Biomedical subjects

Aline Dumuis

Publications and source records attributed to Aline Dumuis.

At least 19 recordsLinked to original sources

PubMed2006

Neuronal 5-HT metabotropic receptors: fine-tuning of their structure, signaling, and roles in synaptic modulation.

Serotonin (5-hydroxytryptamine, 5-HT) is, without doubt, the neurotransmitter for which the number of receptors is the highest. Fifteen genes encoding functional 5-HT receptors have been cloned in mammalian brain. 5-HT(3) receptors are ionotropic receptors, whereas all the others are metabotropic G-protein-coupled receptors (GPCRs). 5-HT receptor diversity is further increased by post-genomic modifications, such as alternative splicing (up to 10 splice variants for the 5-HT(4) receptor) or by mRNA editing in the case of 5-HT(2C) receptors. The cellular and behavioral implications of 5-HT(2C) receptor editing are of great physiological importance. Signaling of 5-HT receptors involves a great variety of pathways, but only some of these have been demonstrated in neurons. The classical view of neurotransmitter receptors localized within the synaptic cleft cannot be applied to 5-HT receptors, which are mostly (but not exclusively) localized at extra-synaptic locations either pre- or post-synaptically. 5-HT receptors are engaged in pre- or post-synaptic complexes composed of many GPCR-interacting proteins. The functions of these proteins are starting to be revealed. These proteins have been implicated in targeting, trafficking to or from the membrane, desensitization, and fine-tuning of signaling.

Alternative Splicing↗

PubMed2006

Adaptive changes in serotonin neurons of the raphe nuclei in 5-HT(4) receptor knock-out mouse.

Decreased serotonin (5-HT) transmission is thought to underlie several mental diseases, including depression and feeding disorders. However, whether deficits in genes encoding G protein-coupled receptors may down-regulate the activity of 5-HT neurons is unknown currently. Based on recent evidence that stress-induced anorexia may involve 5-HT(4)receptors (5-HT(4)R), we measured various aspects of 5-HT function in 5-HT(4)R knock-out (KO) mice. When compared to dorsal raphe nucleus (DRN) 5-HT neurons from wild-type mice, those from 5-HT(4)R KO mice exhibited reduced spontaneous electrical activity. This reduced activity was associated with diminished tissue levels of 5-HT and its main metabolite, 5-hydroxyindole acetic acid (5-HIAA). Cumulative, systemic doses of the 5-HT uptake blocker citalopram, that reduced 5-HT cell firing by 30% in wild-type animals, completely inhibited 5-HT neuron firing in the KO mice. This effect was reversed by administration of the 5-HT(1A) receptor (5-HT(1A)R) antagonist, WAY100635, in mice of both genotypes. Other changes in DRN of the KO mice included increases in the levels of 5-HT plasma membrane transporter sites and mRNA, as well as a decrease in the density of 5-HT(1A)R sites without any change in 5-HT(1A) mRNA content. With the exception of increased 5-HT turnover index in the hypothalamus and nucleus accumbens and a decreased density of 5-HT(1A)R sites in the dorsal hippocampus (CA1) and septum, no major changes were detected in 5-HT territories of projection, suggesting region-specific adaptive changes. The mechanisms whereby 5-HT(4)R mediate a tonic positive influence on the firing activity of DRN 5-HT neurons and 5-HT content remain to be determined.

Animals↗

PubMed2005

5-HT7 receptor is coupled to G alpha subunits of heterotrimeric G12-protein to regulate gene transcription and neuronal morphology.

The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT7 receptor. Expression of 5-HT7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT7 receptors significantly increased neurite length, whereas stimulation of 5-HT4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT7R/G12 and 5-HT4R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter.

Animals↗

PubMed2005

Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements.

