Michel Emerit - Academia.edu (original) (raw)

Papers by Michel Emerit

Research paper thumbnail of Targeting of the 5-HT1A Serotonin Receptor to Neuronal Dendrites Is Mediated by Yif1B

Journal of Neuroscience, 2008

The 5-HT 1A receptor (5-HT 1A R) is the most extensively characterized serotonin (5-HT) receptor ... more The 5-HT 1A receptor (5-HT 1A R) is the most extensively characterized serotonin (5-HT) receptor mainly because of its involvement in the mode of action of antidepressants. The 5-HT 1A R is confined to the somatodendritic domain of central neurons, where it mediates serotonin-evoked hyperpolarization. Our previous studies underlined the role of the short 5-HT 1A R C-terminal domain in receptor targeting to dendrites. We used this 17 aa region as bait in a yeast two-hybrid screen, and identified, for the first time, an intracellular protein interacting with the 5-HT 1A R. This protein is homologous to the yeast Yif1p, previously implicated in vesicular trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus, but not yet characterized in mammals. We confirmed 5-HT 1A R-Yif1B interaction by glutathione S-transferase pull-down experiments using rat brain extracts and transfected cell lines. Yif1B is highly expressed in the brain, and specifically in raphe 5-HT 1A R-expressing neurons. Colocalization of Yif1B and 5-HT 1A R was observed in small vesicles involved in transient intracellular trafficking. Last, inhibition of endogenous expression of Yif1B in primary neuron cultures by small interfering RNA specifically prevented the addressing of 5-HT 1A R to distal portions of the dendrites, without affecting other receptors, such as sst2A, P2X 2 , and 5-HT 3A receptors. Together, our results provide strong evidence that Yif1B is a member of the ER/Golgi trafficking machinery, which plays a key role in specific targeting of 5-HT 1A R to the neuronal dendrites. This finding opens up new pathways for the study of 5-HT 1A R regulation by partner proteins and for the development of novel antidepressant drugs.

Research paper thumbnail of Trophic Effects of Neurotransmitters during Brain Maturation

Neonatology, 1992

Besides their neurotransmitter and/or neuromodulatory roles, many neuroactive substances synthesi... more Besides their neurotransmitter and/or neuromodulatory roles, many neuroactive substances synthesized and released during brain development can also directly influence neuronal differentiation. Transitory expression of neurotransmitters, their metabolic enzymes and their receptors is only one aspect of this trophic role. The most considerable progress in neurotrophic factor research has been made with the use of primary cultures of neuronal cells, and numerous studies have focused on the effects of neurotransmitters on the differentiation of cells at various stages of development. Thus, several neuropeptides like VIP, substance P, enkephalins, somatostatin, and monoamines, can modulate neuronal differentiation, but only during a limited period of fetal life. Among the monoamines, it was shown that, depending on the target, 5-HT stimulates the development of the neuropile, the myelinization of axons, the differentiation of the synaptic contacts, induces markers of monoaminergic neuron differentiation, inhibits the development of the growth cone, decreases the branching of neurites, and influences the survival, cell body size, and neurite outgrowth in several neuronal cultures. 5-HT can also indirectly influence the differentiation of serotonergic neurons by the intermediate of astrocytes, and it was shown in our laboratory that 5-HT1A agonists can stimulate the cholinergic parameters of primary cultures of rat fetal septal neurons. At the molecular level, the events triggered by neurotransmitters that underlie their neurotrophic action probably involve the transmembrane influx of calcium. To date, calcium regulation of cellular processes is one of the most rapidly expanding areas of research in developmental neurobiology.

Research paper thumbnail of A 2(R43Q) Mutation, Linked to Epilepsy in Humans, Alters GABAA Receptor Assembly and Modifies Subunit Composition on the Cell Surface

J Biol Chem, 2006

... Cossette, P., Liu, L., Brisebois, K., Dong, H., Lortie, A., Vanasse, M., Saint-Hilaire, JM, C... more ... Cossette, P., Liu, L., Brisebois, K., Dong, H., Lortie, A., Vanasse, M., Saint-Hilaire, JM, Carmant, L., Verner, A., Lu, WY, Wang, YT, and Rouleau, GA (2002) Nat. Genet. 31, 184-189. ...

