Joël Bockaert
email: joel.bockaert@igf.cnrs.fr

Sylvie Claeysen
email: sylvie.claeysen@igf.cnrs.fr

Aline Dumuis
email: aline.dumuis@igf.cnrs.fr

Philippe Marin
email: philippe.marin@igf.cnrs.fr

CLASSIFICATION AND SIGNALING CHARACTERISTICS OF 5-HT RECEPTORS

Keywords: serotonin, 5-hydroxytryptamine, 5-HT, G-protein coupled receptors, GPCR, signaling, G-protein, G-protein associated proteins, GIPs, pharmacology

come

Introduction

GPCRs are certainly among the oldest devices devoted to signal transduction, being present before plants, fungi and animal. Fungi (for example: Saccharomyces cerevisiae) express pheromone and glucose sensing GPCRs (for further reading see [START_REF] Fredriksson | The Gprotein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints[END_REF][START_REF] Bockaert | Molecular tinkering of G protein-coupled receptors: an evolutionary success[END_REF][START_REF] Bockaert | Molecular and cellular determinants of GPCR splice variant constitutive activity[END_REF]).

Although serotonin (5-hydroxytryptamine: 5-HT) is also one of the oldest cell-cell communication signaling molecule, it seems that both serotonin and associated receptors did not appear before the emergence of excitable cells and simple nervous systems such as those of coelenterates (Cnidaria) [START_REF] Hajj-Ali | Characterization of a serotonin receptor in the cnidarian Renilla koellikeri: a radiobinding analysis[END_REF], tunicates, nematodes, mollusks and insects [START_REF] Kroeze | Molecular Biology and Genomic Organization of G Protein-coupled serotonin receptors[END_REF]. Once present, 5-HT receptors have been "tinkered" to generate a large diversity of molecules, likely to ensure a fine control of intracellular signaling and cellular functions. Indeed, there are at least seven subclasses of 5-HT receptors in the nematode Caenorhabditis elegans and four in Aplysia, Drosophila, and Anopheles mosquito [START_REF] Kroeze | Molecular Biology and Genomic Organization of G Protein-coupled serotonin receptors[END_REF].

The classification of 5-HT receptors can be based on both their primary sequence dendrograms (trees based on sequence alignments) and/or their pharmacology. The pharmacology of invertebrate 5-HT receptors is rather poor for obvious reasons (compounds are not developed by the pharmaceutical industry) and only sequences characteristics can be used for the classification. The dendrograms show few discrete groupings for invertebrate GPCRs except one for 5-HT7-like receptors [START_REF] Kroeze | Molecular Biology and Genomic Organization of G Protein-coupled serotonin receptors[END_REF]. Thus, the classification of invertebrate 5-HT receptors is almost inexistent.

In vertebrates, the classification followed a slow evolution during the last 50 years. Two main periods can be distinguished. A pharmacological period, in which the development of specific 5-HT ligands had permit a fairly good classification and a molecular period in which the cloning of 5-HT receptors had completed the pharmacological classification. It is interesting to note that an excellent matching was found between the two (Figure 1). The pharmacological period defined 4 classes (5-HT1, 2 , 3 and 4) and the molecular cloning period 3 more (5-HT5, 6, 7). Some subclasses had also been defined by molecular biology. The actual dendrogram of vertebrate 5-HT receptors shows groupings, which fit with the classification defined by pharmacology (binding site structure). The historical evolution of the 5-HT receptors classification will be reviewed and we will also show that not only the coding sequences but also the intron/exon structure of the 5-HT receptor genes can be classified in seven classes.

It is evident that the interest of having so many receptors is not to allow development of specific and therapeutic drugs by the pharmaceutical industry. The interest, in a physiological and evolutionary perspective, is that these receptors have different cellular expression and control differently, via different fine signaling events, cell physiology. Indeed, each receptor subclass has a different signaling repertoire. The classical signaling events associated with most of these receptors have been reviewed [START_REF] Hannon | Molecular biology of 5-HT receptors[END_REF]Hoyer, 2008, Bockaert et al., 2006). We will update these data and will insist on the new concepts and data showing that our knowledge of the complexity of signaling cascades of 5-HT receptor classes and subclasses is still in the infancy.

We will not systematically present the different drugs and radioligands characterizing each receptor since this has been recently well done [START_REF] Hannon | Molecular biology of 5-HT receptors[END_REF].

Historical perspective of 5-HT receptor classification: a good ping-pong game between pharmacology and molecular biology

As already said in introduction, there are two main periods, a pharmacological and a molecular biology period.

The pharmacological period -Figure 1 The four pharmacologically defined 5-HT receptor classes This period obviously started in 1950 when Gaddum and colleagues [START_REF] Gaddum | Two kinds of tryptamine receptors[END_REF] found that, in the guinea pig ileum, the 5-HT induced contracting effect could be blocked in part by dibenziline (the D receptor which was finally characterized as a post-synaptic 5-HT2 receptor and in part by morphine (the M receptor which was finally characterized as pre-synaptic 5-HT3 receptor).

In 1975-80, a great effort has been made by Fillion in France [START_REF] Fillion | 5-HT and LSD high affinity binding sites to brain synaptosomal membranes [proceedings[END_REF] and Peroutka and Snyder in USA [START_REF] Peroutka | Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [3H]lysergic acid diethylamide and [3H]spiroperidol[END_REF] data and binding data, Bradley et al. [START_REF] Bradley | Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine[END_REF] proposed the existence of three 5-HT receptors families, the 5-HT1, 5-HT2 and the 5-HT3, the latter being only based, at that time, on the existence of the Gaddum "M" response. It is remarkable that even after 20 years of intensive discovery of new 5-HT receptor ligands and intensive molecular cloning the 3 classes remain pharmacologically the same.

In addition to binding studies and organ responses (such as guinea-pig contractions) another tool was introduced in the eighty: the use of second messenger production in intact cells or membranes following 5-HT stimulation. This tool was superb to study structure-response relationships and had the advantage to clearly distinguish agonists from antagonists. It was at that time, that we discovered the 5-HT4 receptor which was able to stimulate cAMP production in colliculi neurons [START_REF] Bockaert | 5-HT(4) receptors: History, molecular pharmacology and brain functions[END_REF], Dumuis et al., 1988b).

Pharmacologically defined 5-HT1 and 5-HT2 sub-classes

Before molecular cloning started, binding studies (using more and more selective radio-ligands) and second messenger studies were used to characterize subtypes of 5-HT1 receptors.

The 5-HT1A receptor was first characterized using [ 3 H]-8-OH-DPAT [START_REF] Gozlan | Identification of presynaptic serotonin autoreceptors using a new ligand: 3H-PAT[END_REF] and corresponds to the spiperone sensitive [ 3 H]-5-HT binding sites [START_REF] Pedigo | Discrimination of multiple [3H]5-hydroxytryptamine binding sites by the neuroleptic spiperone in rat brain[END_REF]. Few years latter this receptor was found, in neurons, to be negatively coupled to adenylyl-cyclase [START_REF] De Vivo | Characterization of the 5-hydroxytryptamine1A receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes[END_REF]Maayani, 1986, Weiss et al., 1986).

The 5-HT1B was first characterized as the part of [ 3 H]-5-HT binding sites insensitive to spiperone and 8-OH-DPAT and then as binding sites labeled with [ 125 I]-CYP (cyanopindolol). We found that this pre-synaptic receptor was negatively coupled to adenylyl cyclase in substantia nigra [START_REF] Bouhelal | 5-HT1B receptors are negatively coupled with adenylate cyclase in rat substantia nigra[END_REF].

The 5-HT1C receptor had a very transient existence in the 5-HT1 class. This receptor, found at high density in the choroids plexus and labeled with [ 3 H]-5-HT (as other 5-HT1 receptors) but which is coupled to inositol phosphate production and Ca 2+ signaling (as 5-HT2 receptors), definitively joined the 5-HT2 family after its early cloning, its sequence homology with other 5-HT2 receptors and an obvious 5-HT2 pharmacology [START_REF] Hannon | Molecular biology of 5-HT receptors[END_REF] (Figure 1).

