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1.  Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser280 by Hallucinogenic versus Nonhallucinogenic Agonists* 
The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT2A receptor in response to hallucinogenic versus nonhallucinogenic agonists, which underlies their distinct capacity to desensitize the receptor.
doi:10.1074/mcp.M113.036558
PMCID: PMC4014284  PMID: 24637012
2.  5-HT4 Receptors Constitutively Promote the Non-Amyloidogenic Pathway of APP Cleavage and Interact with ADAM10 
ACS Chemical Neuroscience  2012;4(1):130-140.
In addition to the amyloidogenic pathway, amyloid precursor protein (APP) can be cleaved by α-secretases, producing soluble and neuroprotective APP alpha (sAPPα) (nonamyloidogenic pathway) and thus preventing the generation of pathogenic amyloid-β. However, the mechanisms regulating APP cleavage by α-secretases remain poorly understood. Here, we showed that expression of serotonin type 4 receptors (5-HT4Rs) constitutively (without agonist stimulation) induced APP cleavage by the α-secretase ADAM10 and the release of neuroprotective sAPPα in HEK-293 cells and cortical neurons. This effect was independent of cAMP production. Interestingly, we demonstrated that 5-HT4 receptors physically interacted with the mature form of ADAM10. Stimulation of 5-HT4 receptors by an agonist further increased sAPPα secretion, and this effect was mediated by cAMP/Epac signaling. These findings describe a new mechanism whereby a GPCR constitutively stimulates the cleavage of APP by α-secretase and promotes the nonamyloidogenic pathway of APP processing.
doi:10.1021/cn300095t
PMCID: PMC3547471  PMID: 23336052
Alpha-secretase; Alzheimer’s disease; sAPP alpha; serotonin
3.  Gαo Is Required for L-Canavanine Detection in Drosophila 
PLoS ONE  2013;8(5):e63484.
Taste is an essential sense for the survival of most organisms. In insects, taste is particularly important as it allows to detect and avoid ingesting many plant toxins, such as L-canavanine. We previously showed that L-canavanine is toxic for Drosophila melanogaster and that flies are able to detect this toxin in the food. L-canavanine is a ligand of DmXR, a variant G-protein coupled receptor (GPCR) belonging to the metabotropic glutamate receptor subfamily that is expressed in bitter-sensitive taste neurons of Drosophila. To transduce the signal intracellularly, GPCR activate heterotrimeric G proteins constituted of α, β and γ subunits. The aim of this study was to identify which Gα protein was required for L-canavanine detection in Drosophila. By using a pharmacological approach, we first demonstrated that DmXR has the best coupling with Gαo protein subtype. Then, by using genetic, behavioral assays and electrophysiology, we found that Gαo47A is required in bitter-sensitive taste neurons for L-canavanine sensitivity. In conclusion, our study revealed that Gαo47A plays a crucial role in L-canavanine detection.
doi:10.1371/journal.pone.0063484
PMCID: PMC3646046  PMID: 23671680
4.  Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability 
The Journal of Cell Biology  2012;198(2):251-263.
Synaptic activity–dependent remodeling of the glutamate receptor scaffold complex generates a negative feedback loop that limits further NMDA receptor activation.
Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor–mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.
doi:10.1083/jcb.201110101
PMCID: PMC3410417  PMID: 22801779
5.  Early administration of RS 67333, a specific 5-HT4 receptor agonist, prevents amyloidogenesis and behavioral deficits in the 5XFAD mouse model of Alzheimer’s disease 
Amyloid β (Aβ) accumulation is considered the main culprit in the pathogenesis of Alzheimer’s disease (AD). Recent studies suggest that decreasing Aβ production at very early stages of AD could be a promising strategy to slow down disease progression. Serotonin 5-HT4 receptor activation stimulates α-cleavage of the amyloid precursor protein (APP), leading to the release of the soluble and neurotrophic sAPPα fragment and thus precluding Aβ formation. Using the 5XFAD mouse model of AD that shows accelerated Aβ deposition, we investigated the effect of chronic treatments (treatment onset at different ages and different durations) with the 5-HT4 receptor agonist RS 67333 during the asymptomatic phase of the disease. Chronic administration of RS 67333 decreased concomitantly the number of amyloid plaques and the level of Aβ species. Reduction of Aβ levels was accompanied by a striking decrease in hippocampal astrogliosis and microgliosis. RS 67333 also transiently increased sAPPα concentration in the cerebrospinal fluid and brain. Moreover, a specific 5-HT4 receptor antagonist (RS 39604) prevented the RS 67333-mediated reduction of the amyloid pathology. Finally, the novel object recognition test deficits of 5XFAD mice were reversed by chronic treatment with RS 67333. Collectively, these results strongly highlight this 5-HT4 receptor agonist as a promising disease modifying-agent for AD.