Research paper thumbnail of A New Mechanism of Receptor Targeting by Interaction between Two Classes of Ligand-Gated Ion Channels

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 3, 2016

The 5-HT3 receptors are serotonin-gated ion channels that physically couple with purinergic P2X2 ... more The 5-HT3 receptors are serotonin-gated ion channels that physically couple with purinergic P2X2 receptors to trigger a functional cross-inhibition leading to reciprocal channel occlusion. Although this functional receptor-receptor coupling seems to serve a modulatory role on both channels, this might not be its main physiological purpose. Using primary cultures of rat hippocampal neurons as a quantitative model of polarized targeting, we show here a novel function for this interaction. In this model, 5-HT3A receptors did not exhibit by themselves the capability of distal targeting in dendrites and axons but required the presence of P2X2R for their proper subcellular localization. 5-HT3AR distal targeting occurred with a delayed time course and exhibited a neuron phenotype dependency. In the subpopulation of neurons expressing endogenous P2X2R, 5-HT3AR distal neuritic localization correlated with P2X2R expression and could be selectively inhibited by P2X2R RNA interference. Cotransf...

Research paper thumbnail of Intracellular Cross Talk and Physical Interaction between Two Classes of Neurotransmitter-Gated Channels

The Journal of Neuroscience the Official Journal of the Society For Neuroscience, Feb 15, 2003

Fast chemical communications in the nervous system are mediated by several classes of receptor ch... more Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X 2 ATP-gated channels and 5-HT 3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X 2 and 5-HT 3 receptors is disrupted when the intracellular C-terminal domain of the P2X 2 receptor subunit is deleted and when minigenes coding for P2X 2 or 5-HT 3 A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X 2 receptors in myenteric neurons also disrupts the functional interaction between native P2X 2 and 5-HT 3 receptors. Therefore, activity-dependent intracellular coupling of distinct receptor channels underlies ionotropic cross talks that may significantly contribute to the regulation of neuronal excitability and synaptic plasticity.

Research paper thumbnail of Protein interactome mining defines melatonin MT1 receptors as integral component of presynaptic protein complexes of neurons

Journal of pineal research, Jan 29, 2015

In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak leve... more In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT1 and MT2 receptors, belonging to the G protein-coupled receptor (GPCR) super-family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT1 and MT2 receptors, which comprises 378 individual proteins. Among the proteins intera...

Research paper thumbnail of Boue-Grabot E, Barajas-Lopez C, Chakfe Y, et al. Intracellular cross talk and physical interaction between two classes of neurotransmitter-gated channels

The Journal of Neuroscience : The Official Journal of the Society for Neuroscience

Fast chemical communications in the nervous system are mediated by several classes of receptor ch... more Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X2 ATP-gated channels and 5-HT3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X2 and 5-HT3 receptors is disrupted when the intracellular C-terminal domain of the P2X2 receptor subunit is deleted and when minigenes coding for P2X2 or 5-HT3A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X2 receptors in myenteric neurons also disrupts the fun...

Research paper thumbnail of Yif1B is involved in the anterograde traffic pathway and the Golgi architecture

Traffic, 2015

Yif1B is an intracellular membrane-bound protein belonging to the Yip family, shown previously to... more Yif1B is an intracellular membrane-bound protein belonging to the Yip family, shown previously to control serotonin 5-HT1A receptor targeting to dendrites. Because some Yip proteins are involved in the intracellular traffic between the ER and the Golgi, here we investigated the precise localization of Yif1B in HeLa cells. We found that Yif1B is not resident into the Golgi, but rather belongs to the IC compartment. After analyzing the role of Yif1B in protein transport, we showed that the traffic of the VSVG protein marker was accelerated in Yif1B depleted HeLa cells, as well as in hippocampal neurons from Yif1B KO mice. Conversely, Yif1B depletion in HeLa cells did not change the retrograde traffic of ShTx. Interestingly, in long term depletion of Yif1B as in Yif1B KO mice, we observed a disorganized Golgi architecture in CA1 pyramidal hippocampal neurons, which was confirmed by electron microscopy. However, because short term depletion of Yif1B did not change Golgi structure, it is likely that the implication of Yif1B in anterograde traffic does not rely on its role in structural organization of the Golgi apparatus, but rather on its shuttling between the ER, the IC and the Golgi compartments.

Research paper thumbnail of Insights into Serotonin Receptor Trafficking: Cell Membrane Targeting and Internalization

Progress in Molecular Biology and Translational Science, 2015

Serotonin receptors (5-HTRs) mediate both central and peripheral control on numerous physiologica... more Serotonin receptors (5-HTRs) mediate both central and peripheral control on numerous physiological functions such as sleep/wake cycle, thermoregulation, food intake, nociception, locomotion, sexual behavior, gastrointestinal motility, blood coagulation, and cardiovascular homeostasis. Six families of the G-protein-coupled receptors comprise most of serotonin receptors besides the conserved 5-HT3R Cys-loop type which belongs to the family of Cys-loop ligand-gated cation channel receptors. Many of these receptors are targets of pharmaceutical drugs, justifying the importance for elucidating their coupling, signaling and functioning. Recently, special interest has been focused on their trafficking inside cell lines or neurons in conjunction with their interaction with partner proteins. In this review, we describe the trafficking of 5-HTRs including their internalization, desensitization, or addressing to the plasma membrane depending on specific mechanisms which are peculiar for each class of serotonin receptor.

