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

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

Valerie Compan
email: valerie.compan@igf.cnrs.fr

Valérie Compan

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

5-HT4 receptors, a place in the sun: Act two

published or not. The documents may come

Introduction

G protein-coupled receptors (GPCRs) are conserved molecules which have been central, during evolution to recognize and transduce external (e.g. odours, gustative molecules, light) as well as cell-cell communication signals (hormones, neurotransmitters, some growth factors) [START_REF] Bockaert | Molecular tinkering of G protein-coupled receptors: an evolutionary success[END_REF]. They comprise 2% of the human genome and remain central for the understanding of human physiology and human diseases. 40-50% of all marketed drugs act directly to modulate GPCRs even if less than 20% of GPCRs (non-odorant receptors) are currently targeted. 5-HT4 receptors reached their "place in the sun" of GPCR pharmacology almost 20 years ago [2*] and remain the matter of intense research especially in brain and gastro-intestinal systems [3*]. Indeed, the hope that 5-HT4 receptor agonists could be successful drugs in gastro-intestinal pathologies and in particular for chronic idiopathic constipation is high. Several compounds have already been tested [3*]. Some have been withdrawn for adverse but non 5-HT4 receptormediated side effects. For example, cisapride is known to inhibit hERG channels [START_REF] Kamiya | Molecular determinants of hERG channel block by terfenadine and cisapride[END_REF]. However, prucalopride, has just been commercialized [START_REF] Manabe | New-generation 5-HT4 receptor agonists: potential for treatment of gastrointestinal motility disorders[END_REF]. In brain, none 5-HT4 receptor ligand has yet reached such a development level, even if a clinical trial for Alzheimer's disease reached phase IIb [START_REF] Bockaert | Dumuis A: 5-HT4 receptors[END_REF][START_REF] Moser | SL65.0155, a novel 5hydroxytryptamine(4) receptor partial agonist with potent cognition-enhancing properties[END_REF]. In addition to this pathology, important discoveries indicate that two others mental diseases, such as depression and feeding disorders, could benefit from modulation of 5-HT4 receptor signalling [8*-11*]. Recently, it has been proposed that GPCR interacting proteins (GIPs) may be used for modulating GPCR-signalling in pathologies [12*]. For example, p11, a GIP that modulates the trafficking of 5-HT1B and 5-HT4 receptors to the plasma membrane, has been expressed, in nucleus accumbens (NAc) to reverse depression like behaviours in mice [13**]. In this short review, we will only discuss recent studies on new aspects of 5-HT4 receptor signalling including non G protein-mediated events [14*], the nature and functions of the 5-HT4 receptor-associated GIPs [12*] as well as new evidences for a role of those receptors in important aforementioned brain diseases. Is a "place in the sun" for 5-HT4 receptors, in brain diseases treatments close to be reached?

New vistas in 5-HT4 receptors signalling

The capacity of 5-HT4 receptors to couple and initiate signalling pathways through multiple G proteins, especially Gs, G13 and in some cell lines Gq and Gi, has already been documented in precedent reviews [3*,15]. Here, we are highlighting recent studies which provide evidence that 5-HT4 receptor stimulation mediates G protein-independent pathways, the first one being ERK activation through phosphorylation and activation of the 5-HT4 receptor-associated nonreceptor tyrosine kinase Src (Figure 1). 5-HT4 receptor-mediated ERK activation is the first example that involves neither a classical G protein signalling (Gs/Gq/Gi) and associated messengers, nor b-arrestins [14*]. This result does not seem to be cell specific, because 5-HT4 receptors stimulation activates Src in HEK293, neurons [14*] and also in epithelial cell lines. Indeed, in the enterocyte cell line Caco 2, 5-HT4 receptor-induced Src activation was required to activate the PLCg1 pathway and to inhibit Na + /H + exchanger [16] (Figure 1). As nothing was known about desensitization of GPCRs acting in a G protein-independent manner, we investigated this question using 5-HT4 receptors. We reported that the 5-HT4 receptor-operated Src/ERK pathway, but not the Gs pathway, was negatively regulated by GPCR kinase 5 (GRK5) physically pre-associated with the proximal C-terminal region of the receptor in both human HEK293 cells and mouse colliculi neurons [17**]. This inhibition required two sequences of events: the association of b-arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C-terminal domain and the phosphorylation, by GRK5, of b-arrestin1 (at Ser412) bound to the receptor. Phosphorylated b-arrestin1, in turn, prevented activation of Src constitutively bound to 5-HT4 receptors, a necessary step in receptor-stimulated ERK signalling [17**] (Figure 1).

GPCR interacting proteins (GIPs) associated with 5-HT4 receptors

GPCR functions are controlled by numerous protein partners named GIPs [12*]. These interactions create additional opportunities for G protein-dependent but also independent signalling. In a first study using a proteomic approach, we found ten proteins that have been identified to interact directly with the C-terminus of 5-HT4(a) receptor variant and three that associate with the C-terminus of 5-HT4(e) receptor variant, most through PDZ domains. Among the more interesting interacting proteins are the Na + /H + exchanger regulatory factor (NHERF) and sorting nexin 27 (SNX27) [18*]. The group of P. Greengard recently documented the interaction of 5-HT4 receptors with p11. p11 (also called S100A10 or calpactin-1 light chain or annexin-2 light chain) is an inducible 97-amino acid protein member of the S100 superfamily comprising 10-14 kDa, EF hand-containing Ca 2+ -binding proteins, which transmit Ca 2+dependent cell-regulatory signals. This protein is present in a large number of organisms, interact with trafficking proteins, several channels but also with the 5-HT1B and the 5-HT4 receptors [19**]. Interaction of both receptors with p11 increased their cell surface localization and, correspondingly, enhanced their associated signal transduction [20*]. The majority of GIPs associated to 5-HT receptors have so far been isolated by in vitro binding assays using GPCR C-termini as baits [18*,21,22]. Recently, we identified more than 150 new protein partners of the 5-HT4 receptor by a shot gun proteomic approach associating immunoprecipitation of the N-terminally tagged receptor, expressed in an heterologous system (HEK293 cells), and systematic identification using tandem mass spectrometry. Several of the identified proteins are known to act on the trafficking and/or the metabolism of APP (amyloid precursor protein) (unpublished data), reinforcing the proposal that 5-HT4 receptors could be a target for Alzheimer disease [23*].