doi:10.3389/fnagi.2013.00096
PMCID: PMC3871961  PMID: 24399967
alpha-secretase; sAPP alpha; serotonin; amyloid plaques; preventive pharmacotherapy; G protein-coupled receptor
6.  5-HT6 receptor recruitment of mTOR as a mechanism for perturbed cognition in schizophrenia 
EMBO Molecular Medicine  2012;4(10):1043-1056.
Cognitive deficits in schizophrenia severely compromise quality of life and are poorly controlled by current antipsychotics. While 5-HT6 receptor blockade holds special promise, molecular substrates underlying their control of cognition remain unclear. Using a proteomic strategy, we show that 5-HT6 receptors physically interact with several proteins of the mammalian target of rapamycin (mTOR) pathway, including mTOR. Further, 5-HT6 receptor activation increased mTOR signalling in rodent prefrontal cortex (PFC). Linking this signalling event to cognitive impairment, the mTOR inhibitor rapamycin prevented deficits in social cognition and novel object discrimination induced by 5-HT6 agonists. In two developmental models of schizophrenia, specifically neonatal phencyclidine treatment and post-weaning isolation rearing, the activity of mTOR was enhanced in the PFC, and rapamycin, like 5-HT6 antagonists, reversed these cognitive deficits. These observations suggest that recruitment of mTOR by prefrontal 5-HT6 receptors contributes to the perturbed cognition in schizophrenia, offering new vistas for its therapeutic control.
doi:10.1002/emmm.201201410
PMCID: PMC3491835  PMID: 23027611
5-HT6 receptor; cognition; mTORC1; proteomics; schizophrenia
7.  Systems medicine and integrated care to combat chronic noncommunicable diseases 
Genome Medicine  2011;3(7):43.
We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems.
doi:10.1186/gm259
PMCID: PMC3221551  PMID: 21745417
8.  Homer1a-Dependent Crosstalk Between NMDA and Metabotropic Glutamate Receptors in Mouse Neurons 
PLoS ONE  2010;5(3):e9755.
Background
A large number of evidences suggest that group-I metabotropic glutamate receptors (mGluR1a, 1b, 1c, 5a, 5b) can modulate NMDA receptor activity. Interestingly, a physical link exists between these receptors through a Homer-Shank multi-protein scaffold that can be disrupted by the immediate early gene, Homer1a. Whether such a versatile link supports functional crosstalk between the receptors is unknown.
Methodology/Principal Findings
Here we used biochemical, electrophysiological and molecular biological approaches in cultured mouse cerebellar neurons to investigate this issue. We found that Homer1a or dominant negative Shank3 mutants that disrupt the physical link between the receptors allow inhibition of NMDA current by group-I mGluR agonist. This effect is antagonized by pertussis toxin, but not thapsigargin, suggesting the involvement of a G protein, but not intracellular calcium stores. Also, this effect is voltage-sensitive, being present at negative, but not positive membrane potentials. In the presence of DHPG, an apparent NMDA “tail current” was evoked by large pulse depolarization, only in neurons transfected with Homer1a. Co-immunoprecipitation experiments showed interaction between G-protein βγ subunits and NMDA receptor in the presence of Homer1a and group-I mGluR agonist.
Conclusions/Significance
Altogether these results suggest a direct inhibition of NMDA receptor-channel by Gbetagamma subunits, following disruption of the Homer-Shank3 complex by the immediate early gene Homer1a. This study provides a new molecular mechanism by which group-I mGluRs could dynamically regulate NMDA receptor function.
doi:10.1371/journal.pone.0009755
PMCID: PMC2841198  PMID: 20305784
9.  Hyperfunction of Muscarinic Receptor Maintains Long-Term Memory in 5-HT4 Receptor Knock-Out Mice 
PLoS ONE  2010;5(3):e9529.