Research paper thumbnail of Hippocampal Stimulation of Cholinergic Expression in Primary Cultures of Basal Forebrain Neurons

Receptor-Receptor Interactions, 1987

Research paper thumbnail of A new vesicular scaffolding complex mediates the G-protein-coupled 5-HT1A receptor targeting to neuronal dendrites

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 10, 2012

Although essential for their neuronal function, the molecular mechanisms underlying the dendritic... more Although essential for their neuronal function, the molecular mechanisms underlying the dendritic targeting of serotonin G-protein-coupled receptors are poorly understood. Here, we characterized a Yif1B-dependent vesicular scaffolding complex mediating the intracellular traffic of the rat 5-HT(1A) receptor (5-HT(1A)R) toward dendrites. By combining directed mutagenesis, GST-pull down, and surface plasmon resonance, we identified a tribasic motif in the C-tail of the 5-HT(1A)R on which Yif1B binds directly with high affinity (K(D) ≈ 37 nM). Moreover, we identified Yip1A, Rab6, and Kif5B as new partners of the 5-HT(1A)R/Yif1B complex, and showed that their expression in neurons is also crucial for the dendritic targeting of the 5-HT(1A)R. Live videomicroscopy revealed that 5-HT(1A)R, Yif1B, Yip1A, and Rab6 traffic in vesicles exiting the soma toward the dendritic tree, and also exhibit bidirectional motions, sustaining their role in 5-HT(1A)R dendritic targeting. Hence, we propose a n...

Research paper thumbnail of Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels

The Journal of biological chemistry, Jan 10, 2004

ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central syna... more ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We...

Research paper thumbnail of Cross-talk and co-trafficking between rho1/GABA receptors and ATP-gated channels

The Journal of biological chemistry, Jan 20, 2004

Gamma-aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functi... more Gamma-aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory rho1/GABA and the excitatory P2X2 receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X2 is deleted or in competition experiments with minigenes coding for the C-terminal domain of P2X2 or the main intracellular loop of rho1 subunits. We also show a physical interaction between P2X2 and rho1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression wi...

Research paper thumbnail of Intracellular cross talk and physical interaction between two classes of neurotransmitter-gated channels

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2003

Fast chemical communications in the nervous system are mediated by several classes of receptor ch... more Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X2 ATP-gated channels and 5-HT3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X2 and 5-HT3 receptors is disrupted when the intracellular C-terminal domain of the P2X2 receptor subunit is deleted and when minigenes coding for P2X2 or 5-HT3A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X2 receptors in myenteric neurons also disrupts the fun...

Research paper thumbnail of Modulation of 5-HT3 receptor desensitization by the light chain of microtubule-associated protein 1B expressed in HEK 293 cells

The Journal of physiology, 2008

Regulation of ligand-gated ion channel (LGIC) function and trafficking by cytoskeleton proteins h... more Regulation of ligand-gated ion channel (LGIC) function and trafficking by cytoskeleton proteins has been the topic of recent research. Here, we report that the light chain (LC1) of microtubule-associated protein 1B (MAP1B) specifically interacted with the 5-HT(3A) receptor, a predominant serotonin-gated ion channel in the brain. LC1 and 5-HT(3A) receptors were colocalized in central neurons and in HEK 293 cells expressing 5-HT(3A) receptors. LC1 reduced the steady-state density of 5-HT(3A) receptors at the membrane surface of HEK 293 cells and significantly accelerated receptor desensitization time constants from 3.8 +/- 0.3 s to 0.8 +/- 0.1 s. However, LC1 did not significantly alter agonist binding affinity and single-channel conductance of 5-HT(3A) receptors. On the other hand, application of specific LC1 antisense oligonucleotides and nocodazole, a microtubule disruptor, significantly prolonged the desensitization time of the recombinant and native neuronal 5-HT(3) receptors by ...