The 5-HT1D history is even more complex. It was first observed that the [ 125 I]-CYP ligand which binds to 5-HT1B in rodents, fish, opossum was not labeling such an homolog receptor in guinea-pig, pig and human. However, a receptor functionally similar (inhibition of 5-HT release; inhibition of adenylyl-cyclase) was present in those species and thus was called 5-HT1D. It is the cloning period, which bring some light. Indeed, in human two genes coding for receptors having a 5-HT1D pharmacology were cloned: the (5-HT1Da and the 5-HT1Db) having very slight difference in pharmacology. In rat too, two genes are present the a and the b. Thus, the 5-HT1Db gene in both rodent and human code for the now called 5-HT1B receptor (Figure 1). The binding of [ 125 I]-CYP ligand to the rodent receptor and not to the human receptor and more generally the low affinity of the human receptor for bblockers is due to a single residue difference in TM7 (an asparagine, N355, in human and a threonine in rodent) [START_REF] Adham | A single point mutation increases the affinity of serotonin 5-HT1D alpha, 5-HT1D beta, 5-HT1E and 5-HT1F receptors for betaadrenergic antagonists[END_REF].

The 5-HT1Da code for 5-HT1D receptor in both rodent and human. Selective ligands are now available to distinguish between 5-HT1B and 1D receptors [START_REF] Hannon | Molecular biology of 5-HT receptors[END_REF]. All active anti-migraine drugs (triptans) act on 5-HT1B receptor.

Whether or not some may be active via a specific action on 5-HT1D receptors is still a matter of debate [START_REF] Hannon | Molecular biology of 5-HT receptors[END_REF].

The putative 5-ht1E receptor was identified in the human frontal cortex as a residual [ 3 H]-5-HT binding when all others 5-HT1 receptors were blocked. It has not been found in rodent and no specific drugs are available. A gene has been cloned which code for a receptor which could correspond to the pharmacologically 5-ht1E binding site. Its exact signaling properties remain to be found [START_REF] Hannon | Molecular biology of 5-HT receptors[END_REF].

When examining the status of 5-HT2 receptors during the pharmacological period the actual 5-HT2A and 5-HT2C were clearly defined. The 5-HT2A was indeed the D receptor of Gaddum [START_REF] Gaddum | Two kinds of tryptamine receptors[END_REF] as well as the 5-HT2 receptor found in brain by Peroutka and Snyder with binding studies [START_REF] Peroutka | Two distinct central serotonin receptors with different physiological functions[END_REF]. [ 3 H]-Ketanserin became rapidly the reference ligand for 5-HT2A. The 5-HT2C was, in fact, defined as 5-HT1C during this period (see above). Both were found to be coupled to phospholipase C. The existence of the actually characterized 5-HT2B was "present" at a sub-luminal stage during this period. Indeed, a 5-HT2 receptor was present in the fundus (thus called 5-HT2F) in which it induced contraction but no convincing pharmacological tools were available at that time for its definitive characterization [START_REF] Hannon | Molecular biology of 5-HT receptors[END_REF].

The molecular cloning period

G-protein coupled receptors

The molecular cloning period started with the cloning of 5-HT1A receptor. An orphan receptor (G21) isolated by cross-hybridization at reduced stringency with a full length b2-adrenergic receptor probe appeared to code for the 5-HT1A receptor [START_REF] Fargin | The genomic clone G-21 which resembles a beta-adrenergic receptor sequence encodes the 5-HT1A receptor[END_REF]. Rapidly, most of the pharmacologically characterized 5-HT receptors were cloned [START_REF] Hannon | Molecular biology of 5-HT receptors[END_REF] including the fundus receptor first called 5-HT2F (for fundus) [START_REF] Foguet | Cloning and functional characterization of the rat stomach fundus serotonin receptor[END_REF] but which was named 5-HT2B in view of the existence, at that time of a 5-HT2A and a 5-HT2C receptor.

In addition, "new" genes coding for 5-HT receptors were cloned which were not yet pharmacologically characterized. This includes the 5-HT1F, 5-ht5A, 5-HT5B, 5-HT6 and 5-HT7.

The 5-HT1F has been cloned on the basis of its sequence homology with the 5-HT1B/1D receptors [START_REF] Adham | Cloning of another human serotonin receptor (5-HT1F): a fifth 5-HT1 receptor subtype coupled to the inhibition of adenylate cyclase[END_REF]. It is expressed in many brain areas has a high affinity for triptans and LSD. It may be a better target for anti-migraine drugs than 5-HT1B receptors since it could by devoid of vasomotor effects. It is negatively coupled to adenylyl-cyclase.

The cloned 5-ht5A and 5-ht5B proteins [START_REF] Matthes | Mouse 5-hydroxytryptamine5A and 5hydroxytryptamine5B receptors define a new family of serotonin receptors: cloning, functional expression, and chromosomal localization[END_REF] have not yet reached the status of "receptors" since neither their signal transduction nor their physiological function are clearly established.

The 5-HT6 receptor was suspected to be present in the striatum and some glioblastomes such as NCB20. It was positively coupled with adenylyl cyclase and sensitive to antipsychotic drugs. The cloning was realized in 1993 in the rat (Ruat et al., 1993a). It is essentially present in brain and is indeed coupled positively coupled to adenylyl cyclase in striatal neurons [START_REF] Sebben | 5-HT6 receptors positively coupled to adenylyl cyclase in striatal neurones in culture[END_REF].

Another 5-HT receptor positively coupled with adenylyl cyclase was discovered by homology cloning, the 5-HT7 receptor (Ruat et al., 1993b).

Interestingly, this receptor shares a low homology with other members of the 5-HT receptor family (<50%) and a high homology with a drosophila 5-HT receptor positively coupled with an adenylate cyclase (> 94%) [START_REF] Kroeze | Molecular Biology and Genomic Organization of G Protein-coupled serotonin receptors[END_REF].

Ionotropic 5-HT3 receptors

This 5-HT receptor is the only one not being a metabotropic receptor but a ligand-gated ion channel. It is interesting to note that the main neurotransmitters (glutamate, GABA, acetylcholine, 5-HT) have managed, during evolution, to use both rapid (msec) ion-gated channel mediated and slow (sec) GPCR mediated transmission.

Two genes coding for 5-HT3 receptors have been cloned, the 5-HT3A [START_REF] Maricq | Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel[END_REF] and the 5-HT3B [START_REF] Dubin | The pharmacological and functional characteristics of the serotonin 5-HT(3A) receptor are specifically modified by a 5-HT(3B) receptor subunit[END_REF][START_REF] Maricq | Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel[END_REF]. They have high level of identity with other members of the Cys-Cys loop ligand-gated channel superfamily (e.g. nicotinic, GABAA, glycine receptors) especially in the channel domain area.

The native receptor, as revealed by electron microscope, is pentameric [START_REF] Boess | Ultrastructure of the 5hydroxytryptamine3 receptor[END_REF]. The 5-HT3B is unable to form a functional receptor on its own it can co-assemble with 5-HT3A to form a functional 5-HT3A/B channels with properties similar to native 5-HT3 receptors. Other human, but nor rodent, sequences have been cloned the 5-HT3C, D, E but their role as 5-HT3 receptors are obscured although they can have a modulating role associated with 5-HT3A [START_REF] Niesler | Characterization of the novel human serotonin receptor subunits 5-HT3C,5-HT3D, and 5-HT3E[END_REF], Jensen J.B., S.C., A.D. & P.M. 10 et al., 2008). Since 5-HT3A and 5-HT3B receptors are co-distributed in the brain as well as in dorsal root ganglia and trigeminal ganglia hetero-pentameric structures main exist.

Evolutionary consideration

As seen in Figure 1 the classification proposed during the pharmacological period has been, with few exceptions discussed in the previous paragraph, confirmed during the molecular cloning period. This indicates that the pharmacologists did a good job in their classification based on structure-function relationships, binding data as well as the few elements of signal transduction that they had in hands at that time. In addition, this indicates that the binding sites of all these different receptors have co-evolved with the whole primary structure of their corresponding receptor. Indeed, there is a perfect coincidence between the grouping of vertebrates 5-HT receptors as revealed by the dendogram based on sequence alignments and classes and sub-classes defined by the pharmacology [START_REF] Kroeze | Molecular Biology and Genomic Organization of G Protein-coupled serotonin receptors[END_REF]. This was not necessarily expected. Why having such different binding sites to recognize a common neurotransmitter? With the increasing complexity of the neuronal network it was certainly interesting to have an increased diversity of 5-HT receptors signaling (thus of the receptors domains involved in signaling) as well as a diversity in cellular expression. However, their binding site could have been kept similar. Note however that the evolution of the binding sites had permit to have receptors with different affinity for 5-HT.