Patients suffering from dementia of Alzheimer's type express less serotonin 4 receptors (5-HTR4), but whether an absence of these receptors modifies learning and memory is unexplored. In the spatial version of the Morris water maze, we show that 5-HTR4 knock-out (KO) and wild-type (WT) mice performed similarly for spatial learning, short- and long-term retention. Since 5-HTR4 control mnesic abilities, we tested whether cholinergic system had circumvented the absence of 5-HTR4. Inactivating muscarinic receptor with scopolamine, at an ineffective dose (0.8 mg/kg) to alter memory in WT mice, decreased long-term but not short-term memory of 5-HTR4 KO mice. Other changes included decreases in the activity of choline acetyltransferase (ChAT), the required enzyme for acetylcholine synthesis, in the septum and the dorsal hippocampus in 5-HTR4 KO under baseline conditions. Training- and scopolamine-induced increase and decrease, respectively in ChAT activity in the septum in WT mice were not detected in the 5-HTR4 KO animals. Findings suggest that adaptive changes in cholinergic systems may circumvent the absence of 5-HTR4 to maintain long-term memory under baseline conditions. In contrast, despite adaptive mechanisms, the absence of 5-HTR4 aggravates scopolamine-induced memory impairments. The mechanisms whereby 5-HTR4 mediate a tonic influence on ChAT activity and muscarinic receptors remain to be determined.
doi:10.1371/journal.pone.0009529
PMCID: PMC2832007  PMID: 20209108
10.  PICK1 uncoupling from mGluR7a causes absence-like seizures 
Nature neuroscience  2008;11(8):940-948.
Absence epilepsy is a neurological disorder that causes a recurrent loss of consciousness and generalized spike-and-wave discharges on an electroencephalogram (EEG). The role of metabotropic glutamate receptors (mGluRs) and associated scaffolding proteins in absence epilepsy has been unclear to date. We investigated a possible role for these proteins in absence epilepsy, focusing on the mGluR7a receptor and its PDZ-interacting protein, protein interacting with C kinase 1 (PICK1), in rats and mice. Injection of a cell-permeant dominant-negative peptide or targeted mutation of the mGluR7a C terminus, both of which disrupt the interaction between the receptor and PDZ proteins, caused behavioral symptoms and EEG discharges that are characteristic of absence epilepsy. Inactivation of the Pick1 gene also facilitated pharmacological induction of the absence epilepsy phenotype. The cortex and thalamus, which are known to participate in absence epilepsy, were involved, but the hippocampus was not. Our results indicate that disruption of the mGluR7a-PICK1 complex is sufficient to induce absence epilepsy—like seizures in rats and mice, thus providing, to the best of our knowledge, the first animal model of metabotropic glutamate receptor—PDZ protein interaction in absence epilepsy.
doi:10.1038/nn.2142
PMCID: PMC2764992  PMID: 18641645
11.  Plant Insecticide L-Canavanine Repels Drosophila via the Insect Orphan GPCR DmX 
PLoS Biology  2009;7(6):e1000147.
An orphan G-protein-coupled gustatory receptor mediates detection of the plant poison L-canavanine in fruit flies.
For all animals, the taste sense is crucial to detect and avoid ingesting toxic molecules. Many toxins are synthesized by plants as a defense mechanism against insect predation. One example of such a natural toxic molecule is l-canavanine, a nonprotein amino acid found in the seeds of many legumes. Whether and how insects are informed that some plants contain l-canavanine remains to be elucidated. In insects, the taste sense relies on gustatory receptors forming the gustatory receptor (Gr) family. Gr proteins display highly divergent sequences, suggesting that they could cover the entire range of tastants. However, one cannot exclude the possibility of evolutionarily independent taste receptors. Here, we show that l-canavanine is not only toxic, but is also a repellent for Drosophila. Using a pharmacogenetic approach, we find that flies sense food containing this poison by the DmX receptor. DmXR is an insect orphan G-protein–coupled receptor that has partially diverged in its ligand binding pocket from the metabotropic glutamate receptor family. Blockade of DmXR function with an antagonist lowers the repulsive effect of l-canavanine. In addition, disruption of the DmXR encoding gene, called mangetout (mtt), suppresses the l-canavanine repellent effect. To avoid the ingestion of l-canavanine, DmXR expression is required in bitter-sensitive gustatory receptor neurons, where it triggers the premature retraction of the proboscis, thus leading to the end of food searching. These findings show that the DmX receptor, which does not belong to the Gr family, fulfills a gustatory function necessary to avoid eating a natural toxin.