Research paper thumbnail of Serotonergic signaling: multiple effectors and pleiotropic effects

Wiley Interdisciplinary Reviews: Membrane Transport and Signaling, 2012

ABSTRACT

Research paper thumbnail of Neuronal Phenotype Dependency of Agonist-Induced Internalization of the 5-HT1A Serotonin Receptor

Journal of Neuroscience, 2014

Selective serotonin reuptake inhibitors (SSRI) are aimed at increasing brain 5-HT tone; however, ... more Selective serotonin reuptake inhibitors (SSRI) are aimed at increasing brain 5-HT tone; however, this expected effect has a slow onset after starting SSRI treatment because of initial activation of 5-HT(1A) autoreceptor-mediated negative feedback of 5-HT release. After chronic SSRI treatment, 5-HT(1A) autoreceptors desensitize, which allows 5-HT tone elevation. Because 5-HT(1A) receptor (5-HT(1A)R) internalization has been proposed as a possible mechanism underlying 5-HT(1A) autoreceptor desensitization, we examined whether this receptor could internalize under well controlled in vitro conditions in the LLC-CPK1 cell line and in raphe or hippocampal neurons from rat embryos. To this goal, cells were transfected with recombinant lentiviral vectors encoding N-terminal tagged 5-HT(1A)R, and exposed to various pharmacological conditions. Constitutive endocytosis and plasma membrane recycling of tagged-5-HT(1A)R was observed in LLC-PK1 cells as well as in neurons. Acute exposure (for 1 h) to the full 5-HT(1A)R agonists, 5-HT and 5-carboxamido-tryptamine, but not the partial agonist 8-OH-DPAT, triggered internalization of tagged 5-HT(1A)R in serotonergic neurons only. In contrast, sustained exposure (for 24 h) to all agonists induced tagged-5-HT(1A)R endocytosis in raphe serotonergic neurons and a portion of hippocampal neurons, but not LLC-PK1 cells and partial agonist displayed an effect only in serotonergic neurons. In all cases, agonist-induced tagged 5-HT(1A)R endocytosis was prevented by the 5-HT(1A)R antagonist, WAY-100635, which was inactive on its own. These data showed that agonist-induced 5-HT(1A)R internalization does exist in neurons and depends on agonist efficacy and neuronal phenotype. Its differential occurrence in serotonergic neurons supports the idea that 5-HT(1A)R internalization might underlie 5-HT(1A) autoreceptor desensitization under SSRI antidepressant therapy.

Research paper thumbnail of Native and Cloned 5-HT3A(S) Receptors Are Anchored to F-Actin in Clonal Cells and Neurons

Molecular and Cellular Neuroscience, 2002

Using selective antibodies to visualize the short isoform of the 5-HT 3A receptor, we report here... more Using selective antibodies to visualize the short isoform of the 5-HT 3A receptor, we report here that both native and cloned 5-HT 3A (S) receptors formed clusters associated with F-actin in all cell types studied. NG 108-15 cells expressing native 5-HT 3A (S) receptors, COS-7 cells transiently expressing 5-HT 3A (S) subunits, and CHO cells stably transfected with a plasmid encoding the 5-HT 3A (S) sequence all exhibited similar surface receptor topology with 5-HT 3A (S) receptor cluster accumulation in F-actinrich lamellipodia and microspikes. Colocalization and coclustering of 5-HT 3A (S) subunits and F-actin were also observed in transfected hippocampal neurons. Treatment of the neurons with latrunculin-A, a compound altering F-actin polymerization, demonstrated that 5-HT 3A (S) receptor cluster size and topology were dependent on Factin integrity. These results suggest that the anchoring of 5-HT 3A (S) receptor clusters to the cytoskeletal network probably plays a key role in the physiological regulation of the receptor topology and dynamics, as is the case for other members of the 4-TMD ion channel receptor family.

Research paper thumbnail of Hippocampal Membranes Contain a Neurotrophic Activity That Stimulates Cholinergic Properties of Fetal Rat Septal Neurons Cultured Under Serum-Free Conditions

Journal of Neurochemistry, 1989

Primary cultures of fetal rat septal neurons were used to identify a membrane-associated choliner... more Primary cultures of fetal rat septal neurons were used to identify a membrane-associated cholinergic neurotrophic activity. Under serum-free culture conditions, -98% of the septal cells are neurons, and -6% of the neurons are cholinergic as determined immunocytochemically . Crude

Research paper thumbnail of Subunit-specific Coupling between  -Aminobutyric Acid Type A and P2X2 Receptor Channels

Journal of Biological Chemistry, 2004

ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central syna... more ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We previously demonstrated functional and physical cross-talk between rho1 and P2X2 receptors, which induced a retargeting of rho1 channels to surface clusters when co-expressed in hippocampal neurons (Boue-Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X2 and chimeric rho1 receptors with the C-terminal sequences of alpha2, beta3, or gamma2 subunits indicated that only rho1-beta3 and P2X2 channels exhibit both functional cross-inhibition in Xenopus oocytes and co-clustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X2 and the intracellular loop of beta GABAA subunits are required for the functional interaction between ATP- and GABA-gated channels. This gamma subunit-dependent cross-talk may contribute to the regulation of synaptic activity.