Learning and memory and Alzheimer's disease

Pro-cognitive effects of 5-HT4 receptor agonists have been described, both on short-term memory (such as social olfactory memory) or long-term olfactory memory (such as olfactory associative memory, for a review see [START_REF] Bockaert | Dumuis A: 5-HT4 receptors[END_REF]). 5-HT4 receptor-induced increase in memory acquisition has been seen in autoshaping task, object recognition, Morris water-maze, task with long inter-trial intervals (2 h), delayed matching performance and impeded spontaneous alteration scores [START_REF] Bockaert | Dumuis A: 5-HT4 receptors[END_REF]. Such positive effects on learning and memory could rely on a 5-HT4 receptor-mediated LTP (long term potentiation) induction that has been documented in the amygdala [24*] and suggested in the hippocampus [START_REF] Kemp | Hippocampal long-term depression and long-term potentiation encode different aspects of novelty acquisition[END_REF], where 5-HT4 receptor activation cause a long-lasting excitability increase in CA1 pyramidal neurons [START_REF] Mlinar | 5-HT4 receptor activation induces long-lasting EPSP-spike potentiation in CA1 pyramidal neurons[END_REF]. The complexity of 5-HT4 receptor effects on the synaptic plasticity of the Shaffer-collateral/CA1 hippocampal synapses in correlation with the type of learning has been highlighted [START_REF] Kemp | Hippocampal long-term depression and long-term potentiation encode different aspects of novelty acquisition[END_REF]. The conclusion being that 5-HT4 receptor activation may be beneficial for particular types of information acquisition that depend on LTP such as exploration of an empty novel environment and detrimental for other types of learning which required LTD (long term depression) such as exploration of a new environment containing unfamiliar objects. A recent report shows that an increase in cAMP phosphodiesterases (PDEs) activity occurs in the prefrontal cortex (PDE4) and hippocampus, following 5-HT4 receptor stimulation. This may be part of a negative feed-back regulation mediated by cAMP [START_REF] Levallet | Increased particulate phosphodiesterase 4 in the prefrontal cortex supports 5-HT4 receptor-induced improvement of object recognition memory in the rat[END_REF]. Moreover, we contributed to show that the promnesic effect of 5-HT4 receptor activation is also mediated by a potentiation of learning-induced spine growth in the hippocampus [28**]. In 5-HT4 receptor knock-out mice, the loss of function mutation on learning and memory appears to be circumvented by adaptative changes in cholinergic systems [START_REF] Segu | Hyperfunction of muscarinic receptor maintains long-term memory in 5-HT4 receptor knock-out mice[END_REF], supporting the fact that 5-HT4 receptor can have a role to play in Alzheimer's disease (AD). Indeed, 5-HT4 receptors have been reported to promote non-amyloidogenic processing of the amyloid precursor protein (APP) by stimulating the a-secretase [23*,30]. Along these lines, a selective 5-HT4 receptor partial agonist, RS 67333 inhibits the generation of the bamyloid peptide (Ab) in primary cortical neurons [START_REF] Cachard-Chastel | 5-HT4 receptor agonists increase sAPPalpha levels in the cortex and hippocampus of male C57BL/6j mice[END_REF]. Interestingly, two 5-HT4 receptor partial agonists, the SL 65.0155 from Sanofi-Aventis [START_REF] Moser | SL65.0155, a novel 5hydroxytryptamine(4) receptor partial agonist with potent cognition-enhancing properties[END_REF] and VRX-03011 (Epix Pharmaceuticals [START_REF] Mohler | VRX-03011, a novel 5-HT4 agonist, enhances memory and hippocampal acetylcholine efflux[END_REF]) have reached clinical phase II for the treatment of Alzheimer's disease. Both compounds act synergically with cholinesterase inhibitors on rodent memory. All these studies correlate with reports documenting a significant decrease in the density of 5-HT4 receptor binding sites in cortical and hippocampal regions in the Alzheimer brain [START_REF] Reynolds | 5-Hydroxytryptamine (5-HT)4 receptors in post mortem human brain tissue: distribution, pharmacology and effects of neurodegenerative diseases[END_REF][START_REF] Wong | Characterization of [3H]GR 113808 binding to 5-HT4 receptors in brain tissues from patients with neurodegenerative disorders[END_REF][START_REF] Tsang | A serotoninergic basis for hyperphagic eating changes in Alzheimer's disease[END_REF].

5-HT4 receptors for treating feeding disorders

The first evidence that 5-HT4 receptors could be implicated in feeding disorders was obtained using the 5-HT4 receptor knock-out mice [11*]. Indeed, the defective mutation of the mHtr4 gene encoding 5-HT4 receptors reduced hypophagia following restraint stress, a possible animal model for anorexia nervosa [START_REF] Rybkin | Effect of restraint stress on food intake and body weight is determined by time of day[END_REF]. It has been proposed that motivation disorders whereby patients self-impose food refusal despite an energy request (anorexia-like behaviour) could be seen as prominent disturbances in a reward structure (nucleus accumbens: NAc) rather than in the hypothalamus [10**,36]. We have recently described that the rewarding drug MDMA (3,4-N-methylenedioxymethamphetamine) reduces feeding via 5-HT4 receptor activation an their downstream signalling cascade cAMP/PKA synthesis of CART, an anorectic and addiction peptide (CART: Cocaine-and amphetamine-regulated transcript) [10**]. Intra-accumbal injection of a 5-HT4 receptor antagonist (RS 39604) or siRNAmediated 5-HT4 receptors knock-down decrease satiety [10**,34] and indicate a 5-HT4 receptors localization on intrinsic, likely GABAergic neurons of the NAc (Figure 2). The neuronal network of addiction may thus include the neuronal pathway related to anorexia [10**]. Full 5-HT4 receptor antagonists may thus be useful drugs to treat anorexia related disorders. It is interesting to note that oral administration of the 5-HT4 receptor partial agonist, mosapride citrate, increased feeding in an animal model of obesity (ob/ob) mice [START_REF] Asakawa | Mosapride improves food intake, while not worsening glycemic control and obesity, in ob/ob obese mice with decreased gastric emptying[END_REF] (mosapride being a partial agonist, can also partially inhibit 5-HT4 receptors activation by endogenous 5-HT). Current studies of the involvement of 5-HT4 receptors and related mechanisms in stress-induced hypophagia and in addiction to drugs of abuse (e. g. cocaine) are in progress. Studies conducted in mice have found a promising opening in human therapy. Indeed, recently, the density of 5-HT4 receptors was found to be decreased in Alzheimer's disease patients also suffering from hyperphagia [START_REF] Tsang | A serotoninergic basis for hyperphagic eating changes in Alzheimer's disease[END_REF]. New development of PET radioligand such as [ 11 C]SB207145 should facilitate in vivo brain imaging of 5-HT4 receptors in human brain disorders [START_REF] Kornum | Evaluation of the novel 5-HT4 receptor PET ligand [11C]SB207145 in the Gottingen minipig[END_REF].