Finally, the 5-HT receptor dendrogram shows that the nature of the primary coupling with the G protein is not an element of clustering when considering the sequence homologies. The 5-HT6 receptor coupled to Gs is more similar to 5-HT2 receptors coupled to Gq than to 5-HT7 receptors coupled to Gs.

Genomic, post-genomic characteristics of 5-HT receptors -Figure 2 5-HTRs are generated from 17 different genes in the human genome (NCBI, Map Viewer at http://www.ncbi.nlm.nih.gov/mapview/), 12 genes encoding metabotropic 5-HT receptors and 5 genes encoding ionotropic 5-HT3 receptors.

There is one pseudogene: the 5-HT5B.

Metabotropic 5-HT receptor genes G-protein coupled receptor genes diverged from a common ancestor, and their structure reflects their evolutionary relationship [START_REF] Barnes | A review of central 5-HT receptors and their function[END_REF]. 5-HT1Rs are coded by intronless genes and clearly constitute a subgroup of genes that arose by duplication events implicating several chromosomes (1, 3, 5 and 6) (Fig. 2). 5-HT2R genes are also closely related, presenting two similar introns located at the end of transmembrane domain 2 (TM2) and within TM4-coding sequences. The 5-HT2CR gene presents an additional intron in the extracellular domain of the coding sequences and extends over more than 183kb. Alternative splicing of the 5-HT2R genes does not generate any functional splice variant [START_REF] Xie | The human serotonin 5-HT2C receptor: complete cDNA, genomic structure, and alternatively spliced variant[END_REF]. The 5-HT4R gene is far more complex. The coding sequence extends over more than 185 kb and includes at least 14 exons leading to multiple splicing combinations. To date, ten functional splice variants have been described that can be generated by alternative splicing of the human transcript [START_REF] Bender | Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant[END_REF][START_REF] Hiroi | Identification and characterization of the human serotonin-4 receptor gene promoter[END_REF]. For nine of these variants, splicing gives rise to alternate Cterminal domains (Fig. 2). As some exons (such as the h or the i exons) do not present any in-frame stop codons, many additional combinations probably remain to be discovered [START_REF] Brattelid | Cloning, pharmacological characterisation and tissue distribution of a novel 5-HT4 receptor splice variant, 5-HT4(i)[END_REF]. Indeed, a recent publication described 10 novel splice variants of the porcine 5-HT4R including a functional homofusion variant [START_REF] De Maeyer | Alternative splicing and exon duplication generates 10 unique porcine 5-HT 4 receptor splice variants including a functional homofusion variant[END_REF]. The existence of these variants, some of them arising from duplication events, remains to be demonstrated in the human genome. The relevance and the functional role of such a multiplicity of isoforms also remain to be elucidated. The finding that some less-well represented 5-HT4 splice variants (e.g., 5-HT4(a)R and 5-HT4(e)R) interact with specific intracellular proteins supports the view that these isoforms are in charge of the fine regulation of signal transduction and are not the result of transcriptional leakage [START_REF] Joubert | New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting[END_REF]. Two 5-HT5R genes have been described in the rodent, but only the 5-HT5AR can be generated in humans [START_REF] Rees | Cloning and characterisation of the human 5-HT5A serotonin receptor[END_REF]. A 5-HT5BR pseudogene had been localized on chromosome 2 in Build 35.1 of the human genome. However, this annotation has been permanently suppressed in the actual build (36.3) because of insufficient evidence that this locus could be transcribed. The 5-HT6R gene presents two splicing sites: the first one lies in the middle of the intracellular loop 3 and is similar to the splicing site found in the 5-HT5A R gene; the second splicing site separates exons encoding TM6 and TM7 (Fig. 2). A non-functional splice variant has been described for this receptor (Olsen et al. 1999). The human 5-HT7R gene is more complex: different acceptor and donor sites can be used to generate three receptors that differ in the composition and length of their C-terminal domains (5-HT7(a), 5-HT7(b), and 5-HT7(d)). Because of its altered intron-exon organization, the rat gene generates the 5-HT7(c) but not the 5-HT7(d) isoform [START_REF] Heidmann | Four 5hydroxytryptamine7 (5-HT7) receptor isoforms in human and rat produced by alternative splicing: species differences due to altered intron-exon organization[END_REF]. This splicing position giving rise to alternate C-terminus is similar to that found in the 5-HT4R gene. The different C-terminal domains of the 5-HT7R variants can probably recruit specific sets of intracellular proteins that might modulate their function as demonstrated for the 5-HT4R isoforms.

Ionotropic 5-HT3 receptor genes

The subunits of the pentameric 5-HT3 ion channel are encoded by 5 different genes ( [START_REF] Barnes | The 5-HT(3) receptor -the relationship between structure and function[END_REF] and Fig. 2). HTR3A and 5-HT3B genes arose by duplication in the same locus (11q23.1). Other duplication events generated multiple copies of the 5-HT3R gene in the 3q27.1 locus, generating the HTR3C, HTR3D and HTR3E genes, and even a pseudogene (htr3c2). With the exception of HTR3D, all genes presented a common structure of 8 to 9 exons, the TM regions being encoded by the 2 to 3 last exons. An alterative splicing site in the 5-HT3AR gene generates two splice variants of 510 and 478 amino acids, (annotated respectively 5-HT3A(a) and 5-HT3A(b) in NCBI Gene Database), that differ in TM3 and intracellular loop 3 ( [START_REF] Belelli | Cloning and functional expression of a human 5-hydroxytryptamine type 3AS receptor subunit[END_REF][START_REF] Miyake | Molecular cloning of human 5-hydroxytryptamine3 receptor: heterogeneity in distribution and function among species[END_REF] and Fig. 2). The number of 5-HT3AR isoforms is not fixed to date as additional splice variants have been reported ( [START_REF] Bruss | Modified 5-HT3A receptor function by co-expression of alternatively spliced human 5-HT3A receptor isoforms[END_REF]). HTR3D gene has been described as encoding a 4TM-subunit of 279 residues, lacking the major part of the large N-terminal domain ( [START_REF] Niesler | Characterization of the novel human serotonin receptor subunits 5-HT3C,5-HT3D, and 5-HT3E[END_REF] and Fig. 2). However, two alternative transcripts of this gene have been reported that could produce proteins of 454 or 233 amino acids (see accession numbers Q70Z44-1 and Q70Z44-3 in Uniprot Database: http://www.uniprot.org). Further works remain to be done to conclude of the relevance of this observation.

Editing

RNA editing is a dynamic mechanism that generates molecular and functional diversity. The most common type of RNA editing involves nucleotide substitution that consists of either cytidine to uridine (C to U) or, most frequently, adenosine to inosine (A to I). Inosine is read as guanosine by the translational machinery of the cell [START_REF] Higuchi | RNA editing of AMPA receptor subunit GluR-B: a base-paired intronexon structure determines position and efficiency[END_REF]. The 5HT2CR is the only known GPCR to be regulated by editing. Editing takes place at four major positions on exon V (corresponding to the second exon in the coding sequence), termed sites A, B, C, and D, and one minor site, termed E. 5-HT2CR mRNA editing has been identified as A to I discrepancies between genomic and cDNA sequences in the region encoding the second intracellular loop of the receptor. This A to I substitution alters the coding potentials of three codons. Theoretically, 5-HT2CR editing can generate up to 32 different mRNAs that encode 24 receptor isoforms from the non-edited form (INI) to the fully edited form (VGV) [START_REF] Fitzgerald | Messenger RNA editing of the human serotonin 5-HT2C receptor[END_REF]. The editing of 5-HT2C transcripts is tissue-specific. Seven major 5-HT2CR isoforms, encoded by 11 distinct RNA species, are expressed in rat brain compared with 14 receptor isoforms encoded by 23 trancripts in human brain. The most prevalent isoform in human brain is 5-HT2C-VSV, whereas 5-HT2C-VNV is the most abundant in the rat. Editing is regulated by 5-HT itself. Depletion of 5-HT increases the expression of forms having a high affinity for 5-HT, whereas stimulation of 5-HT2C receptors has the opposite effect (Gurevich et al., 2002a). Altered RNA editing of the 5-HT2C receptor may play a role in depression. 5-HT2C receptor mRNA isoforms expressed in human prefrontal cortex differ in tissues obtained from depressed suicide victims compared with healthy controls. In suicide patients, three abundant isoforms, resulting from an increase at the E and C sites and a decrease at the D site and encoding receptors, are expressed. These isoforms exhibit decreased basal activity and decreased agonist affinity and potency (Gurevich et al., 2002b).