Research paper thumbnail of Targeting of the 5-HT1A Serotonin Receptor to Neuronal Dendrites Is Mediated by Yif1B

Journal of Neuroscience, 2008

The 5-HT 1A receptor (5-HT 1A R) is the most extensively characterized serotonin (5-HT) receptor ... more The 5-HT 1A receptor (5-HT 1A R) is the most extensively characterized serotonin (5-HT) receptor mainly because of its involvement in the mode of action of antidepressants. The 5-HT 1A R is confined to the somatodendritic domain of central neurons, where it mediates serotonin-evoked hyperpolarization. Our previous studies underlined the role of the short 5-HT 1A R C-terminal domain in receptor targeting to dendrites. We used this 17 aa region as bait in a yeast two-hybrid screen, and identified, for the first time, an intracellular protein interacting with the 5-HT 1A R. This protein is homologous to the yeast Yif1p, previously implicated in vesicular trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus, but not yet characterized in mammals. We confirmed 5-HT 1A R-Yif1B interaction by glutathione S-transferase pull-down experiments using rat brain extracts and transfected cell lines. Yif1B is highly expressed in the brain, and specifically in raphe 5-HT 1A R-expressing neurons. Colocalization of Yif1B and 5-HT 1A R was observed in small vesicles involved in transient intracellular trafficking. Last, inhibition of endogenous expression of Yif1B in primary neuron cultures by small interfering RNA specifically prevented the addressing of 5-HT 1A R to distal portions of the dendrites, without affecting other receptors, such as sst2A, P2X 2 , and 5-HT 3A receptors. Together, our results provide strong evidence that Yif1B is a member of the ER/Golgi trafficking machinery, which plays a key role in specific targeting of 5-HT 1A R to the neuronal dendrites. This finding opens up new pathways for the study of 5-HT 1A R regulation by partner proteins and for the development of novel antidepressant drugs.

Research paper thumbnail of Trophic Effects of Neurotransmitters during Brain Maturation

Neonatology, 1992

Besides their neurotransmitter and/or neuromodulatory roles, many neuroactive substances synthesi... more Besides their neurotransmitter and/or neuromodulatory roles, many neuroactive substances synthesized and released during brain development can also directly influence neuronal differentiation. Transitory expression of neurotransmitters, their metabolic enzymes and their receptors is only one aspect of this trophic role. The most considerable progress in neurotrophic factor research has been made with the use of primary cultures of neuronal cells, and numerous studies have focused on the effects of neurotransmitters on the differentiation of cells at various stages of development. Thus, several neuropeptides like VIP, substance P, enkephalins, somatostatin, and monoamines, can modulate neuronal differentiation, but only during a limited period of fetal life. Among the monoamines, it was shown that, depending on the target, 5-HT stimulates the development of the neuropile, the myelinization of axons, the differentiation of the synaptic contacts, induces markers of monoaminergic neuron differentiation, inhibits the development of the growth cone, decreases the branching of neurites, and influences the survival, cell body size, and neurite outgrowth in several neuronal cultures. 5-HT can also indirectly influence the differentiation of serotonergic neurons by the intermediate of astrocytes, and it was shown in our laboratory that 5-HT1A agonists can stimulate the cholinergic parameters of primary cultures of rat fetal septal neurons. At the molecular level, the events triggered by neurotransmitters that underlie their neurotrophic action probably involve the transmembrane influx of calcium. To date, calcium regulation of cellular processes is one of the most rapidly expanding areas of research in developmental neurobiology.

Research paper thumbnail of A 2(R43Q) Mutation, Linked to Epilepsy in Humans, Alters GABAA Receptor Assembly and Modifies Subunit Composition on the Cell Surface

J Biol Chem, 2006

... Cossette, P., Liu, L., Brisebois, K., Dong, H., Lortie, A., Vanasse, M., Saint-Hilaire, JM, C... more ... Cossette, P., Liu, L., Brisebois, K., Dong, H., Lortie, A., Vanasse, M., Saint-Hilaire, JM, Carmant, L., Verner, A., Lu, WY, Wang, YT, and Rouleau, GA (2002) Nat. Genet. 31, 184-189. ...