Treating depression

Few years ago, polymorphisms of the Htr4 gene encoding the 5-HT4 receptors have been highly correlated with major depression and/or bipolar disorders and post-mortem examination of brain of depressed suicide victims indicate an altered expression of 5-HT4 receptors (for a review see [8*]). However, the evidence that 5-HT4 receptors may be a direct target for treating depression is more recent [8*]. It is well known that inhibitors of 5-HT and nor-epinephrine reuptake are central in the therapeutic action of antidepressants. In particular, 5-HT reuptake inhibitors (SSRI) are commonly used. The final effect of these drugs seems to be an increase in the tonic 5-HT release and activation of post-synaptic 5-HT receptors, in particular 5-HT1A receptors in limbic structures such as hippocampus. However, this effect is delayed and obtained only after 3-8 weeks. One of the possible explanations may be that this delay is due to the period needed to obtain desensitization of the self-inhibition of 5-HT neurons by somatodendritic 5-HT1A receptors. At the beginning of SSRI treatment, 5-HT neuron activities are quasi suppressed and symptoms may be increased, in particular anxiety. Thus, this period is problematic and drugs rapidly activating 5-HT neurons may facilitate SSRI treatments and even be antidepressant by themselves. 5-HT4 receptors were first shown to increase 5-HT release in the hippocampus [START_REF] Ge | 5-HT4 receptor-mediated modulation of 5-HT release in the rat hippocampus in vivo[END_REF] and then to be a tonic activator of about half of dorsal raphe nucleus (DRN) 5-HT neurons called "responders" neurons. It appears that those neurons have a higher basal firing rate (1.9Hz) than non-responders ones (1.3 Hz) [START_REF] Lucas | 5-HT4 receptors exert a frequency-related facilitatory control on dorsal raphe nucleus 5-HT neuronal activity[END_REF]. Thus, i.p. administration of the 5-HT4 receptor antagonists (such as GR 125487) highly reduced the basal firing rate of "responders" but were inactive on "non-responders" [START_REF] Lucas | 5-HT4 receptors exert a frequency-related facilitatory control on dorsal raphe nucleus 5-HT neuronal activity[END_REF]. Conversely, "responders" but not "non-responders" were activated by i.p. administration of 5-HT4 receptor agonists such as cisapride, prucalopride or RS 67333 indicating that 5-HT4 receptors have also a phasic control of firing [8*,40]. More interestingly, the inhibitory effect of SSRI on 5-HT neuron firing was counteracted by prucalopride in neurons discharging at 2Hz or higher but not when the frequency was < 1Hz [8*]. When an SSRI such as citalopram was co-administrated with prucalopride or RS 67333 for 3 days, the activity of DRN 5-HT neurons was stimulated instead of inhibited when citalopram was given alone [9**]. These results are in accordance with the observation that the average firing rate of DRN 5-HT neurons was reduced by more than 50% in 5-HT4 receptor knock-out mice compared to WT [START_REF] Conductier | Adaptive changes in serotonin neurons of the raphe nuclei in 5-HT(4) receptor knock-out mouse[END_REF]. Since the effect of 5-HT4 receptor agonists on firing rate was observed after i.p. delivery but not after a direct delivery at the DR level, indicates that the 5-HT4 receptors involved were not localized in DRN [42*] (Figure 2). Since 1) the electrical stimulation of medial prefrontal cortex (mPFC) stimulates the electrical activity of 40-45% of DRN 5-HT neurons, 2) the mPFC 5-HT1ARs mediated a "longloop feedback" on about half of DRN 5-HT neurons and 3) the 5-HT4 and 5-HT1A receptors are co-localized on pyramidal mPFC, it was tempting to hypothesized that the 5-HT4 receptordependent control of DRN neuronal activity was originating from mPFC. Indeed, pyramidal cells express 5-HT4 receptors (see [8*,42*,43*] for details) (Figure 2). Viral-induced overexpression of 5-HT4 receptors in the mPFC but not in others discrete brain areas induced a marked increase in DR 5-HT neuronal firing [42*]. In conclusion, those convergent results demonstrated that mPFC 5-HT4 receptors exert an excitatory influence of 5-HT neurons that compensates (at least after 3 days of activation with a 5-HT4 receptor agonist) the initial suppression of DR 5-HT neurons activity by SSRI [9**]. Thus, 5-HT4 receptor agonists may be antidepressant by themselves or reduce the delay of action of SSRI. This has been clearly the case as shown by Lucas et al. [9**] on a series of biological and behavioural parameters in rats. The biological parameters were including neurogenesis of hippocampal granular cells (GCs), which are also expressing 5-HT4 receptors (Figure 2 The fact that chronic SSRI treatments led to 5-HT4 receptor stimulation is also suggested by the observation that such treatments induced a downregulation of those receptors in hippocampus (particularly CA1 region), striatum, and substantia nigra but, interestingly, not in mPFC [START_REF] Licht | The brain 5-HT4 receptor binding is down-regulated in the Flinders Sensitive Line depression model and in response to paroxetine administration[END_REF][START_REF] Vidal | Long-term treatment with fluoxetine induces desensitization of 5-HT4 receptor-dependent signalling and functionality in rat brain[END_REF]. Finally, the p11 saga has highlighted the connection between 5-HT4 receptors and depression. This protein has been identified as a new antidepressant target. p11 is down-regulated in depressive-like state in rodents, in the HelplessH/Rouen mice, and human brain, and is up-regulated following anti-depressant treatments including electroconvulsive treatments [19**]. p11 knock-out mice display a depressive-like phenotype whereas mice over-expressing p11 behave as those treated with antidepressant [19**]. p11 interacts with both 5-HT1B and 5-HT4 receptors (but not with many others GPCRs), facilitates their cell surface localization as well as their signalling and neuronal functions [19**]. Interestingly, 5-HT1B receptors are pre-synaptically localized and their effects on the inhibition of cortico-accumbal glutamatergic transmission require p11 [19**]. This observation, as well as human functional imaging or results from therapeutic deep brain stimulation of depression corroborating animal studies suggesting that reduce activity in this region (see [13**]) can promote depression, led Alexander et al. [13**] to investigate the possibility that adeno-associated virus (AAV) p11 gene-transfer to NAc could reverse their depression like behaviours. This was indeed the case and was associated with an increase in 5-HT1B receptors in density [13**]. The data did not indicate if this increase is taking place on NAc presynaptic (glutamatergic?) terminals or on the intrinsic NAc neurons or both. This will be important to know. Is the p11-mediated increase in 5-HT4 receptor membrane targeting also involved in this gene therapy? The presence of 5-HT4 receptors on intrinsic NAc neurons as well as their ability to increase cAMP and CREB phosphorylation may suggest such a role [10**]. However note that p11 is also controlling the trafficking of several ionic channels see [19**]. Is p11 gene-transfer in human treatment of severe depression, a science fiction story or a reasonable hope? The future will tell us.