Recently, it has been reported that the small nucleolar RNA (snoRNA) HBII-52, which exhibits sequence complementarity to the alternative spliced exon V encoding for a functional 5-HT2C receptor, regulates receptor mRNA splicing.

Notably, Prader-Willy syndrome patients (characterized by neonatal muscular hypotonia, early childhood obesity, hypogonadism, and mental retardation) who do not express HBII-52 snoRNA exhibit abnormally low levels of the non-edited 5-HT2C receptor.

G protein-mediated signaling

The pleiotropic functions of 5-HT are not only exerted by concerted actions of multiple receptor subtypes, splicing and editing, but also by multiple coupling of each receptor specie. Since we have written two recent reviews [START_REF] Millan | Signaling at Gprotein-coupled serotonin receptors: recent advances and future research directions[END_REF], Bockaert et al., 2006) on the subject, we will focus here on the more recent and original aspects of this important question. In addition, we will try to make the distinction between signaling events established in native cells versus those observed when the receptor is expressed in cell lines. 3A) This is an inhibitory receptor coupled to Gi/Go inhibiting the adenylyl-cyclase but also opening G protein-gated inwardly rectifying gK + (GIRK) and inhibiting gCa 2+ . Interestingly, 5-HT1A receptors have different properties depending on their localization on 5-HT neurons (auto-receptors) versus on post-synaptic heterologous neurons. In particular, drugs have different efficacies and the auto-receptors have a much greater ability to desensitize. The reasons are still obscure but one possibility may be that auto-receptors are preferentially coupled to Gi3 and post-synaptic receptors to Go (Mannoury la [START_REF] La Cour | Regional differences in the coupling of 5-hydroxytryptamine-1A receptors to G proteins in the rat brain[END_REF]. Other signaling events mediated through activation of Gi/Go in native tissues (and in some cases in vivo) include 1) activation or inhibition of ERK (extracellular signal-regulated kinase), depending of the brain area [START_REF] Crane | 5-HT1A receptors mediate (+)8-OH-DPAT-stimulation of extracellular signal-regulated kinase (MAP kinase) in vivo in rat hypothalamus: time dependence and regional differences[END_REF], 2) activation of the Akt pathway [START_REF] Cowen | 5-HT receptors couple to activation of Akt, but not extracellular-regulated kinase (ERK), in cultured hippocampal neurons[END_REF] and 3) inhibition of NMDA transmission in prefrontal cortex, an effect potentiated by dampening RGS4 function [START_REF] Gu | RGS4 modulates serotonin signaling in prefrontal cortex and links to serotonin dysfunction in a rat model of schizophrenia[END_REF]. RGS4 is an RGS gene associated with schizophrenia [START_REF] Gu | RGS4 modulates serotonin signaling in prefrontal cortex and links to serotonin dysfunction in a rat model of schizophrenia[END_REF]. In COS7 cells, 5-HT1A receptors also stimulate both an ERK-dependent anti-apoptotic pathway and a Janus-kinase 2 (Jak2)-dependent pro-apoptotic pathway as well as c-jun N-terminal kinase (Turner et al., 2007a, Turner et al., 2007b).

5-HT1A receptor -(Figure

5-HT1B/1D/1E/1F receptors

5-HT1B receptors are negatively coupled to adenylyl-cyclase via Gi/Go in substantia nigra (Bouhelal et al., 1988, Hamblin and[START_REF] Hamblin | Primary structure and functional characterization of a human 5-HT1D-type serotonin receptor[END_REF]. In transfected cells, 5-HT1B receptors regulate PLC, PLD, Akt kinase, neuronal nitric oxide synthase (nNOS), ERK pathway. They also activate K + channels and inhibit Ca 2+ channels. The negative coupling of 5-HT1D/1E/1F receptors to adenylyl-cyclase has only been demonstrated in transfected cells, but likely occurs in vivo (Bockaert et al., 2006).

5-HT2A receptors -(Figure 3 B) 5-HT2A receptors activate PLC, PLA2, and the ERK pathway (downstream of PLC) in neurons [START_REF] Bockaert | Neuronal 5-HT metabotropic receptors: fine-tuning of their structure, signaling, and roles in synaptic modulation[END_REF]. Recently, an original signaling pathway has been associated with 5-HT2A receptor stimulation in platelets and a rat cortical cell line: the covalent link of serotonin to a glutamine residue of small G proteins such as RhoA and Rab4 as well as Rac1, a process which render them constitutively active [START_REF] Walther | Serotonylation of small GTPases is a signal transduction pathway that triggers platelet alpha-granule release[END_REF][START_REF] Dai | Transglutaminasecatalyzed transamidation: a novel mechanism for Rac1 activation by 5hydroxytryptamine2A receptor stimulation[END_REF]. Physical interaction of 5-HT-bound, activated Rab4 (Rab4-GTP) with the serotonin transporter (SERT) has been found to "paralyze" its translocation from intracellular compartments to the plasma membrane [START_REF] Ahmed | Serotonin transamidates Rab4 and facilitates its binding to the C terminus of serotonin transporter[END_REF]. 5-HT2A receptors inhibit the 5-HT1A-mediated modulation of NMDA current in pyramidal neurons via an arrestin/Src/ERK pathway [START_REF] Yuen | Activation of 5-HT2A/C receptors counteracts 5-HT1A regulation of n-methyl-D-aspartate receptor channels in pyramidal neurons of prefrontal cortex[END_REF]. In C6 glioma cells, 5-HT2A activates matrix metalloproteinases (MMPs) via the Src pathway, the release of FGF receptor 2 ligand, the activation of FGFR2 receptor, the activation of downstream ERK and transcription of GDNF [START_REF] Tsuchioka | Serotonin (5-HT) induces glial cell line-derived neurotrophic factor (GDNF) mRNA expression via the transactivation of fibroblast growth factor receptor 2 (FGFR2) in rat C6 glioma cells[END_REF]. Thus, 5-HT2A receptors activate ERK through several pathways. They also activate the Janus kinase-2 (Jak2/STAT) transcription pathway in skeletal and smooth muscles [START_REF] Guillet-Deniau | Identification and localization of a skeletal muscle secrotonin 5-HT2A receptor coupled to the Jak/STAT pathway[END_REF][START_REF] Banes | Activation of the JAK/STAT pathway in vascular smooth muscle by serotonin[END_REF] and PLD in nonneuronal cells.

5-HT2B receptors

As 5-HT2A receptors, 5-HT2B receptors are coupled to Gq/G11 proteins and activate PLC. However, to date, no study has reported such a coupling within neurons. 5-HT2B receptors have also been reported to stimulate Ca 2+ mobilization in astrocytes derived from various rat cerebral regions. Additional 5-HT2B receptormediated signaling pathways have been described in non-neuronal cells expressing endogenous receptors. Stimulation of 5-HT2B receptors is associated with an increase in cyclic GMP through the dual activation of constitutive and inducible NOS. Gq/11-coupled 5-HT2B receptors also activate PI3K/Akt and ERK1/2 signaling pathways [START_REF] Bockaert | Neuronal 5-HT metabotropic receptors: fine-tuning of their structure, signaling, and roles in synaptic modulation[END_REF]. 3C) 5-HT2C receptors have been reported to activate PLC via Gq/11 in many brain regions, including the choroid plexus [START_REF] Sanders-Bush | Putative selective 5-HT-2 antagonists block serotonin 5-HT-1c receptors in the choroid plexus[END_REF]. The nonedited form (5-HT2CINI) is more potent in stimulating PLC and exhibits higher constitutive activity than the edited forms [START_REF] Niswender | RNA editing of the human serotonin 5-hydroxytryptamine 2C receptor silences constitutive activity[END_REF]. Moreover, 5-HT2CINI receptors are capable of coupling to the G13 protein, activating PLD signaling pathway and increasing Rho GTPase activity [START_REF] Mcgrew | Phospholipase D activation by endogenous 5-hydroxytryptamine 2C receptors is mediated by Galpha13 and pertussis toxin-insensitive Gbetagamma subunits[END_REF], whereas the fully edited isoform (5-HT2CVGV) fails to activate G13. RNA editing has an important implication for signal transduction in vivo [START_REF] Price | RNA editing of the human serotonin 5-HT2C receptor alters receptor-mediated activation of G13 protein[END_REF].

5-HT2C receptors -(Figure

In CHO cells, 5-HT2C receptors also activate Gi/Go, modulating PLA2 and Akt/GSK3-b [START_REF] Cussac | Differential activation of Gq/11 and Gi(3) proteins at 5hydroxytryptamine(2C) receptors revealed by antibody capture assays: influence of receptor reserve and relationship to agonist-directed trafficking[END_REF].