Research paper thumbnail of A New Mechanism of Receptor Targeting by Interaction between Two Classes of Ligand-Gated Ion Channels

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 3, 2016

The 5-HT3 receptors are serotonin-gated ion channels that physically couple with purinergic P2X2 ... more The 5-HT3 receptors are serotonin-gated ion channels that physically couple with purinergic P2X2 receptors to trigger a functional cross-inhibition leading to reciprocal channel occlusion. Although this functional receptor-receptor coupling seems to serve a modulatory role on both channels, this might not be its main physiological purpose. Using primary cultures of rat hippocampal neurons as a quantitative model of polarized targeting, we show here a novel function for this interaction. In this model, 5-HT3A receptors did not exhibit by themselves the capability of distal targeting in dendrites and axons but required the presence of P2X2R for their proper subcellular localization. 5-HT3AR distal targeting occurred with a delayed time course and exhibited a neuron phenotype dependency. In the subpopulation of neurons expressing endogenous P2X2R, 5-HT3AR distal neuritic localization correlated with P2X2R expression and could be selectively inhibited by P2X2R RNA interference. Cotransf...

Research paper thumbnail of Intracellular Cross Talk and Physical Interaction between Two Classes of Neurotransmitter-Gated Channels

The Journal of Neuroscience the Official Journal of the Society For Neuroscience, Feb 15, 2003

Fast chemical communications in the nervous system are mediated by several classes of receptor ch... more Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X 2 ATP-gated channels and 5-HT 3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X 2 and 5-HT 3 receptors is disrupted when the intracellular C-terminal domain of the P2X 2 receptor subunit is deleted and when minigenes coding for P2X 2 or 5-HT 3 A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X 2 receptors in myenteric neurons also disrupts the functional interaction between native P2X 2 and 5-HT 3 receptors. Therefore, activity-dependent intracellular coupling of distinct receptor channels underlies ionotropic cross talks that may significantly contribute to the regulation of neuronal excitability and synaptic plasticity.

Research paper thumbnail of Protein interactome mining defines melatonin MT1 receptors as integral component of presynaptic protein complexes of neurons

Journal of pineal research, Jan 29, 2015

In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak leve... more In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT1 and MT2 receptors, belonging to the G protein-coupled receptor (GPCR) super-family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT1 and MT2 receptors, which comprises 378 individual proteins. Among the proteins intera...

Research paper thumbnail of Boue-Grabot E, Barajas-Lopez C, Chakfe Y, et al. Intracellular cross talk and physical interaction between two classes of neurotransmitter-gated channels

The Journal of Neuroscience : The Official Journal of the Society for Neuroscience

Fast chemical communications in the nervous system are mediated by several classes of receptor ch... more Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X2 ATP-gated channels and 5-HT3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X2 and 5-HT3 receptors is disrupted when the intracellular C-terminal domain of the P2X2 receptor subunit is deleted and when minigenes coding for P2X2 or 5-HT3A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X2 receptors in myenteric neurons also disrupts the fun...

Research paper thumbnail of Yif1B is involved in the anterograde traffic pathway and the Golgi architecture

Traffic, 2015

Yif1B is an intracellular membrane-bound protein belonging to the Yip family, shown previously to... more Yif1B is an intracellular membrane-bound protein belonging to the Yip family, shown previously to control serotonin 5-HT1A receptor targeting to dendrites. Because some Yip proteins are involved in the intracellular traffic between the ER and the Golgi, here we investigated the precise localization of Yif1B in HeLa cells. We found that Yif1B is not resident into the Golgi, but rather belongs to the IC compartment. After analyzing the role of Yif1B in protein transport, we showed that the traffic of the VSVG protein marker was accelerated in Yif1B depleted HeLa cells, as well as in hippocampal neurons from Yif1B KO mice. Conversely, Yif1B depletion in HeLa cells did not change the retrograde traffic of ShTx. Interestingly, in long term depletion of Yif1B as in Yif1B KO mice, we observed a disorganized Golgi architecture in CA1 pyramidal hippocampal neurons, which was confirmed by electron microscopy. However, because short term depletion of Yif1B did not change Golgi structure, it is likely that the implication of Yif1B in anterograde traffic does not rely on its role in structural organization of the Golgi apparatus, but rather on its shuttling between the ER, the IC and the Golgi compartments.

Research paper thumbnail of Insights into Serotonin Receptor Trafficking: Cell Membrane Targeting and Internalization

Progress in Molecular Biology and Translational Science, 2015

Serotonin receptors (5-HTRs) mediate both central and peripheral control on numerous physiologica... more Serotonin receptors (5-HTRs) mediate both central and peripheral control on numerous physiological functions such as sleep/wake cycle, thermoregulation, food intake, nociception, locomotion, sexual behavior, gastrointestinal motility, blood coagulation, and cardiovascular homeostasis. Six families of the G-protein-coupled receptors comprise most of serotonin receptors besides the conserved 5-HT3R Cys-loop type which belongs to the family of Cys-loop ligand-gated cation channel receptors. Many of these receptors are targets of pharmaceutical drugs, justifying the importance for elucidating their coupling, signaling and functioning. Recently, special interest has been focused on their trafficking inside cell lines or neurons in conjunction with their interaction with partner proteins. In this review, we describe the trafficking of 5-HTRs including their internalization, desensitization, or addressing to the plasma membrane depending on specific mechanisms which are peculiar for each class of serotonin receptor.