Conclusion

Drug development is a long and a non-linear process with many go and no periods. Several 5-HT4 receptor drugs have been on the market for gastro-intestinal health problems such as gastro-oesophageal reflux and irritable bowel syndrome associated with constipation and recently constipation in women in whom laxatives were inefficient. This experience should be beneficial for development of brain 5-HT4 receptor acting drugs. Here again, the process is very slow. Some have been tested in clinic for Alzheimer's disease but the processes were stopped either for lack of significant efficacy in phase IIb (SL 65.0155 from Sanofi-Aventis) or for economical reasons (VRX-03011 from Epix Pharmaceuticals). However, the clinical trials for Alzheimer's disease are problematic due to heterogeneity of patients and lack of biomarkers. More work is needed on the role of 5-HT4 receptors on cellular biology of APP. We have recently performed a deep proteomic analysis of 5-HT4 receptors "receptosome" and found that it includes several important proteins known to be implicated in the trafficking and metabolism of APP (in preparation). The hope is also that 5-HT4 receptors will reach clinical evaluation for feeding-associated diseases such as anorexia, the third chronic illness of adolescent in industrialized countries and major depressive disorders identified as the leading cause of disability in the Western World for those aged 15 to [START_REF] Kobayashi | Reversal of hippocampal neuronal maturation by serotonergic antidepressants[END_REF] 5-HT4 receptors are mainly located on neurons containing g-aminobutyric acid (GABA) in the nucleus accumbens and striatum [START_REF] Compan | Lesion study of the distribution of serotonin 5-HT4 receptors in rat basal ganglia and hippocampus[END_REF], pyramidal cells expressing the glutamate (GLU) in the medial prefrontal cortex [43*] and hippocampus (CA1 [START_REF] Roychowdhury | 5-HT1A and 5-HT4 receptor colocalization on hippocampal pyramidal cells[END_REF], CA3: [START_REF] Vilaro | Serotonin 5-HT4 receptors and their mRNAs in rat and guinea pig brain: distribution and effects of neurotoxic lesions[END_REF] and granule cells [44**,49]). 5-HT4 receptors are unlikely located on dorsal raphe nucleus (DRN) [START_REF] Vilaro | Serotonin 5-HT4 receptors and their mRNAs in rat and guinea pig brain: distribution and effects of neurotoxic lesions[END_REF] (Compan, unpublished). However, autoradiographic studies have shown moderated densities of 5-HT4 receptors in the rat DRN, using both the radiolabeled antagonists [ 3 H]GR 113808 [START_REF] Waeber | Regional distribution and ontogeny of 5-HT4 binding sites in rodent brain[END_REF] or [ 125 I]SB207710 [START_REF] Vilaro | Serotonin 5-HT4 receptors and their mRNAs in rat and guinea pig brain: distribution and effects of neurotoxic lesions[END_REF] suggesting that these receptors are possibly localized on DRN afferents neurons. 5-HT4 receptors are heteroreceptors, located on the somatodendritic and axon terminals of the spiny efferent neurons of the striatum containing GABA to the substantia nigra and globus pallidus [START_REF] Compan | Lesion study of the distribution of serotonin 5-HT4 receptors in rat basal ganglia and hippocampus[END_REF]. 5-HT4 receptors are not located on the nigro-striatal dopaminergic neurons [START_REF] Compan | Lesion study of the distribution of serotonin 5-HT4 receptors in rat basal ganglia and hippocampus[END_REF].

In brain areas of the reward system, 5-HT4 receptors are located on the pyramidal cells of the medial prefrontal cortex [43*] that also expressed 5-HT1A and 5-HT2A receptors. More recently, it has been shown that 5-HT4 receptors are likely expressed by efferent neurons of the nucleus accumbens to the lateral hypothalamus [10**].

Medial Prefrontal Cortex

  ) [9**]. Interestingly, Kobayashi et al. [44**] described recently an other effect of fluoxetine, a classical SSRI, on GCs. Fluoxetine induced GC "dematuration" which developed along with increases in efficacy of 5-HT4 receptor-dependent neuromodulation of the GCs to CA3 synapses and was almost suppressed in mice lacking those receptors [44**].