The 5-hydroxytryptamine type 4 receptors (5-HT4Rs) are involved in memory, cognition, feeding, respiratory control, and gastrointestinal motility through activation of a G(s)/cAMP pathway. We have shown that 5-HT4R undergoes rapid and profound homologous uncoupling in neurons. However, no significant uncoupling was observed in COS-7 or HEK293 cells, which expressed either no or a weak concentration of GRK2, respectively. High expression of GRK2 in neurons is likely to be the reason for this difference because overexpression of GRK2 in COS-7 and HEK293 cells reproduced rapid and profound uncoupling of 5-HT4R. We have also shown, for the first time, that GRK2 requirements for uncoupling and endocytosis were very different. Indeed, beta-arrestin/dynamin-dependent endocytosis was observed in HEK293 cells without any need of GRK2 overexpression. In addition to this difference, uncoupling and beta-arrestin/dynamin-dependent endocytosis were mediated through distinct mechanisms. Neither uncoupling nor beta-arrestin/dynamin-dependent endocytosis required the serine and threonine residues localized within the specific C-terminal domains of the 5-HT4R splice variants. In contrast, a cluster of serines and threonines, common to all variants, was an absolute requirement for beta-arrestin/dynamin-dependent receptor endocytosis, but not for receptor uncoupling. Furthermore, beta-arrestin/dynamin-dependent endocytosis and uncoupling were dependent on and independent of GRK2 kinase activity, respectively. These results clearly demonstrate that the uncoupling and endocytosis of 5-HT4R require different GRK2 concentrations and involve distinct molecular events.

Alternative Splicing↗

PubMed2005

Molecular characterization of a purified 5-HT4 receptor: a structural basis for drug efficacy.

Serotonin 5-HT(4(a)) receptor, a G-protein-coupled receptor (GPCR), was produced as a functional isolated protein using Escherichia coli as an expression system. The isolated receptor was characterized at the molecular level by circular dichroism (CD) and steady-state fluorescence. A specific change in the near-UV CD band associated with the GPCR disulfide bond connecting the third transmembrane domain to the second extracellular loop (e2) was observed upon agonist binding to the purified receptor. This is a direct experimental evidence for a change in the conformation of the e2 loop upon receptor activation. Different variations were obtained depending whether the ligand was an agonist (partial or full) or an inverse agonist. In contrast, antagonist binding did not induce any variation. These observations provide a first direct evidence for the fact that free (or antagonist-occupied), active (partial- or full agonist-occupied) and silent (inverse agonist-occupied) states of the receptor involve different arrangements of the e2 loop. Finally, ligand-induced changes in the fluorescence emission profile of the purified receptor confirmed that the partial agonist stabilized a single, well-defined, conformational state and not a mixture of different states. This result is of particular interest in a pharmacological perspective since it directly demonstrates that the efficacy of a drug is likely due to the stabilization of a ligand-specific state rather than selection of a mixture of different conformational states of the receptor.

Animals↗

PubMed2005

Palmitoylation of the 5-hydroxytryptamine4a receptor regulates receptor phosphorylation, desensitization, and beta-arrestin-mediated endocytosis.

The mouse 5-hydroxytryptamine4a (5-HT4a) receptor is an unusual member of the G protein-coupled receptor superfamily because it possesses two separate carboxyl-terminal palmitoylation sites, which may allow the receptor to adopt different conformations in an agonist-dependent manner (J Biol Chem 277:2534-2546, 2002). By targeted mutation of the proximal (Cys-328/329) or distal (Cys-386) palmitoylation sites, or a combination of both, we generated 5-HT4a receptor variants with distinct functional characteristics. In this study, we showed that upon 5-HT stimulation, the 5-HT4a receptor undergoes rapid (t(1/2) approximately 2 min) and dose-dependent (EC50 approximately 180 nM) phosphorylation on serine residues by a staurosporine-insensitive receptor kinase. Overexpression of GRK2 significantly reduced the receptor-promoted cAMP formation. The Cys328/329-Ser mutant, which is constitutively active in the absence of ligand, exhibited enhanced receptor phosphorylation under both basal and agonist-stimulated conditions and was more effectively desensitized and internalized via a beta-arrestin-2 mediated pathway compared with the wild-type 5-HT4a. In contrast, G protein activation, phosphorylation, desensitization, and internalization of the other palmitoylation-deficient receptor mutants were affected differently. These findings suggest that palmitoylation plays an important role in modulating 5-HT4a receptor functions and that G protein activation, phosphorylation, desensitization, and internalization depend on the different receptor conformations.

Animals↗

PubMed2004

New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting.