5-HT4 receptors -(Figure 3D)

Since 5-HT4 receptor has been discovered from its capacity to activate adenylyl cyclase, the 5-HT4Rs mediated Gs/cAMP/PKA signaling pathway is obviously the best characterized. In transfected cell lines, other couplings have been found including coupling to Gq and G13 (see [START_REF] Bockaert | Neuronal 5-HT metabotropic receptors: fine-tuning of their structure, signaling, and roles in synaptic modulation[END_REF], Bockaert et al., 2008). Following PKA activation a series of ionic currents are modulated. These include a long-lasting inhibition of K + currents (mediated by a PKA-dependent inhibition of phosphatases), which enhances neuronal excitability, and a decrease in spike accommodation [START_REF] Ansanay | A cyclic AMPdependent, long-lasting inhibition of a K + current in mammalian neurons[END_REF], inhibition of AHP, activation of IH, and activation or inhibition of GABAA receptor [START_REF] Bockaert | Neuronal 5-HT metabotropic receptors: fine-tuning of their structure, signaling, and roles in synaptic modulation[END_REF], Bockaert et al., 2008). cAMP produced upon 5-HT4 receptor activation also stimulates the exchange factor Epac, which activates a-secretase and the release of sAPPa (amyloid precursor protein) via an Epac/Rap1/Ras pathway [START_REF] Lezoualc'h | The serotonin 5-HT4 receptor and the amyloid precursor protein processing[END_REF].

5-ht5 receptor

The signaling pathways associated with 5-ht5 putative receptors are still obscure. No data are available in native cells [START_REF] Hannon | Molecular biology of 5-HT receptors[END_REF].

5-HT6 receptors

As 5-HT4 receptors and 5-HT7 receptors, 5-HT6 receptors are positively coupled to adenylyl-cyclase in neurons [START_REF] Sebben | 5-HT6 receptors positively coupled to adenylyl cyclase in striatal neurones in culture[END_REF]. 5-HT6 receptors also activate the ERK pathway in CHO cells via a mechanism partially dependent on PKA and requiring the tyrosine kinase Fyn [START_REF] Yun | The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn[END_REF].

5-HT7 receptors -(Figure 3E)

The coupling of 5-HT7 receptors is more documented than that of the 5-HT6 receptor. The 5-HT7 receptor has been recognized as the receptor coupled to adenylyl cyclase exhibiting a high affinity for 5-carboxamidotryptamine (5-CT) in the guinea pig hippocampus (Dumuis et al., 1988a[START_REF] Shenker | Pharmacological characterization of two 5-hydroxytryptamine receptors coupled to adenylate cyclase in guinea pig hippocampal membranes[END_REF]. 5-HT7 receptors also engage the ERK1/2 pathway in hippocampal neurons via activation of Epac [START_REF] Lin | Coupling of neuronal 5-HT7 receptors to activation of extracellular-regulated kinase through a protein kinase A-independent pathway that can utilize Epac[END_REF]. Activation of ERK via PKA is also possible. 5-HT7 receptor activation increases neurite length through a Ga12/RhoA/Cdc42 signaling pathway in hippocampal neurons [START_REF] Kvachnina | 5-HT7 receptor is coupled to G alpha subunits of heterotrimeric G12-protein to regulate gene transcription and neuronal morphology[END_REF][START_REF] Lin | Coupling of neuronal 5-HT7 receptors to activation of extracellular-regulated kinase through a protein kinase A-independent pathway that can utilize Epac[END_REF]. In astrocytoma cells, 5-HT7 receptors activate p38 and PKCe. The implication of G proteins in this pathway is not demonstrated [START_REF] Lieb | Serotonin via 5-HT7 receptors activates p38 mitogen-activated protein kinase and protein kinase C epsilon resulting in interleukin-6 synthesis in human U373 MG astrocytoma cells[END_REF].

Non-G protein dependent signaling

Recently, many GPCRs have also been shown to signal without activation of G proteins. These non-G protein-mediated signaling generally implicate interactions with GPCR interacting proteins (see chapter xx) [START_REF] Bockaert | GPCR-interacting proteins (GIPs): nature and functions[END_REF]. A classic non-G signaling pathway, which implicates b-arrestins, is the ERK pathway. Long lasting activation (> 5 min, up to several hrs) of ERK induced by many GPCRs, including b-adrenergic receptors or AT1A angiotensin II receptors, is due to their cointernalization with b-arrestin after their uncoupling from G proteins and takes place in the cytosol. Internalized GPCR-b-arrestin complex recruits many proteins including the Src-Raf-MEK-ERK module. This contrasts with the rapid (< 5 min) activation of ERK evoked by GPCRs, which is generally G protein-dependent and leads to nuclear translocation of ERK. However, several GPCRs, including 5-HT receptors, differ from this general scheme. For instance, both early and sustained phases of ERK activation mediated by the 5-HT2C receptor are independent of receptor's cognate G proteins (Gq, G13 and Gi/o) and require physical interaction of calmodulin to the receptor C-terminal domain and b-arrestin1 & 2 recruitment by the receptor [START_REF] Labasque | Physical interaction of calmodulin with the 5-hydroxytryptamine2C receptor Cterminus is essential for G protein-independent, arrestin-dependent receptor signaling[END_REF] (Figure 3C). This non-G signaling pathway, established not only in transfected HEK-293 cells but also in cortical neurons and choroid plexus epithelial cells expressing native 5-HT2C receptor, might be involved in neurogenesis induced by chronic treatment with 5-HT2C receptor agonists and their antidepressant-like activity. Activation of 5-HT4 receptors likewise induces a sustained ERK phosphorylation in both transfected HEK-293 cells and cultured colliculi neurons [START_REF] Barthet | 5-hydroxytryptamine 4 receptor activation of the extracellular signal-regulated kinase pathway depends on Src activation but not on G protein or beta-arrestin signaling[END_REF]. Again, this activation was mostly Gs/cAMP/PKA-independent and did not involve any other G proteins (Gq, Gi, Go) and associated downstream messengers. Interestingly, 5-HT4 receptor-mediated ERK activation did not either involve b-arrestins but was dependent on Src tyrosine kinase. This receptor-operated pathway might be important for 5-HT4 receptormediated long-term potentiation (LTP) [START_REF] Huang | 5-Hydroxytryptamine induces a protein kinase A/mitogen-activated protein kinase-mediated and macromolecular synthesisdependent late phase of long-term potentiation in the amygdala[END_REF]. Collectively, these findings demonstrated that multiple non-G mechanisms, requiring or not recruitment of b-arrestins and additional partners (see also next chapter), can contribute to the activation of the ERK pathway by 5-HT receptors.

Constitutive activity

Some GPCRs are capable of activating G proteins in the absence of agonists. This intrinsic activity was mainly established for heterogeneously expressed GPCRs but also in authentic cellular contexts for some GPCRs. Some antagonists can inhibit these "constitutive" activities and thus behave as "inverse agonists". Several 5-HT receptors exhibit constitutive activity in neurons. Constitutive activity at postsynaptic 5-HT1A receptors expressed in hippocampus has been described. Such an intrinsic activity has not been yet demonstrated for 5-HT1A autoreceptors [START_REF] Martel | Native rat hippocampal 5-HT1A receptors show constitutive activity[END_REF]. As previously mentioned, constitutive activity of 5-HT2C receptors is highly dependent of the degree of mRNA edition: non edited or partially edited versions of the receptor exhibit the highest levels of constitutive activity whereas the fully edited one is devoid of constitutive activity [START_REF] Berg | Physiological relevance of constitutive activity of 5-HT2A and 5-HT2C receptors[END_REF][START_REF] Chanrion | Inverse agonist and neutral antagonist actions of antidepressants at recombinant and native 5hydroxytryptamine2C receptors: differential modulation of cell surface expression and signal transduction[END_REF].

Constitutive activation of non-edited or partially edited isoforms is associated with a constitutive internalization [START_REF] Marion | RNA editing induces variation in desensitization and trafficking of 5-hydroxytryptamine 2c receptor isoforms[END_REF]. Physiological constitutive activity of 5-HT2A receptors might explain impairment of associative learning by inverse agonists [START_REF] Berg | Physiological relevance of constitutive activity of 5-HT2A and 5-HT2C receptors[END_REF]. 5-HT4 receptor display a particularly high constitutive activity in transfected cells, depending of the splice variant analyzed. The shorter ones exhibit a higher constitutive activity than the longer ones [START_REF] Bockaert | 5-HT(4) receptors: History, molecular pharmacology and brain functions[END_REF]. Unfortunately, no data are available concerning a possible in vivo existence of constitutive activity. Constitutive activity of native 5-HT7 receptors also remains to be demonstrated [START_REF] Millan | Signaling at Gprotein-coupled serotonin receptors: recent advances and future research directions[END_REF].