Author Summary
Plants evolve to fend off the insects that attack them, often by synthesizing compounds toxic to insects. In turn, insects develop strategies to avoid these plants or resist their toxins. Some plant toxins are nonprotein amino acids. For example, seeds from numerous legumes contain high amounts of l-canavanine, a nonprotein amino acid that is structurally related to l-arginine and is highly toxic to most insects. How insects can detect l-canavanine remains to be elucidated. Using pharmacology, genetics, and behavioral approaches, we show that flies sense l-canavanine using the receptor DmX, an orphan G-protein–coupled receptor that has diverged in its ligand binding pocket from metabotropic glutamate receptors. Disruption of the DmXR gene, called mangetout (mtt), suppresses the l-canavanine repellent effect. DmXR is expressed and required in aversive gustatory receptor neurons, where it triggers the premature retraction of the proboscis, thus leading to the end of food searching. Our results indicate a mechanism by which some insects may detect and avoid a plant toxin.
doi:10.1371/journal.pbio.1000147
PMCID: PMC2695807  PMID: 19564899
12.  Engineering GPCR signaling pathways with RASSLs 
Nature methods  2008;5(8):673-678.
We are creating families of designer G-protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (Gs, Gi, Gq). These new advances are reviewed here to help facilitate the use of these powerful and diverse tools.
doi:10.1038/nmeth.1232
PMCID: PMC2703467  PMID: 18668035
13.  Physical Interaction of Calmodulin with the 5-Hydroxytryptamine2C Receptor C-Terminus Is Essential for G Protein-independent, Arrestin-dependent Receptor Signaling 
Molecular Biology of the Cell  2008;19(11):4640-4650.
The serotonin (5-hydroxytryptamine; 5-HT)2C receptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT2C receptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca2+-dependent “1-10” CaM-binding motif located in the proximal region of the 5-HT2C receptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both β-arrestin recruitment by 5-HT2C receptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on β-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT2C receptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT2C receptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT2C receptor agonists and their antidepressant-like activity.
doi:10.1091/mbc.E08-04-0422
PMCID: PMC2575147  PMID: 18768750
14.  BAY36-7620: a potent non-competitive mGlu1 receptor antagonist with inverse agonist activity 
Molecular Pharmacology  2001;59(5):965-973.
L-glutamate (Glu) activates at least eight different G protein-coupled receptors, the metabotropic glutamate (mGlu) receptors, which mostly act as regulators of synaptic transmission. These receptors consist of two domains: an extracellular one where agonists bind, and a transmembrane heptahelix region involved in G-protein activation. Although new mGlu receptor agonists and antagonists have been described, few are selective for a single mGlu subtype. Here, we have examined the effects of a novel compound BAY36-7620 [(3aS,6aS)-6a-Naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on], on mGlu receptors (mGlu1-8), transiently expressed in HEK 293 cells. BAY36-7620 is a potent (IC50 = 0.16 μM) and selective antagonist at mGlu1 receptors and inhibits >60% of mGlu1a receptor constitutive activity (IC50 = 0.38 μM). BAY36-7620 is therefore the first described mGlu1 receptor inverse agonist. To address the mechanism of action of BAY36-7620, Glu dose-response curves were performed in the presence of increasing concentrations of BAY36-7620. The results show that BAY36-7620 largely decreases the maximal effect of Glu. Moreover, BAY36-7620 did not displace the [3H]quisqualate binding from the Glu-binding pocket., further indicating that BAY36-7620 is a non-competitive mGlu1 antagonist. We then looked for its site of action. Studies of chimeric receptors containing regions of mGlu1, and regions of DmGluA, mGlu2 or mGlu5, revealed that the transmembrane region of mGlu1 is necessary for activity of BAY36-7620. Transmembrane helices 4–7 are shown to play a critical role in the selectivity of BAY36-7620. This specific site of action of BAY36-7620 differs from that of competitive antagonists, and indicates that the transmembrane region plays a pivotal role in the agonist-independent activity of this receptor. BAY36-7620 will be useful to further delineate the functional importance of the mGlu1 receptor, including its putative agonist-independent activity.