  years. *8. Lucas G: Serotonin receptors, type 4: a new hope? Curr Drug Targets 2009, 10:1085-1095. A comprehensive review on the potential use of 5-HT4 receptors as a target for treating depression **9. Lucas G, Rymar VV, Du J, Mnie-Filali O, Bisgaard C, Manta S, Lambas-Senas L, Wiborg O, Haddjeri N, Pineyro G, et al.: Serotonin(4) (5-HT(4)) receptor agonists are putative antidepressants with a rapid onset of action. Neuron 2007, 55:712-725. This report is giving compelling and very important evidence, from preclinical animal models, that agonists acting at the 5-HT4 receptors be fast-acting antidepressants and may act in synergy with classical antidepressants. **10. Jean A, Conductier G, Manrique C, Bouras C, Berta P, Hen R, Charnay Y, Bockaert J, Compan V: Anorexia induced by activation of serotonin 5-HT4 receptors is mediated by increases in CART in the nucleus accumbens. Proc Natl Acad Sci U S A 2007, 104:16335-16340. Although 5-HT receptors, in the hypothalamus, has been central for the 5-HT-mediated hypophagia, this article describes an important role of 5-HT4 receptors expressed in the Nucleus accumbens (NAc) in the anorectic effects of 3,4-N-methylene-dioxymethamphetamine (MDMA, the active principle of ectasy). Direct activation of 5-HT4 receptors of the NAc reduced the physiological drive to eat in food deprived mice and increases the synthesis of the CART peptide (CART:cocaine-and amphetamine-regulated transcript) via a cAMP/PKA signalling pathway *11. Compan V, Zhou M, Grailhe R, Gazzara RA, Martin R, Gingrich J, Dumuis A, Brunner D, Bockaert J, Hen R: Attenuated response to stress and novelty and hypersensitivity to seizures in 5-HT4 receptor knock-out mice. J Neurosci 2004, 24:412-419. Generation of 5-HT4 receptor knock-out mice and description of the first phenotypes. *12. Bockaert J, Perroy J, Becamel C, Marin P, Fagni L: GPCR interacting proteins (GIPs) in the nervous system: Roles in physiology and pathologies. Annu Rev Pharmacol Toxicol 2010, 50:89-109. A review analysing the nature, the physiological functions, pathological implications and the putative roles as drug targets of GPCR interacting proteins (GIPs). **13. Alexander B, Warner-Schmidt J, Eriksson T, Tamminga C, Arango-Llievano M, Ghose S, Vernov M, Stavarche M, Musatov S, Flajolet M, et al.: Reversal of depressed behaviors in mice by p11 gene therapy in the nucleus accumbens. Sci Transl Med 2010, 2:54ra76.A provocative report describing, in mice, data supporting a brain region-specific gene therapy for depression: adeno-associated virus (AAV)-mediated transfer of the gene encoding p11 to the NAC. p11 interacts with several channels, 5-HT1B and 5-HT4 receptors increasing their cell surface expression. p11 is increased in rodent brain following antidepressant and electroconvulsive therapies. Here, the authors found that local NAc knockdown of p11 resulted in behavioural deficits related to depression and that AAV-mediated p11 gene transfer to the NAc rescued the depression-related behaviours in p11 knock-out mice. Is gene therapy for severe psychiatric disorders a reasonable hope? *14. Barthet G, Framery B, Gaven F, Pellissier L, Reiter E, Claeysen S, Bockaert J, Dumuis A: 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. Mol Biol Cell 2007, 18:1979-1991. 5-HT4 receptors, as many GPCRs, can signal without activation of a G protein. Many GPCRs activate ERK pathway in a non-G but b-arrestin dependent manner whereas 5-HT4 receptors activate this pathway via the activation of Src and without the implication of b-arrestin. 15. Woehler A, Ponimaskin EG: G protein--mediated signaling: same receptor, multiple effectors. Curr Mol Pharmacol 2009, 2:237-248. 16. Gill RK, Saksena S, Tyagi S, Alrefai WA, Malakooti J, Sarwar Z, Turner JR, Ramaswamy K, Dudeja PK: Serotonin inhibits Na+/H+ exchange activity via 5-HT4 receptors and activation of PKC alpha in human intestinal epithelial cells. Gastroenterology 2005, 128:962-974. **17. Barthet G, Carrat G, Cassier E, Barker B, Gaven F, Pillot M, Framery B, Pellissier LP, Augier J, Kang DS, et al.: Beta-arrestin1 phosphorylation by GRK5 regulates G protein-independent 5-HT4 receptor signalling. Embo J 2009, 28:2706-2718.

Figure 1 :

 1 Figure 1: Gs-independent signalling and desensitization of 5-HT4 receptors. In addition to G proteins (mainly Gs) activation, 5-HT4 receptors activated the tyrosine kinase Src in a G-protein independent manner [13*]. Src is associated with the inactive 5-HT4 receptors and phosphorylated, therefore activated, upon receptor agonist stimulation. Activation of Src led to a b-arrestin-independent ERK activation in neurons or PLCg1 activation and finaly downstream Na + /H + exchanger inhibition in intestinal cells [16]. Desensitization of the 5-HT4 receptor-induced Src activation first involved GRK5 recruitment. GRK5 phosphorylated b-arrestin. Phosphorylated b-arrestin inhibited, via an unknown mechanism the 5-HT4 receptor-induced Src phosphorylation [17**].

Figure 2 :

 2 Figure 2: Cellular localization of 5-HT4 receptors in the forebrain.5-HT4 receptors are mainly located on neurons containing g-aminobutyric acid (GABA) in the nucleus accumbens and striatum[START_REF] Compan | Lesion study of the distribution of serotonin 5-HT4 receptors in rat basal ganglia and hippocampus[END_REF], pyramidal cells expressing the glutamate (GLU) in the medial prefrontal cortex [43*] and hippocampus (CA1[START_REF] Roychowdhury | 5-HT1A and 5-HT4 receptor colocalization on hippocampal pyramidal cells[END_REF], CA3:[START_REF] Vilaro | Serotonin 5-HT4 receptors and their mRNAs in rat and guinea pig brain: distribution and effects of neurotoxic lesions[END_REF] and granule cells [44**,49]). 5-HT4 receptors are unlikely located on dorsal raphe nucleus (DRN)[START_REF] Vilaro | Serotonin 5-HT4 receptors and their mRNAs in rat and guinea pig brain: distribution and effects of neurotoxic lesions[END_REF] (Compan, unpublished). However, autoradiographic studies have shown moderated densities of 5-HT4 receptors in the rat DRN, using both the radiolabeled antagonists [ 3 H]GR 113808[START_REF] Waeber | Regional distribution and ontogeny of 5-HT4 binding sites in rodent brain[END_REF] or [ 125 I]SB207710[START_REF] Vilaro | Serotonin 5-HT4 receptors and their mRNAs in rat and guinea pig brain: distribution and effects of neurotoxic lesions[END_REF] suggesting that these receptors are possibly localized on DRN afferents neurons. 5-HT4 receptors are heteroreceptors, located on the somatodendritic and axon terminals of the spiny efferent neurons of the striatum containing GABA to the substantia nigra and globus pallidus[START_REF] Compan | Lesion study of the distribution of serotonin 5-HT4 receptors in rat basal ganglia and hippocampus[END_REF]. 5-HT4 receptors are not located on the nigro-striatal dopaminergic neurons[START_REF] Compan | Lesion study of the distribution of serotonin 5-HT4 receptors in rat basal ganglia and hippocampus[END_REF]. In brain areas of the reward system, 5-HT4 receptors are located on the pyramidal cells of the medial prefrontal cortex[43*] that also expressed 5-HT1A and 5-HT2A receptors. More recently, it has been shown that 5-HT4 receptors are likely expressed by efferent neurons of the nucleus accumbens to the lateral hypothalamus [10**].


