons to promote avoidance of the EAA-deficient diet. Using real-time calcium imaging in larval brains, we show that amino acid imbalance induces a rapid and reversible activation of three DA neurons that are necessary and sufficient for food rejection. Taken together, these data identify a central amino-acid-sensing mechanism operating in specific DA neurons and controlling food intake.

Substrate-induced unlocking of the inner gate determines the catalytic efficiency of a neurotransmitter:sodium symporter.

Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na(+)-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na(+)-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.

The oligomeric state sets GABA(B) receptor signalling efficacy.

G protein-coupled receptors (GPCRs) have key roles in cell-cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA(B) receptor is one of the most abundant, being distributed in most brain regions, on either pre- or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers.

TrkB receptor interacts with mGlu2 receptor and mediates antipsychotic-like effects of mGlu2 receptor activation in the mouse.

Metabotropic glutamate receptor 2 (mGlu2) attracts particular attention as a possible target for a new class of antipsychotics. However, the signaling pathways transducing the effects of mGlu2 in the brain remain poorly characterized. Here, we addressed this issue by identifying native mGlu2 interactome in mouse prefrontal cortex. Nanobody-based affinity purification and mass spectrometry identified 149 candidate mGlu2 partners, including the neurotrophin receptor TrkB. The later interaction was confirmed both in cultured cells and prefrontal cortex. mGlu2 activation triggers phosphorylation of TrkB on Tyr816 in primary cortical neurons and prefrontal cortex. Reciprocally, TrkB stimulation enhances mGlu2-operated Gi/o protein activation. Furthermore, TrkB inhibition prevents the rescue of behavioral deficits by glutamatergic antipsychotics in phencyclidine-treated mice. Collectively, these results reveal a cross-talk between TrkB and mGlu2, which is key to the behavioral response to glutamatergic antipsychotics.

Stability of GABAB receptor oligomers revealed by dual TR-FRET and drug-induced cell surface targeting.

The function of cell surface proteins likely involves the formation and dissociation of oligomeric complexes. However, the dynamics of this process are unknown. Here we examined this process for the GABA(B) receptors that assemble into oligomers of heterodimers through the association of their GABA(B1) subunit. We report a method to study oligomer dynamics based on a drug-controlled cell surface targeting of intracellularly retained receptors and a parallel measurement of two FRET signals in HEK293 cells. GABA(B1) subunits at the cell surface (4.0 ± 0.6 a.u.) are labeled with a pair of fluorophores (donor and red acceptor). New receptors are then targeted to the cell surface during 3h treatment with AP21967 such that the number of receptors is doubled (9.1 ± 0.7 a.u.). After labeling these new receptors with a second acceptor (green), the red FRET remained unchanged (5189 ± 36 vs. 4783 ± 32 cps), supporting the stability of the preformed oligomers. However, new oligomers are detected by the green FRET signal indicating both receptor populations are in the same microdomains. As a control, we confirmed the strict stability of the GABA(B) heterodimer itself. Herein, using a novel method to monitor the dynamics of cell surface complexes, we provide evidence for the stability of GABA(B) oligomers.

The binding sites for cocaine and dopamine in the dopamine transporter overlap.

Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog LeuT. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed mutagenesis and by trapping the radiolabeled cocaine analog [3H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn2+-binding site that was situated extracellularly to the predicted common binding pocket. Our data demonstrate the molecular basis for the competitive inhibition of dopamine transport by cocaine.

[G-protein-coupled receptors dimers and oligomers: for which purpose? The GABA(B) receptor under investigation]. / Des dimères et des oligomères de récepteurs couplés aux protéines G, oui mais pourquoi ? - Le récepteur GABA(B) sous interrogatoire.

G protein-coupled receptors are membrane receptors that are involved in most of the physiological processes. The large variety of their functions arises from both the number of receptors and the formation of dimers or oligomers having specific properties. The precise consequences of the oligomerization are not well understood yet but it has been proposed to affect the protein trafficking, the ligand binding and the signaling. In this review, we explore the functional consequences of receptor dimers and oligomers using as a model the GABA(B) receptor, which is activated by the inhibitory neurotransmitter GABA.

Chronic sodium bromide treatment relieves autistic-like behavioral deficits in three mouse models of autism.