PMCID: PMC2573968  PMID: 11306677
Animals; Cells, Cultured; Humans; Inositol Phosphates; metabolism; Membrane Proteins; drug effects; metabolism; Naphthalenes; pharmacology; Rats; Receptors, Metabotropic Glutamate; agonists; metabolism; Transfection
15.  Muscle Dystroglycan Organizes the Postsynapse and Regulates Presynaptic Neurotransmitter Release at the Drosophila Neuromuscular Junction 
PLoS ONE  2008;3(4):e2084.
Background
The Dystrophin-glycoprotein complex (DGC) comprises dystrophin, dystroglycan, sarcoglycan, dystrobrevin and syntrophin subunits. In muscle fibers, it is thought to provide an essential mechanical link between the intracellular cytoskeleton and the extracellular matrix and to protect the sarcolemma during muscle contraction. Mutations affecting the DGC cause muscular dystrophies. Most members of the DGC are also concentrated at the neuromuscular junction (NMJ), where their deficiency is often associated with NMJ structural defects. Hence, synaptic dysfunction may also intervene in the pathology of dystrophic muscles. Dystroglycan is a central component of the DGC because it establishes a link between the extracellular matrix and Dystrophin. In this study, we focused on the synaptic role of Dystroglycan (Dg) in Drosophila.
Methodology/Principal Findings
We show that Dg was concentrated postsynaptically at the glutamatergic NMJ, where, like in vertebrates, it controls the concentration of synaptic Laminin and Dystrophin homologues. We also found that synaptic Dg controlled the amount of postsynaptic 4.1 protein Coracle and alpha-Spectrin, as well as the relative subunit composition of glutamate receptors. In addition, both Dystrophin and Coracle were required for normal Dg concentration at the synapse. In electrophysiological recordings, loss of postsynaptic Dg did not affect postsynaptic response, but, surprisingly, led to a decrease in glutamate release from the presynaptic site.
Conclusion/Significance
Altogether, our study illustrates a conservation of DGC composition and interactions between Drosophila and vertebrates at the synapse, highlights new proteins associated with this complex and suggests an unsuspected trans-synaptic function of Dg.
doi:10.1371/journal.pone.0002084
PMCID: PMC2323113  PMID: 18446215
16.  5-Hydroxytryptamine4 Receptor Activation of the Extracellular Signal-regulated Kinase Pathway Depends on Src Activation but Not on G Protein or β-Arrestin Signaling 
Molecular Biology of the Cell  2007;18(6):1979-1991.
The 5-hydroxytryptamine4 (5-HT4) receptors have recently emerged as key modulators of learning, memory, and cognitive processes. In neurons, 5-hydroxytryptamine4 receptors (5-HT4Rs) activate cAMP production and protein kinase A (PKA); however, nothing is known about their ability to activate another key signaling pathway involved in learning and memory: the extracellular signal-regulated kinase (ERK) pathway. Here, we show that 5-HT4R stimulation, in primary neurons, produced a potent but transient activation of the ERK pathway. Surprisingly, this activation was mostly PKA independent. Similarly, using pharmacological, genetic, and molecular tools, we observed that 5-HT4Rs in human embryonic kidney 293 cells, activated the ERK pathway in a Gs/cAMP/PKA-independent manner. We also demonstrated that other classical G proteins (Gq/Gi/Go) and associated downstream messengers were not implicated in the 5-HT4R–activated ERK pathway. The 5-HT4R–mediated ERK activation seemed to be dependent on Src tyrosine kinase and yet totally independent of β-arrestin. Immunocytofluorescence revealed that ERK activation by 5-HT4R was restrained to the plasma membrane, whereas p-Src colocalized with the receptor and carried on even after endocytosis. This phenomenon may result from a tight interaction between 5-HT4R and p-Src detected by coimmunoprecipitation. Finally, we confirmed that the main route by which 5-HT4Rs activate ERKs in neurons was Src dependent. Thus, in addition to classical cAMP/PKA signaling pathways, 5-HT4Rs may use ERK pathways to control memory process.