Lucie P Pellissier

Jessica Sallander

Mercedes Campillo

Florence Gaven

Emilie Queffeulou

Marion Pillot

Aline Dumuis

Sylvie Claeysen

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

Leonardo Pardo

Conformational Toggle Switches Implicated in Basal Constitutive and Agonist-Induced Activated States of 5-Hydroxytryptamine-4 Receptors

Keywords: 5-HT, 5-hydroxytryptamine, GPCRs, G protein-coupled receptors, TM, transmembrane, b2-AR, b2

come

INTRODUCTION

G-protein-coupled receptors (GPCRs) are allosteric molecules in equilibrium with many different conformational states [START_REF] Bond | Physiological effects of inverse agonists in transgenic mice with myocardial overexpression of the beta 2-adrenoceptor[END_REF][START_REF] Samama | A mutation-induced activated state of the b2-adrenergic receptor. Extending the ternary complex model[END_REF]. GPCRs adopt at least one inactive (R) and several active (R*) states [START_REF] Alves | Direct observation of G-protein binding to the human delta-opioid receptor using plasmon-waveguide resonance spectroscopy[END_REF][START_REF] Baneres | Molecular characterization of a purified 5-HT4 receptor: a structural basis for drug efficacy[END_REF][START_REF] Ghanouni | Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor[END_REF][START_REF] Okada | Activation of rhodopsin: new insights from structural and biochemical studies[END_REF][START_REF] Swaminath | Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states[END_REF]. R* states can be reached, even in the absence of agonists or mutations (called here basal constitutively active R* or R*basal), whereas chemically different ligands can stabilize different R*s. Partial agonists may stabilize a specific receptor state different from the R* of full agonist. For example, using circular dichroism difference spectra of the purified 5-HT4 receptor, we have previously found that free (or neutral antagonist-occupied), agonist-occupied (partial or full agonist) and silent (inverse agonist-occupied) receptors involved different arrangement of the e2 loop [START_REF] Baneres | Molecular characterization of a purified 5-HT4 receptor: a structural basis for drug efficacy[END_REF]. Inactive R state of family A GPCRs is generally not the equivalent to the "silent" state of the cis-retinal-rhodopsin receptor complex called the ground state (Rg) [START_REF] Palczewski | Crystal structure of rhodopsin: A G protein-coupled receptor[END_REF][START_REF] Park | Crystal structure of the ligand-free G-protein-coupled receptor opsin[END_REF]. Recent insights into the b2-adrenergic receptor (b2-AR) structure indicate that, even in the presence of the partial inverse agonist carazolol, this receptor is certainly not under the Rg state and adopts a structural feature of the R*s (a relatively open "ionic lock") [START_REF] Cherezov | High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor[END_REF][START_REF] Rosenbaum | GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function[END_REF]. Therefore, the Rg state may only be reached with full inverse agonists such as the cis-retinal and the R state may be, in some receptors, already a R* [START_REF] Palczewski | Crystal structure of rhodopsin: A G protein-coupled receptor[END_REF].

One of the main questions in GPCR molecular pharmacology is to understand the structural arrangements of the 7 transmembrane (TM) helices that occur to stabilize either Rg or the different R*s. Notably, some main arrangement and rearrangement events during activation seem to be common, at least in family A GPCRs [START_REF] Kobilka | G protein coupled receptor structure and activation[END_REF][START_REF] Ruprecht | Electron crystallography reveals the structure of metarhodopsin I[END_REF][START_REF] Schertler | Structure of rhodopsin and the metarhodopsin I photointermediate[END_REF][START_REF] Smit | Pharmacogenomic and structural analysis of constitutive g protein-coupled receptor activity[END_REF].

The recent crystal structure of the ligand-free opsin, which contains several features characteristic of R*s [START_REF] Park | Crystal structure of the ligand-free G-protein-coupled receptor opsin[END_REF], has shown that the intracellular part of TM6 is tilted outwards by 6-7 Å, while TM5 moves towards TM6 by 2-3 Å. In addition, conformational changes occurring to generate either Rg or R* states are accomplished by rearrangement of side chains forming different networks of interactions between helices [START_REF] Rosenbaum | GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function[END_REF]Shi et al., 2002;[START_REF] Smit | Pharmacogenomic and structural analysis of constitutive g protein-coupled receptor activity[END_REF]. The structure of metarhodopsin I by electron crystallography [START_REF] Ruprecht | Electron crystallography reveals the structure of metarhodopsin I[END_REF][START_REF] Schertler | Structure of rhodopsin and the metarhodopsin I photointermediate[END_REF], experimental studies of rhodopsin [START_REF] Lin | Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state[END_REF], as well as computer simulations associated with mutagenesis of b2-adrenergic (Shi et al., 2002), 5-HT2A [START_REF] Visiers | Three-dimensional representations of G proteincoupled receptor structures and mechanisms[END_REF], CB1 (McAllister et al., 2004) and histamine H1 receptors [START_REF] Jongejan | Linking agonist binding to histamine H1 receptor activation[END_REF] have shown that W6.48 of the conserved CWxPFF motif of TM6 undergoes a conformational transition, from pointing toward TM7 in the inactive state, to pointing toward TM5 in the agonist-induced R* [START_REF] Ruprecht | Electron crystallography reveals the structure of metarhodopsin I[END_REF][START_REF] Schertler | Structure of rhodopsin and the metarhodopsin I photointermediate[END_REF]. The side chain at position 3.36 has also been suggested to act as a rotamer toggle switch simultaneously with W6.48 in the histamine H1 and cannabinoid (CB1) receptors [START_REF] Jongejan | Linking agonist binding to histamine H1 receptor activation[END_REF][START_REF] Mcallister | Structural mimicry in class A G protein-coupled receptor rotamer toggle switches: the importance of the F3.36(201)/W6.48(357) interaction in cannabinoid CB1 receptor activation[END_REF].