Autism Spectrum Disorders (ASD) are neurodevelopmental disorders whose diagnosis relies on deficient social interaction and communication together with repetitive behavior. To date, no pharmacological treatment has been approved that ameliorates social behavior in patients with ASD. Based on the excitation/inhibition imbalance theory of autism, we hypothesized that bromide ions, long used as an antiepileptic medication, could relieve core symptoms of ASD. We evaluated the effects of chronic sodium bromide (NaBr) administration on autistic-like symptoms in three genetic mouse models of autism: Oprm1-/-, Fmr1-/- and Shank3Δex13-16-/- mice. We showed that chronic NaBr treatment relieved autistic-like behaviors in these three models. In Oprm1-/- mice, these beneficial effects were superior to those of chronic bumetanide administration. At transcriptional level, chronic NaBr in Oprm1 null mice was associated with increased expression of genes coding for chloride ions transporters, GABAA receptor subunits, oxytocin and mGlu4 receptor. Lastly, we uncovered synergistic alleviating effects of chronic NaBr and a positive allosteric modulator (PAM) of mGlu4 receptor on autistic-like behavior in Oprm1-/- mice. We evidenced in heterologous cells that bromide ions behave as PAMs of mGlu4, providing a molecular mechanism for such synergy. Our data reveal the therapeutic potential of bromide ions, alone or in combination with a PAM of mGlu4 receptor, for the treatment of ASDs.

Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations.

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu5 receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes.

The different aspects of the GABAB receptor allosteric modulation.

The GABAB receptor is activated by the main inhibitory neurotransmitter of the central nervous system, the γ-aminobutyric acid (GABA). The receptor is expressed in almost all neuronal and glial cells and plays a central role in the modulation of many physiological and pathological processes. The GABAB receptor has been considered for years as an interesting target for the treatment of spasticity, pain, addiction, anxiety or depression. This has prompted many studies aiming at understanding the activation of the receptor and its modulation. While it belongs to the super-family of G protein-coupled receptors (GPCRs), it was rapidly evident that the GABAB receptor is peculiar in the variety of allosteric modulations governing its activation. Here, I wish to gather the different aspects of the GABAB receptor allosteric modulation. After presenting the main small molecule allosteric modulators known to date, the intramolecular transitions controlling the receptor activation will be summarized. In addition, recent findings obtained in the last decade on the existence of GABAB receptor complexes and their influence on the receptor function will be introduced, including the GABAB receptor oligomers and the auxiliary proteins associated with the receptor. These new concepts will certainly be of major interest in the future analysis of GABAB receptor allosteric modulation.

Closed state of both binding domains of homodimeric mGlu receptors is required for full activity.

Membrane receptors, key components in signal transduction, often function as dimers. These include some G protein-coupled receptors such as metabotropic glutamate (mGlu) receptors that have large extracellular domains (ECDs) where agonists bind. How agonist binding in dimeric ECDs activates the effector domains remains largely unknown. The structure of the dimeric ECDs of mGlu(1) solved in the presence of agonist revealed two specific conformations in which either one or both protomers are in an agonist-stabilized closed form. Here we examined whether both conformations correspond to an active form of the full-length receptor. Using a system that allows the formation of dimers made of a wild-type and a mutant subunit, we show that the closure of one ECD per dimer is sufficient to activate the receptor, but the closure of both ECDs is required for full activity.

GPCR interaction as a possible way for allosteric control between receptors.

For more than twenty years now, GPCR dimers and larger oligomers have been the subject of intense debates. Evidence for a role of such complexes in receptor trafficking to and from the plasma membrane have been provided. However, one main issue is of course to determine whether or not such a phenomenon can be responsible for an allosteric and reciprocal control (allosteric control) of the subunits. Such a possibility would indeed add to the possible ways a cell integrates various signals targeting GPCRs. Among the large GPCR family, the class C receptors that include mGlu and GABAB receptors, represent excellent models to examine such a possibility as they are mandatory dimers. In the present review, we will report on the observed allosteric interaction between the subunits of class C GPCRs, both mGluRs and GABABRs, and on the structural bases of these interactions. We will then discuss these findings for other GPCR types such as the rhodopsin-like class A receptors. We will show that many of the observations made with class C receptors have also been reported with class A receptors, suggesting that the mechanisms involved in the allosteric control between subunits in GPCR dimers may not be unique to class C GPCRs.

Modeling the binding and function of metabotropic glutamate receptors.