Research paper thumbnail of Hippocampal Stimulation of Cholinergic Expression in Primary Cultures of Basal Forebrain Neurons

Receptor-Receptor Interactions, 1987

Research paper thumbnail of A new vesicular scaffolding complex mediates the G-protein-coupled 5-HT1A receptor targeting to neuronal dendrites

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 10, 2012

Although essential for their neuronal function, the molecular mechanisms underlying the dendritic... more Although essential for their neuronal function, the molecular mechanisms underlying the dendritic targeting of serotonin G-protein-coupled receptors are poorly understood. Here, we characterized a Yif1B-dependent vesicular scaffolding complex mediating the intracellular traffic of the rat 5-HT(1A) receptor (5-HT(1A)R) toward dendrites. By combining directed mutagenesis, GST-pull down, and surface plasmon resonance, we identified a tribasic motif in the C-tail of the 5-HT(1A)R on which Yif1B binds directly with high affinity (K(D) ≈ 37 nM). Moreover, we identified Yip1A, Rab6, and Kif5B as new partners of the 5-HT(1A)R/Yif1B complex, and showed that their expression in neurons is also crucial for the dendritic targeting of the 5-HT(1A)R. Live videomicroscopy revealed that 5-HT(1A)R, Yif1B, Yip1A, and Rab6 traffic in vesicles exiting the soma toward the dendritic tree, and also exhibit bidirectional motions, sustaining their role in 5-HT(1A)R dendritic targeting. Hence, we propose a n...

Research paper thumbnail of Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels

The Journal of biological chemistry, Jan 10, 2004

ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central syna... more ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We...

Research paper thumbnail of Cross-talk and co-trafficking between rho1/GABA receptors and ATP-gated channels

The Journal of biological chemistry, Jan 20, 2004

Gamma-aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functi... more Gamma-aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory rho1/GABA and the excitatory P2X2 receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X2 is deleted or in competition experiments with minigenes coding for the C-terminal domain of P2X2 or the main intracellular loop of rho1 subunits. We also show a physical interaction between P2X2 and rho1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression wi...

Research paper thumbnail of Intracellular cross talk and physical interaction between two classes of neurotransmitter-gated channels

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2003

Fast chemical communications in the nervous system are mediated by several classes of receptor ch... more Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X2 ATP-gated channels and 5-HT3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X2 and 5-HT3 receptors is disrupted when the intracellular C-terminal domain of the P2X2 receptor subunit is deleted and when minigenes coding for P2X2 or 5-HT3A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X2 receptors in myenteric neurons also disrupts the fun...

Research paper thumbnail of Modulation of 5-HT3 receptor desensitization by the light chain of microtubule-associated protein 1B expressed in HEK 293 cells

The Journal of physiology, 2008

Regulation of ligand-gated ion channel (LGIC) function and trafficking by cytoskeleton proteins h... more Regulation of ligand-gated ion channel (LGIC) function and trafficking by cytoskeleton proteins has been the topic of recent research. Here, we report that the light chain (LC1) of microtubule-associated protein 1B (MAP1B) specifically interacted with the 5-HT(3A) receptor, a predominant serotonin-gated ion channel in the brain. LC1 and 5-HT(3A) receptors were colocalized in central neurons and in HEK 293 cells expressing 5-HT(3A) receptors. LC1 reduced the steady-state density of 5-HT(3A) receptors at the membrane surface of HEK 293 cells and significantly accelerated receptor desensitization time constants from 3.8 +/- 0.3 s to 0.8 +/- 0.1 s. However, LC1 did not significantly alter agonist binding affinity and single-channel conductance of 5-HT(3A) receptors. On the other hand, application of specific LC1 antisense oligonucleotides and nocodazole, a microtubule disruptor, significantly prolonged the desensitization time of the recombinant and native neuronal 5-HT(3) receptors by ...