The 5-hydroxytryptamine type 4 receptor (5-HT4R) is involved in learning, feeding, respiratory control and gastrointestinal transit. This receptor is one of the G-protein-coupled receptors for which alternative mRNA splicing generates the most variants that differ in their C-terminal extremities. Some 5-HT4R variants (a, e and f) express canonical PDZ ligands at their C-termini. Here, we have examined whether some mouse 5-HT4R variants associate with specific sets of proteins, using a proteomic approach based on peptide-affinity chromatography, two-dimensional electrophoresis and mass spectrometry. We have identified ten proteins that interact specifically with the 5-HT4(a)R and three that only associate with the 5-HT4(e)R. Most of them are PDZ proteins. Among the proteins that associated specifically with the 5-HT4(a)R variant, NHERF greatly modified its subcellular localization. Moreover, NHERF recruited the 5-HT4(a)R to microvilli, where it localized with activated ezrin, consistent with the role of 5-HT4(a)R in cytoskeleton remodelling. The 5-HT4(a)R also interacted with both the constitutive and inducible (upon methamphetamine treatment) forms of the recently cloned sorting nexin 27 (SNX27a and b, respectively). We found that SNX27a redirected part of 5-HT4(a)R to early endosomes. The interaction of the 5-HT4R splice variants with distinct sets of PDZ proteins might specify their cellular localization as well as their signal transduction properties.

Alternative Splicing↗

PubMed2004

The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins.

The 5-hydroxytryptamine type 2A (5-HT(2A)) receptor and the 5-HT(2C) receptor are closely related members of the G-protein-coupled receptors activated by serotonin that share very similar pharmacological profiles and cellular signaling pathways. These receptors express a canonical class I PDZ ligand (SXV) at their C-terminal extremity. Here, we have identified proteins that interact with the PDZ ligand of the 5-HT(2A) and 5-HT(2C) receptors by a proteomic approach associating affinity chromatography using immobilized synthetic peptides encompassing the PDZ ligand and mass spectrometry. We report that both receptor C termini interact with specific sets of PDZ proteins in vitro. The 5-HT(2C) receptor but not the 5-HT(2A) receptor binds to the Veli-3.CASK.Mint1 ternary complex and to SAP102. In addition, the 5-HT(2C) receptor binds more strongly to PSD-95 and MPP-3 than the 5-HT(2A) receptor. In contrast, a robust interaction between the 5-HT(2A) receptor and the channel-interacting PDZ protein CIPP was found, whereas CIPP did not significantly associate with the 5-HT(2C) receptor. We also show that residues located at the -1 position and upstream the PDZ ligand in the C terminus of the 5-HT(2A) and 5-HT(2C) receptors are major determinants in their interaction with specific PDZ proteins. Immunofluorescence and electron microscopy studies strongly suggested that these specific interactions also take place in living cells and that the 5-HT(2) receptor-PDZ protein complexes occur in intracellular compartments. The interaction of the 5-HT(2A) and the 5-HT(2C) receptor with specific sets of PDZ proteins may contribute to their different signal transduction properties.

Animals↗

PubMed2004

Attenuated response to stress and novelty and hypersensitivity to seizures in 5-HT4 receptor knock-out mice.

To study the functions of 5-HT4 receptors, a null mutation was engineered in the corresponding gene. 5-HT4 receptor knock-out mice displayed normal feeding and motor behaviors in baseline conditions but abnormal feeding and locomotor behavior in response to stress and novelty. Specifically, stress-induced hypophagia and novelty-induced exploratory activity were attenuated in the knock-out mice. In addition, pentylenetetrazol-induced convulsive responses were enhanced in the knock-out mice, suggesting an increase in neuronal network excitability. These results provide the first example of a genetic deficit that disrupts the ability of stress to reduce feeding and body weight and suggest that 5-HT4 receptors may be involved in stress-induced anorexia and seizure susceptibility.

Animals↗

PubMed2004

GPCR interacting proteins (GIP).