In this study, we have investigated the 5-HT4 receptor (5-HT4R), and its ability to stabilize different R*s. The focus was mainly put on the roles of the W6.48 rotamer toggle switch and its associated toggle switch at position 3.36. We found that both toggle switches were required for stabilizing the basal R* state (called R*basal), none of them were fully implicated in stabilizing the R* reached in the presence of 5-HT (called R*-5-HT), whereas the T3.36 and the W6.48 toggle switches were implicated in stabilizing the R* in the presence of synthetic agonists belonging to either the benzamide or aryl ketone classes (called R*-benzamides), or to a benzimidazolone class (called R*-BIMU 8).

MATERIALS AND METHODS

Plasmid construct

HA-tagged-5-HT4 receptor cDNAs in pRK5 were generated by fusing the sequence of the HA epitope (YPYDVPDYA) to a cleavable signal peptide (MVLLLILSVLLLKEDVRGSAQS) derived from the metabotropic glutamate receptor 5 (mGlu5R). This sequence was inserted into the pRK5 vector using Xba I and BsrG I restriction sites. Then, full-length mouse 5-HT4(a)R cDNAs were subcloned in frame using BsrG I and Hind III. HA-5-HT4-T3.36A, HA-5-HT4-T3.36S, HA-5-HT4-T3.36C, HA-5-HT4-W6.48A receptor mutants were generated from HA-5-HT4(a)R, cloned in pRK5, with the Quick Change Site-Directed Mutagenesis Kit (Stratagene, Amsterdam, Netherlands).

Membrane preparation and radioligand binding assay

Membranes were prepared from transiently transfected cells plated on 15-cm dishes and grown in DMEM with 10% dialyzed foetal calf serum (dFCS) as described in Claeysen et al. [START_REF] Claeysen | A Single Mutation in the 5-HT4 Receptor (5-HT4-R D100(3.32)A) Generates a Gs-coupled Receptor Activated Exclusively by Synthetic Ligands (RASSL)[END_REF]. The membrane solubilized in 50 mM HEPES (pH 7.4; 5 mg of protein in 1 ml of solution) were stored at -80°C until use. Membrane suspension (about 10 µg), diluted in 100 µl of 50 mM HEPES containing 10 mM pargyline and 0.01% ascorbic acid, was incubated at 20°C for 30 min with 100 µl [ 3 H]GR 113808 (specific activity: 82 Ci/mmole) and 50 µl of buffer or competing drugs. For saturation analysis assays, various concentration of [ 3 H]GR 113808 (0.001-0.8 nM) were used. BIMU8 (10 µM) was used to determine non-specific binding. Protein concentration was determined by using the bicinchoninic acidic method.

Cell surface ELISA

COS-7 cells were transfected with HA-tagged-5-HT4R, WT or mutants cDNAs. Cells were grown on 96-well plates in DMEM medium with 10% dFCS and fixed as described previously [START_REF] Barthet | Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements[END_REF]. Cells were then incubated with anti-HA antibody at 0.6 µg/ml for 60 min in the same buffer and incubated with anti-rabbit/HRP conjugate (Amersham Pharmacia Biotech) at 1 µg/ml for 60 min.

Chromogenic substrate was added (Supersignal® ELISA femto-maximum sensitivity (Pierce, Perbio-Brebières, France). Chemiluminescence was detected and quantified by a Wallac Victor 2 luminescence counter.

Determination of cAMP production in transfected cells

COS-7 cells were transfected with the appropriate cDNA and seeded into 24-well plates (100 000 cells/well). 24 hours post-transfection, a 10 min-stimulation with the appropriate concentrations of drugs was performed as previously described [START_REF] Barthet | Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements[END_REF]. Quantification of cAMP production was performed by Homogenous Time Resolved Fluorescence (htrf®) using the cAMP Dynamic kit (Cisbio International, Bagnols-sur-Cèze, France), according to the manufacturer's instructions.

Data analysis

The dose-response curves were fitted using GraphPad Prism and the following equation for monophasic dose-response curves: y = (ymax -ymin) / 1 + [(x / EC50) nH] + ymin, where EC50 is the concentration of the compound necessary to obtain 50% of the maximal effect and nH is the Hill coefficient. Competition and saturation experiments were analysed by non-linear regression using GraphPad Prism as previously described [START_REF] Ansanay | Pharmacological comparison between [ 3 H]-GR113808 binding sites and functional 5-HT4 receptors in neurons[END_REF]. All data represented correspond to the mean ± SEM of three independent experiments performed in triplicate. Statistical analysis was carried out with the t test using GraphPad Prism 3.0 software.

Nomenclature of side chain conformation

The side chain conformation has been categorized into gauche-(g-: 0°< c1<120°), trans (t: 120°< c1<240°), or gauche+ (g+: 240°< c1<360°) depending on the value of the torsional c1 angle.

Numbering scheme of GPCRs

Residues are identified by the generic numbering scheme of [START_REF] Ballesteros | Integrated methods for the construction of three dimentional models and computational probing of structure-function relations in G protein coupled receptors[END_REF] that allows easy comparison among residues in the 7TM segments of different receptors.

Computational model of the ligand-receptor complexes

A model of the 5-HT4R was constructed by homology modelling using the crystal structure of the b2adrenergic receptor (PDB code 2RH1) [START_REF] Cherezov | High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor[END_REF][START_REF] Rosenbaum | GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function[END_REF] as template.

The 5-HT, BIMU8, and S-zacopride, ligands were docked, by interactive computer graphics, into the receptor model with their protonated side chain interacting with D3.32 in TM3 (Shi and Javitch, 2002). The [START_REF] Duan | A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations[END_REF] force field [START_REF] Duan | A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations[END_REF] was used for peptides and the general Amber force field (GAFF) [START_REF] Wang | Development and testing of a general amber force field[END_REF] and HF/6-31G*-derived RESP atomic charges were used for the ligands. Molecular dynamics simulations, in a explicit lipidic bilayer, of the ligandreceptor complexes were performed with the Sander module of AMBER 9 [START_REF] Case | AMBER 9[END_REF] using the protocol previously described [START_REF] Jongejan | Linking agonist binding to histamine H1 receptor activation[END_REF].