Research paper thumbnail of Serotonergic signaling: multiple effectors and pleiotropic effects

Wiley Interdisciplinary Reviews: Membrane Transport and Signaling, 2012

ABSTRACT

Research paper thumbnail of Neuronal Phenotype Dependency of Agonist-Induced Internalization of the 5-HT1A Serotonin Receptor

Journal of Neuroscience, 2014

Selective serotonin reuptake inhibitors (SSRI) are aimed at increasing brain 5-HT tone; however, ... more Selective serotonin reuptake inhibitors (SSRI) are aimed at increasing brain 5-HT tone; however, this expected effect has a slow onset after starting SSRI treatment because of initial activation of 5-HT(1A) autoreceptor-mediated negative feedback of 5-HT release. After chronic SSRI treatment, 5-HT(1A) autoreceptors desensitize, which allows 5-HT tone elevation. Because 5-HT(1A) receptor (5-HT(1A)R) internalization has been proposed as a possible mechanism underlying 5-HT(1A) autoreceptor desensitization, we examined whether this receptor could internalize under well controlled in vitro conditions in the LLC-CPK1 cell line and in raphe or hippocampal neurons from rat embryos. To this goal, cells were transfected with recombinant lentiviral vectors encoding N-terminal tagged 5-HT(1A)R, and exposed to various pharmacological conditions. Constitutive endocytosis and plasma membrane recycling of tagged-5-HT(1A)R was observed in LLC-PK1 cells as well as in neurons. Acute exposure (for 1 h) to the full 5-HT(1A)R agonists, 5-HT and 5-carboxamido-tryptamine, but not the partial agonist 8-OH-DPAT, triggered internalization of tagged 5-HT(1A)R in serotonergic neurons only. In contrast, sustained exposure (for 24 h) to all agonists induced tagged-5-HT(1A)R endocytosis in raphe serotonergic neurons and a portion of hippocampal neurons, but not LLC-PK1 cells and partial agonist displayed an effect only in serotonergic neurons. In all cases, agonist-induced tagged 5-HT(1A)R endocytosis was prevented by the 5-HT(1A)R antagonist, WAY-100635, which was inactive on its own. These data showed that agonist-induced 5-HT(1A)R internalization does exist in neurons and depends on agonist efficacy and neuronal phenotype. Its differential occurrence in serotonergic neurons supports the idea that 5-HT(1A)R internalization might underlie 5-HT(1A) autoreceptor desensitization under SSRI antidepressant therapy.

Research paper thumbnail of Native and Cloned 5-HT3A(S) Receptors Are Anchored to F-Actin in Clonal Cells and Neurons

Molecular and Cellular Neuroscience, 2002

Using selective antibodies to visualize the short isoform of the 5-HT 3A receptor, we report here... more Using selective antibodies to visualize the short isoform of the 5-HT 3A receptor, we report here that both native and cloned 5-HT 3A (S) receptors formed clusters associated with F-actin in all cell types studied. NG 108-15 cells expressing native 5-HT 3A (S) receptors, COS-7 cells transiently expressing 5-HT 3A (S) subunits, and CHO cells stably transfected with a plasmid encoding the 5-HT 3A (S) sequence all exhibited similar surface receptor topology with 5-HT 3A (S) receptor cluster accumulation in F-actinrich lamellipodia and microspikes. Colocalization and coclustering of 5-HT 3A (S) subunits and F-actin were also observed in transfected hippocampal neurons. Treatment of the neurons with latrunculin-A, a compound altering F-actin polymerization, demonstrated that 5-HT 3A (S) receptor cluster size and topology were dependent on Factin integrity. These results suggest that the anchoring of 5-HT 3A (S) receptor clusters to the cytoskeletal network probably plays a key role in the physiological regulation of the receptor topology and dynamics, as is the case for other members of the 4-TMD ion channel receptor family.

Research paper thumbnail of Hippocampal Membranes Contain a Neurotrophic Activity That Stimulates Cholinergic Properties of Fetal Rat Septal Neurons Cultured Under Serum-Free Conditions

Journal of Neurochemistry, 1989

Primary cultures of fetal rat septal neurons were used to identify a membrane-associated choliner... more Primary cultures of fetal rat septal neurons were used to identify a membrane-associated cholinergic neurotrophic activity. Under serum-free culture conditions, -98% of the septal cells are neurons, and -6% of the neurons are cholinergic as determined immunocytochemically . Crude

Research paper thumbnail of Subunit-specific Coupling between  -Aminobutyric Acid Type A and P2X2 Receptor Channels

Journal of Biological Chemistry, 2004

ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central syna... more ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We previously demonstrated functional and physical cross-talk between rho1 and P2X2 receptors, which induced a retargeting of rho1 channels to surface clusters when co-expressed in hippocampal neurons (Boue-Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X2 and chimeric rho1 receptors with the C-terminal sequences of alpha2, beta3, or gamma2 subunits indicated that only rho1-beta3 and P2X2 channels exhibit both functional cross-inhibition in Xenopus oocytes and co-clustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X2 and the intracellular loop of beta GABAA subunits are required for the functional interaction between ATP- and GABA-gated channels. This gamma subunit-dependent cross-talk may contribute to the regulation of synaptic activity.