Agonist-directed signaling

It has been recently proposed that a given GPCR can be stabilized under different active conformational states depending of the nature of the agonist, a phenomenon called "ligand-directed signaling (LDS)" [START_REF] Kenakin | Functional selectivity through protean and biased agonism: who steers the ship?[END_REF]. LDS has been demonstrated for several 5-HT receptor subtypes. 5-HT1A receptor agonists show differential efficacies to stimulate various G proteins and to activate different channels such as GIRKs versus smooth inward-current channels (Ismooth). The different efficacies of agonists at autoreceptors versus post-synaptic receptors might reflect their coupling to Gi3 versus Go, respectively [START_REF] Millan | Signaling at Gprotein-coupled serotonin receptors: recent advances and future research directions[END_REF]. Rank order of efficacy for recruiting human 5-HT2C receptors differ for PLC versus PLA2 and for Gq/11 versus Gi3. Similarly, contrasting agonist efficacies at PLC versus PLA2 have been observed for human 5-HT2A receptors [START_REF] Millan | Signaling at Gprotein-coupled serotonin receptors: recent advances and future research directions[END_REF]. A particularly interesting example of LDS at 5-HT receptors is the different signaling induced by psychedelic hallucinogens (such as LSD, DOI and (-)DOB) versus non-hallucinogen agonists (such as lisuride) of 5-HT2A receptors. Both ligand categories engage the Gq/PLC pathway, leading to c-fos induction in the somato-sensoriel cortex and the medial-prefrontal cortex (Figure 4) [START_REF] Benneyworth | 31 glutamate receptor subtype 2 blocks a hallucinogenic drug model of psychosis[END_REF][START_REF] Gonzalez-Maeso | Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior[END_REF]. In contrast, only hallucinogenic drugs activate a Gi/o-Src pathway, leading to induction of egr-2 expression in somato-sensoriel cortex and cortical neurons in primary culture [START_REF] Gonzalez-Maeso | Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior[END_REF][START_REF] Benneyworth | 31 glutamate receptor subtype 2 blocks a hallucinogenic drug model of psychosis[END_REF].

Recent reports add to the complexity and interest of this hallucinogenic ligand signaling. A physiological antagonism exists between 5-HT2A and mGlu2/3 receptors in these cortical areas [START_REF] Marek | Physiological antagonism between 5-hydroxytryptamine(2A) and group II metabotropic glutamate receptors in prefrontal cortex[END_REF]. Activation of mGlu2/3 receptors attenuates behavioral effects of hallucinogens known to produce a state of drug intoxication that resembles many of the symptoms of acute schizophrenia [START_REF] Marek | Physiological antagonism between 5-hydroxytryptamine(2A) and group II metabotropic glutamate receptors in prefrontal cortex[END_REF]. Consistent with this hypothesis, data from a recent phase II clinical trial show that an mGlu2/3 receptor agonist is an effective anti-psychotic therapy (Schoepp, 2006, Aghajanian and[START_REF] Aghajanian | Serotonin, via 5-HT2A receptors, increases EPSCs in layer V pyramidal cells of prefrontal cortex by an asynchronous mode of glutamate release[END_REF]. The mechanism of mGlu2/3 receptor agonist effect is controversial. mGlu2/3 receptors are supposed to be localized on glutamatergic terminals connecting pyramidal neurons. 5-HT2A receptors are postsynaptically localized on layer V pyramidal neurons. Since activation of mGlu2/3 receptors inhibit the 5-HT2A receptor-induced excitatory post-synaptic currents, the following hypothesis has been proposed to explain the effects of mGlu2/3 receptors: 1) activation of post-synaptic layer V pyramidal neurons is mediating induction of glutamate release from pre-synaptic nerve terminals connecting these neurons via a retrograde messenger 2) glutamate induced post-synaptic excitatory currents in layer V pyramidal neurons and it is this release which is inhibited following activation of pre-synaptic mGlu2/3 receptors (Aghajanian andMarek, 1999, Benneyworth et al., 2007). However, this hypothesis has been recently challenged by a recent report showing that mGlu2 receptors can form heterodimers with 5-HT2A receptors in postsynaptic pyramidal neurons [START_REF] Gonzalez-Maeso | Identification of a serotonin/glutamate receptor complex implicated in psychosis[END_REF]. Activation of mGlu2 receptors will "directly" inhibit the hallucinogen-induced activation of the Gi/o-Src pathway, preventing induction of egr-2 (Figure 4), without affecting the Gq/ PLC pathway and c-fos induction [START_REF] Gonzalez-Maeso | Identification of a serotonin/glutamate receptor complex implicated in psychosis[END_REF]. This absence of inhibition of hallucinogen-induced Gq/c-fos pathway by mGlu2 receptors observed in the somato-sensoriel cortex by Gonzales-Maseo et al., contrast with the observation made in the pre-frontal cortex by [START_REF] Benneyworth | 31 glutamate receptor subtype 2 blocks a hallucinogenic drug model of psychosis[END_REF].

Further work is obviously to be done to resolve this important paradox.

Toward the notion of 5-HT receptor complex

An increasing body of evidence indicates that 5-HT receptors are not "isolated islands" floating within the plasma membrane, but that they are part of multiprotein complexes that incorporate a dimer (or an oligomer) of receptors associated with numerous intracellular proteins in addition to G proteins and the ubiquitous GPCR signaling modulators b-arrestins. To date, 5-HT receptors are among the GPCRs for which interacting proteins have been the most extensively characterized (Table 1), often by means of yeast two-hybrid screens or proteomic strategies. The list is still growing and will continue to grow during the following years, as the majority of interactions certainly remain to be discovered. Although the functional consequences of most of them remain to be established in authentic cellular contexts, a set of recent studies highlight their impact in CNS physiology and suggest their possible therapeutic exploitation for the treatment of CNS disorders.

We will not provide here an extensive description of 5-HT receptor interacting proteins (see Table 1). The majority of these interactions have been reviewed elsewhere [START_REF] Bockaert | GPCR-interacting proteins (GIPs): nature and functions[END_REF], Bockaert et al., 2006). We will only illustrate from selected examples their influence on receptor signal transduction and subcellular compartmentation and their possible implication in psychiatric disorders such as depression and addiction. 5-HT receptor-interacting proteins can modulate G protein-operated signaling by influencing localization of receptor at the plasma membrane, their desensitization and their coupling to G proteins. 5-HT2A/2C receptors express PDZ binding motifs at their C-terminal extremity, which recruit specific sets of PDZ domain-containing proteins (PDZ proteins) [START_REF] Becamel | The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins[END_REF], Gavarini et al., 2004[START_REF] Becamel | Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach[END_REF]. These interactions govern receptor trafficking in and out of the plasma membrane and receptor desensitization. These effects depend on both the nature of the PDZ protein bound to the receptor and the receptor subtype. Association of the 5-HT2A receptor with PSD-95 stabilizes receptor at the plasma membrane (Xia et al., 2003a). Accordingly, this interaction amplifies receptor-operated signals. In contrast, interaction of PSD-95 with 5-HT2C receptors increases spontaneous and agonistdependent receptor internalization, whereas association of the receptor with another PDZ protein, MAGUK p55 subfamily member 3 (MPP3), prevents both receptor desensitization and internalization [START_REF] Gavarini | Opposite effects of PSD-95 and MPP3 PDZ proteins on serotonin 5-hydroxytryptamine2C receptor desensitization and membrane stability[END_REF]. Overall disruption of 5-HT2C receptor/PDZ protein interactions in cortical neurons increases receptor desensitization, indicating that PDZ proteins predominantly inhibit receptor desensitization and internalization in neurons. Besides PDZ proteins, p11 (also called S100A10 or calpactin I light chain or annexin II light chain), is a member of the S100 Ca 2+ -binding protein superfamily that binds to several 5-HT receptor subtypes and affects receptor trafficking. Moreover, p11 is unique among the S100 family members for its capacity to interact with 5-HT receptors [START_REF] Warner-Schmidt | Role of p11 in cellular and behavioral effects of 5-HT4 receptor stimulation[END_REF]. P11 was first identified in yeast two-hybrid screen as a 5-HT1B receptor interacting protein [START_REF] Svenningsson | Alterations in 5-HT1B receptor function by p11 in depression-like states[END_REF]. P11/5-HT1B receptor interaction promotes receptor expression at the plasma membrane and thereby enhances receptor signal transduction efficacy [START_REF] Svenningsson | Alterations in 5-HT1B receptor function by p11 in depression-like states[END_REF]. More recently, a physical interaction between p11 with 5-HT4 receptors has been described [START_REF] Warner-Schmidt | Role of p11 in cellular and behavioral effects of 5-HT4 receptor stimulation[END_REF]. This interaction likewise increases 5-HT4 receptor cell surface expression and downstream signaling.