A mathematical model for the binding and function of metabotropic glutamate receptors was developed, with the aim to gain new insights into the functioning of these complex receptors. These receptors are homodimers, and each subunit is composed of a ligand binding [Venus flytrap (VFT)] domain and a heptahelical domain (HD) responsible for G-protein activation. Our mechanistic model integrates all structural information available so far: the various states of the VFT dimer (open-open, closed-open, and closed-closed), as well as the fact that a single HD is active at a time. To provide the model with parameters with biological meaning, two published experimental studies were reanalyzed. The first one reports a negative cooperativity in agonist binding (J Biol Chem 279:35526-35534, 2004), whereas the other indicates a positive cooperativity in agonist-mediated response (Nat Struct Mol Biol 11:706-713, 2004). The former study allowed us to explain the mechanistic features associated with VFT recognition by agonists and antagonists integrating a negative allosteric interaction for agonist binding. The second study helped us to quantitatively describe the functional dynamics of transduction of the VFT occupation into functional response, confirming a putative positive cooperativity at the level of receptor coupling efficacy. This model will help both to better understand the functioning of these receptors and to characterize the mechanism of action of various types of allosteric modulators. Moreover, this model may be of general utility for oligomeric systems in which the ligand binding and effector domains correspond to distinct structural domains.

Allosteric interactions between GABAB1 subunits control orthosteric binding sites occupancy within GABAB oligomers.

The GABAB receptor was the first G protein-coupled receptor identified as an obligate heterodimer. It is composed of two subunits, GABAB1 containing the agonist binding site and GABAB2 responsible for G protein activation. The GABAB receptor was found to associate into larger complexes through GABAB1-GABAB1 interactions, both in transfected cells and in brain membranes. Here we assessed the possible allosteric interactions between GABAB heterodimers by analyzing the effect of mutations located at the putative interface between the extracellular binding domains. These mutations decrease, but do not suppress, the Förster resonance energy transfer (FRET) signal measured between GABAB1 subunits. Further analysis of one of these mutations revealed an increase in G protein-coupling efficacy and in the maximal antagonist binding by approximately two-fold. Hypothesizing that a tetramer is an elementary unit within oligomers, additional FRET data using fluorescent ligands and tagged subunits suggest that adjacent binding sites within the GABAB oligomers are not simultaneously occupied. Our data show a strong negative effect between GABAB1 binding sites within GABAB oligomers. Accordingly, GABAB receptor assembly appears to limit receptor signaling to G proteins, a property that may offer novel regulatory mechanism for this important neuronal receptor. This article is part of the "Special Issue Dedicated to Norman G. Bowery".

Oligomerization of a G protein-coupled receptor in neurons controlled by its structural dynamics.

G protein coupled receptors (GPCRs) play essential roles in intercellular communication. Although reported two decades ago, the assembly of GPCRs into dimer and larger oligomers in their native environment is still a matter of intense debate. Here, using number and brightness analysis of fluorescently labeled receptors in cultured hippocampal neurons, we confirm that the metabotropic glutamate receptor type 2 (mGlu2) is a homodimer at expression levels in the physiological range, while heterodimeric GABAB receptors form larger complexes. Surprisingly, we observed the formation of larger mGlu2 oligomers upon both activation and inhibition of the receptor. Stabilizing the receptor in its inactive conformation using biochemical constraints also led to the observation of oligomers. Following our recent observation that mGlu receptors are in constant and rapid equilibrium between several states under basal conditions, we propose that this structural heterogeneity limits receptor oligomerization. Such assemblies are expected to stabilize either the active or the inactive state of the receptor.

Class C G protein-coupled receptors: reviving old couples with new partners.

G protein-coupled receptors (GPCRs) are key players in cell communication and are encoded by the largest family in our genome. As such, GPCRs represent the main targets in drug development programs. Sequence analysis revealed several classes of GPCRs: the class A rhodopsin-like receptors represent the majority, the class B includes the secretin-like and adhesion GPCRs, the class F includes the frizzled receptors, and the class C includes receptors for the main neurotransmitters, glutamate and GABA, and those for sweet and umami taste and calcium receptors. Class C receptors are far more complex than other GPCRs, being mandatory dimers, with each subunit being composed of several domains. In this review, we summarize our actual knowledge regarding the activation mechanism and subunit organization of class C GPCRs, and how this brings information for many other GPCRs.

RgIA4 Potently Blocks Mouse α9α10 nAChRs and Provides Long Lasting Protection against Oxaliplatin-Induced Cold Allodynia.