doi:10.1091/mbc.E06-12-1080
PMCID: PMC1877087  PMID: 17377064
17.  Opposite Effects of PSD-95 and MPP3 PDZ Proteins on Serotonin 5-Hydroxytryptamine2C Receptor Desensitization and Membrane Stability 
Molecular Biology of the Cell  2006;17(11):4619-4631.
PSD-95/Disc large/Zonula occludens 1 (PDZ) domain-containing proteins (PDZ proteins) play an important role in the targeting and the trafficking of transmembrane proteins. Our previous studies identified a set of PDZ proteins that interact with the C terminus of the serotonin 5-hydroxytryptamine (5-HT)2C receptor. Here, we show that the prototypic scaffolding protein postsynaptic density-95 (PSD-95) and another membrane-associated guanylate kinase, MAGUK p55 subfamily member 3 (MPP3), oppositely regulate desensitization of the receptor response in both heterologous cells and mice cortical neurons in primary culture. PSD-95 increased desensitization of the 5-HT2C receptor-mediated Ca2+ response, whereas MPP3 prevented desensitization of the Ca2+ response. The effects of the PDZ proteins on the desensitization of the Ca2+ response were correlated with a differential regulation of cell surface expression of the receptor. Additional experiments were performed to assess how PDZ proteins globally modulate desensitization of the 5-HT2C receptor response in neurons, by using a peptidyl mimetic of the 5-HT2C receptor C terminus fused to the human immunodeficiency virus type-1 Tat protein transduction domain, which disrupts interaction between the 5-HT2C receptor and PDZ proteins. Transduction of this peptide inhibitor into cultured cortical neurons increased the desensitization of the 5-HT2C receptor-mediated Ca2+ response. This indicates that, overall, interaction of 5-HT2C receptors with PDZ proteins inhibits receptor desensitization in cortical neurons.
doi:10.1091/mbc.E06-03-0218
PMCID: PMC1635381  PMID: 16914526
18.  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).
doi:10.1251/bpo39
PMCID: PMC145562  PMID: 12734563
proteomics; spectrum analysis, mass
19.  PAC1 receptor–deficient mice display impaired insulinotropic response to glucose and reduced glucose tolerance 
Journal of Clinical Investigation  2000;105(9):1307-1315.
Pituitary adenylate cyclase–activating polypeptide (PACAP) is a ubiquitous neuropeptide of the vasoactive intestinal peptide (VIP) family that potentiates glucose-stimulated insulin secretion. Pancreatic β cells express two PACAP receptor subtypes, a PACAP-preferring (PAC1) and a VIP-shared (VPAC2) receptor. We have applied a gene targeting approach to create a mouse lacking the PAC1 receptor (PAC1–/–). These mice were viable and normoglycemic, but exhibited a slight feeding hyperinsulinemia. In vitro, in the isolated perfused pancreas, the insulin secretory response to PACAP was reduced by 50% in PAC1–/– mice, whereas the response to VIP was unaffected. In vivo, the insulinotropic action of PACAP was also acutely reduced, and the peptide induced impairment of glucose tolerance after an intravenous glucose injection. This demonstrates that PAC1 receptor is involved in the insulinotropic action of the peptide. Moreover, PAC1–/– mice exhibited reduced glucose-stimulated insulin secretion in vitro and in vivo, showing that the PAC1 receptor is required to maintain normal insulin secretory responsiveness to glucose. The defective insulinotropic action of glucose was associated with marked glucose intolerance after both intravenous and gastric glucose administration. Thus, these results are consistent with a physiological role for the PAC1 receptor in glucose homeostasis, notably during food intake.
PMCID: PMC315446  PMID: 10792006

Results 1-19 (19)