G protein-coupled receptors (GPCR) interact not only with heterotrimeric G proteins but also with accessory proteins called GPCR interacting proteins (GIP). These proteins have important functions. They are implicated in GPCR targeting to specific cellular compartments, in their assembling into large functional complexes called "receptosomes," in their trafficking to and from the plasma membrane, and in the fine-tuning of their signaling properties. There are several types of GIPs. Some are transmembrane proteins such as another GPCR (homodimerization and heterodimerization), ionic channels, ionotropic receptors, and single transmembrane proteins. The latter is implicated in the fine-tuning of receptor pharmacology or signaling. Other GIPs are soluble proteins interacting mainly with the "magic" C-terminal tail. Among them, PDZ domain-containing proteins are the most abundant. They generally, but not always, interact with the extreme C-terminal domain of GPCRs. Some GIPs interact with specific sequences of the C-terminal such as the Homer binding sequence (-PPxxFR-), the dopamine receptor interacting protein (DRIP) binding sequence (-FxxxFxxxF-), etc. Finally, only few GIPs have been found thus far to interact with the third intracellular loop of GPCRs. The future will tell us if this situation is only due to technical reasons.

Animals↗

PubMed2004

GPCR-GIP networks: a first step in the discovery of new therapeutic drugs?

G protein-coupled receptors (GPCRs) are transmembrane molecules that, on interaction with G proteins upon ligand binding, can associate with a large variety of transmembrane or soluble proteins, termed 'GPCR-interacting proteins' (GIPs). Some special transmembrane GIPs are themselves GPCRs that form homo- or heterodimers, while other transmembrane GIPs are ionic channels, ionotropic receptors and single transmembrane proteins that control GPCR pharmacology and trafficking. Most soluble GIPs interact with the C-termini of GPCRs and often physically link GPCRs to large protein networks, called 'receptosomes', that include ionic channels, protein kinases, small G proteins, cytoskeletal proteins and adhesion molecules. Here, we review the nature and functions of some of these networks, such as the glutamate and serotonin receptosomes, and focus on the fine-tuning of GPCR signaling by GIPs. Finally, we discuss the possibilities for developing new therapeutic drugs capable of modulating GPCR signaling by disrupting or reinforcing specific GPCR-GIP interactions.

Animals↗

PubMed2003

The 5-hydroxytryptamine(1A) receptor is stably palmitoylated, and acylation is critical for communication of receptor with Gi protein.

In the present study, we verified that the mouse 5-hydroxytryptamine(1A) (5-HT(1A)) receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT(1A) receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal C-terminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT(1A) receptor acylation, we have analyzed the ability of acylation-deficient mutants to interact with heterotrimeric G(i) protein and to modulate downstream effectors. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with G(i) protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G alpha(i) subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT(1A) receptor is critical for the enabling of G(i) protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G beta gamma-mediated pathway, in addition to the G alpha(i)-mediated signaling.

Amino Acid Sequence↗

PubMed2003

The 'magic tail' of G protein-coupled receptors: an anchorage for functional protein networks.

All cell types express a great variety of G protein-coupled receptors (GPCRs) that are coupled to only a limited set of G proteins. This disposition favors cross-talk between transduction pathways. However, GPCRs are organized into functional units. They promote specificity and thus avoid unsuitable cross-talk. New methodologies (mostly yeast two-hybrid screens and proteomics) have been used to discover more than 50 GPCR-associated proteins that are involved in building these units. In addition, these protein networks participate in the trafficking, targeting, signaling, fine-tuning and allosteric regulation of GPCRs. To date, proteins that interact with the GPCR C-terminus are the most abundant and are the focus of this review.

Allosteric Regulation↗

PubMed2003

New benzo[h][1,6]naphthyridine and azepino[3,2-c]quinoline derivatives as selective antagonists of 5-HT4 receptors: binding profile and pharmacological characterization.

A series of benzo[h][1,6]naphthyridine and azepino[3,2-c]quinoline derivatives were prepared and evaluated to determine the necessary requirements for high affinity on the 5-HT(4) receptors and high selectivity versus other receptors. The compounds were synthesized by substituting the chlorine atom of benzonaphthyridines and azepinoquinolines with various N-alkyl-4-piperidinylmethanolates. They were evaluated in binding assays with [(3)H]GR 113808 as the 5-HT(4) receptor radioligand. The affinity values (K(i) or inhibition percentages) depended upon the substituent on the aromatic ring on one hand and the substituent on the lateral piperidine chain on the other hand. A chlorine atom produced a marked drop in activity while a N-propyl or N-butyl group gave compounds with nanomolar affinities (1 < K(i) < 10 nM). Among the most potent ligands (3a, 4a, 5a), 4a was selected on the basis of its high affinity and selectivity for pharmacological screening and was evaluated in vivo in specific tests. This compound reveals itself as an antagonist/low partial agonist in the COS-7 cells stably expressing the 5-HT(4(a)) receptor. Derivative 4a also showed in vivo potent analgesic activity in the writhing test at very low doses.