Transcripts for α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits are found in diverse tissues. The function of α9α10 nAChRs is best known in mechanosensory cochlear hair cells, but elsewhere their roles are less well-understood. α9α10 nAChRs have been implicated as analgesic targets and α-conotoxins that block α9α10 nAChRs produce analgesia. However, some of these peptides show large potency differences between species. Additionally several studies have indicated that these conotoxins may also activate GABAB receptors (GABABRs). To further address these issues, we cloned the cDNAs of mouse α9 and α10 nAChR subunits. When heterologously expressed in Xenopus oocytes, the resulting α9α10 nAChRs had the expected pharmacology of being activated by acetylcholine and choline but not by nicotine. A conotoxin analog, RgIA4, potently, and selectively blocked mouse α9α10 nAChRs with low nanomolar affinity indicating that RgIA4 may be effectively used to study murine α9α10 nAChR function. Previous reports indicated that RgIA4 attenuates chemotherapy-induced cold allodynia. Here we demonstrate that RgIA4 analgesic effects following oxaliplatin treatment are sustained for 21 days after last RgIA4 administration indicating that RgIA4 may provide enduring protection against nerve damage. RgIA4 lacks activity at GABAB receptors; a bioluminescence resonance energy transfer assay was used to demonstrate that two other analgesic α-conotoxins, Vc1.1 and AuIB, also do not activate GABABRs expressed in HEK cells. Together these findings further support the targeting of α9α10 nAChRs in the treatment of pain.

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

The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABAB, which is a target for various therapeutic applications. It is an obligatory heterodimer made of GB1 and GB2 that can be regulated by positive allosteric modulators (PAMs). The molecular mechanism of activation of the GABAB receptor remains poorly understood. Here, we have developed FRET-based conformational GABAB sensors compatible with high-throughput screening. We identified conformational changes occurring within the extracellular and transmembrane domains upon receptor activation, which are smaller than those observed in the related metabotropic glutamate receptors. These sensors also allow discrimination between agonists of different efficacies and between PAMs that have different modes of action, which has not always been possible using conventional functional assays. Our study brings important new information on the activation mechanism of the GABAB receptor and should facilitate the screening and identification of new chemicals targeting this receptor.

No ligand binding in the GB2 subunit of the GABA(B) receptor is required for activation and allosteric interaction between the subunits.

The GABA(B) receptor plays important roles in the tuning of many synapses. Although pharmacological differences have been observed between various GABA(B)-mediated effects, a single GABA(B) receptor composed of two subunits (GB1 and GB2) has been identified. Although GB1 binds GABA, GB2 plays a critical role in G-protein activation. Moreover, GB2 is required for the high agonist affinity of GB1. Like any other family 3 G-protein-coupled receptors, GB1 and GB2 are composed of a Venus Flytrap module (VFTM) that usually contains the agonist-binding site and a heptahelical domain. So far, there has been no direct demonstration that GB2 binds GABA or another endogenous ligand. Here, we have further refined the GABA-binding site of GB1 and characterized the putative-binding site in the VFTM of GB2. None of the residues important for GABA binding in GB1 appeared to be conserved in GB2. Moreover, mutation of 10 different residues, alone or in combination, within the possible binding pocket of GB2 affects neither GABA activation of the receptor nor the ability of GB2 to increase agonist affinity on GB1. These data indicate that ligand binding in the GB2 VFTM is not required for activation. Finally, although in either GB1 or the related metabotropic glutamate receptors most residues of the binding pocket are conserved from Caenorhabditis elegans to human, no such conservation is observed in GB2. This suggests that the GB2 VFTM does not constitute a binding site for a natural ligand.

Locking the dimeric GABA(B) G-protein-coupled receptor in its active state.

G-protein-coupled receptors (GPCRs) play a major role in cell-cell communication in the CNS. These proteins oscillate between various inactive and active conformations, the latter being stabilized by agonists. Although mutations can lead to constitutive activity, most of these destabilize inactive conformations, and none lock the receptor in an active state. Moreover, GPCRs are known to form dimers, but the role of each protomer in the activation process remains unclear. Here, we show that the heterodimeric GPCR for the main inhibitory neurotransmitter, the GABA(B) receptor, can be locked in its active state by introducing two cysteines expected to form a disulphide bridge to maintain the binding domain of the GABA(B1) subunit in a closed form. This constitutively active receptor cannot be inhibited by antagonists, but its normal functioning, activation by agonists, and inhibition by antagonists can be restored after reduction with dithiothreitol. These data show that the closed state of the binding domain of GABA(B1) is sufficient to turn ON this heterodimeric receptor and illustrate for the first time that a GPCR can be locked in an active conformation.