Interaction of 5-HT receptors with accessory proteins also affect receptor phosphorylation and thereby receptor desensitization. 5-HT2A receptors (third intracellular loop) bind to p90 ribosomal S6 kinase 2 (RSK2), which induces receptor phosphorylation at Ser 314 located in the second intracellular loop [START_REF] Sheffler | p90 ribosomal S6 kinase 2 exerts a tonic brake on G protein-coupled receptor signaling[END_REF][START_REF] Strachen | Ribosomal S6 kinase 2 directly phosphorylates the 5-HT2A serotonin receptor thereby modulating 5-HT2A signaling[END_REF]. Ser 314 phosphorylation leads to a tonic inhibition of receptor-operated signaling. Moreover, 5-HT2A receptor phosphorylation by RSK2 is required for epidermal growth factor-mediated heterologous desensitization of the 5-HT2A receptor. Another partner that might influence receptor phosphorylation and desensitization is calmodulin (CaM). CaM binding site on 5-HT2A receptor Cterminus overlaps with a putative PKC phosphorylation site. In fact, CaM binding to the receptor C-terminus and receptor phosphorylation by PKC were found to be antagonistic, suggesting a role of CaM in the regulation of receptor desensitization [START_REF] Turner | Interaction of calmodulin with the serotonin 5hydroxytryptamine2A receptor. A putative regulator of G protein coupling and receptor phosphorylation by protein kinase C[END_REF]. CaM association with 5-HT1A receptor likewise interferes with receptor phosphorylation by PKC [START_REF] Turner | Calmodulin interacts with the third intracellular loop of the serotonin 5-hydroxytryptamine1A receptor at two distinct sites: putative role in receptor phosphorylation by protein kinase C[END_REF].

Interacting proteins might also directly influence coupling of 5-HT receptors to G proteins. For instance, interaction of CaM with 5-HT2A receptors (second intracellular loop) prevents their coupling to Gq [START_REF] Turner | Interaction of calmodulin with the serotonin 5hydroxytryptamine2A receptor. A putative regulator of G protein coupling and receptor phosphorylation by protein kinase C[END_REF], whereas receptor association with caveolins facilitates coupling to Gq [START_REF] Bhatnagar | Caveolin-1 interacts with 5-HT2A serotonin receptors and profoundly modulates the signaling of selected Galphaq-coupled protein receptors[END_REF].

Beyond G protein-dependent signal transduction, interaction of 5-HT receptors with accessory proteins might promote non-G signaling. As already mentioned, constitutive interaction of 5-HT4 receptor with Src is essential for receptor-operated ERK1,2 signaling, which is G protein-and arrestin-independent [START_REF] Barthet | 5-hydroxytryptamine 4 receptor activation of the extracellular signal-regulated kinase pathway depends on Src activation but not on G protein or beta-arrestin signaling[END_REF]. Activation of the ERK pathway by 5-HT2C receptor is likewise independent of the G proteins known to be activated by the receptor but is intimately related to arrestin recruitment, a process facilitated by CaM bound to the receptor C-terminal domain [START_REF] Labasque | Physical interaction of calmodulin with the 5-hydroxytryptamine2C receptor Cterminus is essential for G protein-independent, arrestin-dependent receptor signaling[END_REF]. Thus, this non-G signaling seems to involve a tripartite "signalosome" including the 5-HT2C receptor, CaM and b-arrestin. Finally, activation of the ERK pathway by 5-HT6 receptor, which is largely insensitive to PKA inhibition, is mediated by a direct interaction of Fyn (via its SH3 domain) with the receptor C-terminal domain [START_REF] Yun | The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn[END_REF]. 5-HT receptor-interacting proteins are not only important for signal transduction but they are also key determinants of sub-cellular distribution of 5-HT receptors. The 5-HT2A receptor PDZ binding motif is essential for the preferential receptor targeting to the dendritic compartment of pyramidal cortical neurons (Xia et al., 2003b). The nature of the PDZ protein(s) that govern dendritic targeting of the receptor remains to be elucidated. PSD-95, the major receptor PDZ partner, is a possible candidate. In dendrites, the receptor is mainly confined in intracellular compartments. This intracellular localization seems to be related to its ability to interact with the microtubule-associated protein MAP1A [START_REF] Cornea-Hebert | Similar ultrastructural distribution of the 5-HT(2A) serotonin receptor and microtubule-associated protein MAP1A in cortical dendrites of adult rat[END_REF]. Interaction of 5-HT1A receptor C-terminus with Yif1b, a protein of the endoplasmic reticulum/Golgi trafficking machinery, is essential for receptor targeting in the somatodendritic compartment [START_REF] Carrel | Targeting of the 5-HT1A serotonin receptor to neuronal dendrites is mediated by Yif1B[END_REF].

The association of 5-HT2A receptor with caveolin-1, detected in both C6 glioma cells and rat brain synaptic membrane preparations, is essential for targeting the receptor to lipid rafts and, as mentioned above, for receptor-mediated signal transduction [START_REF] Bhatnagar | Caveolin-1 interacts with 5-HT2A serotonin receptors and profoundly modulates the signaling of selected Galphaq-coupled protein receptors[END_REF]. In pulmonary artery smooth muscle cells, the 5-HT2A receptor forms a ternary complex with caveolin-1 and voltage-gated K + channels (Kv1.5), which was found to be critical for 5-HT2A receptor-mediated inhibition of Kv1.5 channels [START_REF] Cogolludo | Serotonin Inhibits Voltage-Gated K+ Currents in Pulmonary Artery Smooth Muscle Cells. Role of 5-HT2A Receptors, Caveolin-1, and KV1.5 Channel Internalization[END_REF].

The essential role of 5-HT receptor-interacting proteins in modulating signal transduction suggests that these interactions might be physiologically relevant or contribute to pathological situations. Interaction of p11 with both 5-HT1B and 5-HT4 receptors seems to be required for antidepressant actions of 5-HT1B and 5-HT4 agonists [START_REF] Svenningsson | Alterations in 5-HT1B receptor function by p11 in depression-like states[END_REF][START_REF] Warner-Schmidt | Role of p11 in cellular and behavioral effects of 5-HT4 receptor stimulation[END_REF]. Indeed, p11 KO mice exhibit depression-like phenotype and reduced responsiveness to 5-HT1B and 5-HT4 agonists in rodent behavioral models of depression. Further supporting a role of p11 in depression-like states, p11 brain expression is increased by antidepressant treatments, whereas the level of the protein is reduced in an animal model of depression as well as in the brain of patients who had suffered from unipolar major depression [START_REF] Svenningsson | Alterations in 5-HT1B receptor function by p11 in depression-like states[END_REF]. Moreover, over-expression of p11 in neurons produces behavioral features reminiscent of those observed after antidepressant treatment. Collectively, these data suggest that alteration of 5-HT1B receptor/p11 and 5-HT4 receptor/p11 interactions might contribute, at least in part, to the etiology of depressed states. 5-HT2C receptors interact with the tumor suppressor PTEN, an enzyme exhibiting both lipid and protein phosphatase activities [START_REF] Ji | Disruption of PTEN coupling with 5-HT2C receptors suppresses behavioral responses induced by drugs of abuse[END_REF]. This interaction was detected in dopaminergic neurons of the ventral tegmental area (VTA) innervating the accumbens nucleus, which are tonically inhibited by 5-HT2C receptors, and prevented agonist-induced receptor phosphorylation [START_REF] Ji | Disruption of PTEN coupling with 5-HT2C receptors suppresses behavioral responses induced by drugs of abuse[END_REF]. Disruption of 5-HT2C receptor/PTEN interaction in the VTA reproduced the inhibitory effect of 5-HT2C agonists on VTA dopaminergic neuron firing and prevented the rewarding effects of cannabinoids that were mediated by increased activity of VTA dopaminergic neurons [START_REF] Ji | Disruption of PTEN coupling with 5-HT2C receptors suppresses behavioral responses induced by drugs of abuse[END_REF].