Animals↗

PubMed2002

A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors.

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002).

Journal Article↗

PubMed2002

A single mutation in the 5-HT4 receptor (5-HT4-R D100(3.32)A) generates a Gs-coupled receptor activated exclusively by synthetic ligands (RASSL).

To better understand G-protein-coupled receptor (GPCRs) signaling, cellular and animal physiology, as well as gene therapy, a new tool has recently been proposed. It consists of GPCR mutants that are insensitive to endogenous ligands but sensitive to synthetic ligands. These GPCRs are called receptor activated solely by synthetic ligands (RASSL). Only two examples of such engineered receptors have been described so far: one G(i)-coupled (opioid receptors) and one G(s)-coupled (beta(2)-adrenergic receptors). Here, we describe the first RASSL related to serotonin receptors (D100(3.32)A G(s)-coupled 5-HT(4) receptor or 5-HT(4)-RASSL). 5-HT(4)-RASSL is generated by a single mutation, is totally insensitive to serotonin (5-HT), and still responds to synthetic ligands. These ligands have affinities in the range of nanomolar concentrations for the mutant receptor and exhibit full efficacy. More interestingly, two synthetic ligands behave as antagonists on the wild type but as agonists on the 5-HT(4)-RASSL.

Animals↗

PubMed2002

Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach.

Membrane-bound receptors such as tyrosine kinases and ionotropic receptors are associated with large protein networks structured by protein-protein interactions involving multidomain proteins. Although these networks have emerged as a general mechanism of cellular signalling, much less is known about the protein complexes associated with G-protein-coupled receptors (GPCRs). Using a proteomic approach based on peptide affinity chromatography followed by mass spectrometry and immunoblotting, we have identified 15 proteins that interact with the C- terminal tail of the 5-hydroxytryptamine 2C (5-HT(2C)) receptor, a GPCR. These proteins include several synaptic multidomain proteins containing one or several PDZ domains (PSD95 and the proteins of the tripartite complex Veli3-CASK-Mint1), proteins of the actin/spectrin cytoskeleton and signalling proteins. Coimmunoprecipitation experiments showed that 5-HT(2C) receptors interact with PSD95 and the Veli3-CASK-Mint1 complex in vivo. Electron microscopy also indicated a synaptic enrichment of Veli3 and 5-HT(2C) receptors and their colocalization in microvilli of choroidal cells. These results indicate that the 5-HT(2C) receptor is associated with protein networks that are important for its synaptic localization and its coupling to the signalling machinery.

Amino Acid Sequence↗

PubMed2002

A 5-HT4 receptor transmembrane network implicated in the activity of inverse agonists but not agonists.

Activation of G protein-coupled receptors is thought to involve disruption of intramolecular interactions that stabilize their inactive conformation. Such disruptions are induced by agonists or by constitutively active mutations. In the present study, novel potent inverse agonists are described to inhibit the constitutive activity of 5-HT(4) receptors. Using these compounds and specific receptor mutations, we investigated the mechanisms by which inverse agonists may reverse the disruption of intramolecular interactions that causes constitutive activation. Two mutations (D100(3.32)A in transmembrane domain (TMD)-III and F275(6.51)A in TMD-VI) were found to completely block inverse agonist effects without impairing their binding properties nor the molecular activation switches induced by agonists. Based on the rhodopsin model, we propose that these mutated receptors are in equilibrium between two states R and R* but are unable to reach a third "silent" state stabilized by inverse agonists. We also found another mutation in TMD-VI (W272(6.48)A) that stabilized this silent state. This mutant remained fully activated by agonists. Molecular modeling indicated that Asp-100, Phe-275, and Trp-272 might constitute a network required for stabilization of the silent state by the described inverse agonists. However, this network is not necessary for agonist activity.

Animals↗