Importantly, disconnecting 5-HT2C receptors from PTEN did not reproduce the side effects evoked by 5-HT2C agonists, such as anxiety, penile erection, hypophagia and suppression of locomotor activity. These data suggest that 5-HT2C receptor/PTEN interaction might contribute to the reinforcing role of drugs and thereby constitutes a potential target for the treatment of drug addiction-related behaviors.

5-HT3 receptor signaling

The signaling events associated with 5-HT3 receptors are "in principle" very simple. Being cationic channels permeable Na + , Ca 2+ and K + , activation of 5-HT3 receptors result in cell depolarization. 5-HT3 receptors mediate fast synaptic response post-synaptically and stimulate the release of 5-HT and some others neurotransmitters pre-synaptically. There are discrepancies between the biophysical properties of the homo-pentameric 5-HT3A receptors and native 5-HT3 receptors.

Following the cloning of 5-HT3B receptor, these discrepancies have been, at least in part, solved by the observation that the 5-HT3AB receptors have properties more similar to native receptors. The complexity of native channels is likely to be much higher due to the existence of splices variants of 5-HT3A and 5-HT3B receptors and the possible formation of hetero-pentamers between 5-HT3A and with the newly cloned 5-HT3C, D, E forms [START_REF] Jensen | 3B but which 3B? And that's just one of the questions: the heterogeneity of human 5-HT(3) receptors[END_REF]. A) In somato-sensoriel cortex, [START_REF] Gonzalez-Maeso | Identification of a serotonin/glutamate receptor complex implicated in psychosis[END_REF] showed a heterodimerization between mGluR2 and 5-HT2A receptors. In cortical cultures and somato-sensoriel cortex, stimulation by non-hallucinogenic such as lisuride, 5-HT itself and hallucinogenic drugs, such as LSD and DOI, engaged the Gq/G11/PLC/c-fos pathway [START_REF] Gonzalez-Maeso | Identification of a serotonin/glutamate receptor complex implicated in psychosis[END_REF][START_REF] Gonzalez-Maeso | Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior[END_REF]. B) In cortical cultures, [START_REF] Gonzalez-Maeso | Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior[END_REF]) and somato-sensoriel cortex [START_REF] Gonzalez-Maeso | Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior[END_REF][START_REF] Gonzalez-Maeso | Identification of a serotonin/glutamate receptor complex implicated in psychosis[END_REF], hallucinogenic drugs such as LSD and DOI, but not non-hallucinogenic drugs, activated in addition to the Gq/G11/PLC/c-fos pathway, the Gi/Go/Src/erg-2 pathway. In the somato-sensoriel cortex, activation of mGluR2 with LY 341495 inhibited the Gi/Go/Src/erg-2 pathway but not the Gq/G11/PLC/c-fos. LY 341495, an mGluR2 othosteric agonist, inhibited the stereotyped head twitch behavior induced by hallucinogens [START_REF] Gonzalez-Maeso | Identification of a serotonin/glutamate receptor complex implicated in psychosis[END_REF].

In the medial frontal cortex, the allosteric agonist BINA inhibited the stereotyped head twitch behavior and c-fos induction of hallucinogens. Whether or not there is a regional difference (somato-sensoriel versus medial frontal cortex) between the control triggered by mGluR2 on the c-fos induction by hallucinogens, remain to be seen [START_REF] Benneyworth | 31 glutamate receptor subtype 2 blocks a hallucinogenic drug model of psychosis[END_REF].

Table 1: Proteins identified as specific partners of 5-HT receptors and their role at the cellular level and in vivo.

Only receptor subtypes for which interacting proteins have been unambiguously identified are mentioned. * Refers to experiments using receptors mutated on their PDZ binding motif. $ P11 binds to the third intracellular loop of 5-HT4 receptors and therefore might equally interact with all receptor splice variants, including 5-HT4a and 5-HT4e receptors.

  to use radiolabeled [ 3 H]-5-HT, [ 3 H]-LSD and [ 3 H]-spiperone a dopamine/5-HT antagonist to label brain 5-HT receptors. Two types of receptors were pharmacologically characterized with those binding studies, the 5-HT1 (labeled with [ 3 H]-5-HT and [ 3 H]-LSD) and the 5-HT2 receptors (labeled with [ 3 H]-spiperone and [ 3 H]-LSD. Based on both Gaddum et al.

FIGURE LEGENDS Figure 1 :

 LEGENDS1 FIGURE LEGENDS

Figure 2 :

 2 Figure 2: Human gene structures of 5-HT receptors coding sequences

Figure 3 :

 3 Figure 3: Signaling pathways of major 5-HT receptors

Figure 4 :

 4 Figure 4: Hallucinogens effects of some 5-HT2A agonists such as LSD, DOI…: implication of "Ligand directed signaling" of 5-HT2A receptors and inhibition of

Signaling, trafficking Dendritic targeting [START_REF] Turner | Calmodulin interacts with the third intracellular loop of the serotonin 5-hydroxytryptamine1A receptor at two distinct sites: putative role in receptor phosphorylation by protein kinase C[END_REF][START_REF] Della Rocca | Serotonin 5-HT1A receptor-mediated Erk activation requires calcium/calmodulin-dependent receptor endocytosis[END_REF]) [START_REF] Carrel | Targeting of the 5-HT1A serotonin receptor to neuronal dendrites is mediated by Yif1B[END_REF] [START_REF] Ullmer | Cloning and characterization of MUPP1, a novel PDZ domain protein[END_REF][START_REF] Becamel | Interaction of serotonin 5-hydroxytryptamine type 2C receptors with PDZ10 of the multi-PDZ domain protein MUPP1[END_REF][START_REF] Parker | Agonistinduced phosphorylation of the serotonin 5-HT2C receptor regulates its interaction with multiple PDZ protein 1[END_REF]) [START_REF] Becamel | Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach[END_REF], Becamel et al., 2004[START_REF] Gavarini | Opposite effects of PSD-95 and MPP3 PDZ proteins on serotonin 5-hydroxytryptamine2C receptor desensitization and membrane stability[END_REF]) [START_REF] Becamel | The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins[END_REF]) [START_REF] Becamel | The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins[END_REF]) [START_REF] Becamel | The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins[END_REF]) [START_REF] Becamel | Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach[END_REF], Becamel et al., 2004[START_REF] Gavarini | Opposite effects of PSD-95 and MPP3 PDZ proteins on serotonin 5-hydroxytryptamine2C receptor desensitization and membrane stability[END_REF]) [START_REF] Becamel | Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach[END_REF], Becamel et al., 2004) [START_REF] Becamel | Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach[END_REF][START_REF] Labasque | Physical interaction of calmodulin with the 5-hydroxytryptamine2C receptor Cterminus is essential for G protein-independent, arrestin-dependent receptor signaling[END_REF]) [START_REF] Becamel | Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach[END_REF]) [START_REF] Becamel | Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach[END_REF]) [START_REF] Ji | Disruption of PTEN coupling with 5-HT2C receptors suppresses behavioral responses induced by drugs of abuse[END_REF] 5-HT4 P11 $ + Trafficking, signaling, antidepressant response to receptor stimulation [START_REF] Warner-Schmidt | Role of p11 in cellular and behavioral effects of 5-HT4 receptor stimulation[END_REF] 5-HT4(a) SNX27

NHERF-1 (EBP50) MAGI2 MPP3 Veli 1-3 CRMP2 Src

Targeting Targeting Signaling [START_REF] Joubert | New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting[END_REF]) [START_REF] Joubert | New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting[END_REF]) [START_REF] Joubert | New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting[END_REF]) [START_REF] Joubert | New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting[END_REF]) [START_REF] Joubert | New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting[END_REF]) [START_REF] Joubert | New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting[END_REF]) [START_REF] Barthet | 5-hydroxytryptamine 4 receptor activation of the extracellular signal-regulated kinase pathway depends on Src activation but not on G protein or beta-arrestin signaling[END_REF]) 5-HT4(e) CIPP nNOS Sec23 + + + [START_REF] Joubert | New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting[END_REF]) [START_REF] Joubert | New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting[END_REF]) [START_REF] Joubert | New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting[END_REF]) 5-HT6 Fyn + Trafficking, signaling [START_REF] Yun | The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn[END_REF]