Drugs

The following compounds were used: BIMU8

N-[(1R,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl]-2,3- dihydro-3-iso-propyl-2-oxo-1H-benzimidazol-1-carboxamide hydrochloride, S-zacopride (S)-N-(1- azabicyclo[2.2.2]oct-3-yl)-4-amino-5-chloro-2-methoxybenzamide monohydrochloride, Cisapride cis- 4-amino-5-chloro-N-{1-[3-(4-fluoro-phenoxy)propyl]-3-methoxy-4-piperidinyl}-2-methoxy benzamide, RS 67333 1-(4-amino-5-chloro-2-methoxy-phenyl)-3-(1-butyl-4-piperidinyl)-1- propanone, RO116-1148 2,3-dihydrobenzo-(1,4)-dioxine-5-carboxylic acid 1-butylpiperidin-4- ylmethylamide hydrochloride

RESULTS

The role of T3.36 and W6.48 in stabilizing the basal constitutive activity of 5-HT4 receptors (R*basal).

We have previously shown that the constitutive activity of the histamine H1 receptor was dependent on the nature of the residue at position 3.36 [START_REF] Jongejan | Linking agonist binding to histamine H1 receptor activation[END_REF]. When serine, present in the WT H1

receptor was substituted by threonine or cysteine, the basal constitutive activity increased dramatically, occluding the histamine activation; whereas the constitutive activity was inexistent when serine was substituted by alanine. In biogenic amine receptors, the 3.36 position is rarely occupied by threonine (1%) and more often by cysteine (56%) or serine (31%). Interestingly, position 3.36 is occupied by threonine in 5-HT4Rs. This may explain why 5-HT4Rs have such a high constitutive activity. We engineered the T3.36A/S/C mutant receptors to study the role of the short and polar side chain of T3.36 in the constitutive activity of the 5-HT4R. By using different plasmid concentrations, we managed to obtain similar cell surface expression of the WT 5-HT4R and T3.36A/S/C mutants.

This was confirmed by using an Elisa assay (Fig. 1A) but also by Scatchard analysis using the specific labelled 5-HT4R ligand [ 3 H]GR 113808, as described previously [START_REF] Claeysen | Novel brain-specific 5-HT4 receptor splice variants show marked constitutive activity: role of the C-terminal intracellular domain[END_REF] (see Fig. 1A and its legend). The T3.36A mutation almost abolished the constitutive activity (Fig. 1B), indicating that the hydrogen bond capability of T3.36 plays an important role in stabilizing R* adopted by constitutively active 5-HT4Rs. T3.36S/C mutations provide a way of identifying the conformation of T3.36 in the active state of the R*basal because of the different side chain rotamer distribution of threonine from those of serine or cysteine, when located in an a-helical structure (Fig. 1C) [START_REF] Ballesteros | Serine and threonine residues bend alpha-helices in the chi(1) = g(-) conformation[END_REF]. The conformations of the short and b-branched side chain of threonine are limited to the g+ (85% of the side chains) and g-(15%) because the t conformation is unfavourable due to the steric clash of the side chain methyl group with the backbone carbonyl at the i-3 position [START_REF] Mcgregor | Analysis of the relationship between side-chain conformation and secondary structure in globular proteins[END_REF]. In contrast, serine can adopt either the g+ (52%), g-(20%) or t (28%) rotamer conformation. The fact that T3.36S reduces the constitutive activity of the receptor to a level close to the value observed in the T3.36A mutation precludes the serine-specific t conformation as the active conformation. Cysteine is restricted to the g+ (71%) or t (29%) conformation because of the steric clash between the Sg atom and the carbonyl oxygen of residue i-3 in the g-conformation [START_REF] Mcgregor | Analysis of the relationship between side-chain conformation and secondary structure in globular proteins[END_REF]. The unchanged constitutive activity of the T3.36C mutation, relative to WT, points to the common g+ conformation of cysteine and threonine as the active conformation, in agreement with previous proposals [START_REF] Jongejan | Linking agonist binding to histamine H1 receptor activation[END_REF]. We, thus, propose that T3.36 in the 5-HT4R

undergoes a conformational transition from the inactive g-to the active (R*basal) g+ conformation.

Modecular modelling showed that this conformational transition is only feasible if the side chain of W6.48 modifies its conformation simultaneously, in a concerted manner, from the inactive g+ to the active t conformation (Fig. 1D). Both the W6.48 and the T3.36 concerted toggle switches were absolutely necessary to adopt the R* constitutive active state (R*basal). Indeed, as previously described, the W6.48A mutant [START_REF] Joubert | A 5-HT4 receptor transmembrane network implicated in the activity of inverse agonists but not agonists[END_REF] as well as the T3.36A mutant were under the Rg state (Fig. 1B).

In order to be certain that both mutations (T3.36A and W6.48A) were stabilizing the 5-HT4R under a

Rg state, we studied the activities of these mutants as a function of the receptor density. As seen in Fig. 2A the basal activity of the WT receptor increased proportionally to the plasmid concentration and up to 31 pmol/mg whereas the basal activity of both T3.36A and W6.48A mutants remained totally in the Rg state whatever the receptor density (Fig. 2A). As expected, the inverse agonist RO 116-1148 had no effect on the basal activity in both W6.48A [START_REF] Joubert | A 5-HT4 receptor transmembrane network implicated in the activity of inverse agonists but not agonists[END_REF] and T3.36A mutants whereas it totally blocked the basal activity of the WT receptor (Fig. 2B). In conclusion, both the T3.36 and the W6.48 toggle switches were necessary to reach a R*basal.

Reaching the maximal R*-5-HT state does not require the W6.48 toggle switch whereas the T3.36 toggle switch is partially involved.

When measured at similar cell surface density, the maximal activity (Emax) of the R*-5-HT state in WT and W6.48A mutant were not different (Fig. 1B, Table 1). In the W6.48 mutant, the EC50 was shifted to the right by a factor of 25, despite the fact that the binding affinity of 5-HT for the W6.48A mutant receptor remained the same. Thus, the mutation did not affected 5-HT binding, weakly the coupling and not at all the maximal activity of the R*-5-HT state. In contrast, when measured at similar cell surface density, the maximal activity (Emax) of the R*-5-HT state, in the T3.36A mutant, dropped by 26 % compared to that of the WT, whereas its EC50 shifted to the right by a factor of 24 and its binding affinity was also reduced by a factor of 18. We constructed a three-dimensional model of the complex between 5-HT and a b2-AR-based model of the 5-HT4R (see Methods). This computational model considers that agonists bind the active conformation of the 5-HT4R, in which T3.36 is pointing towards TM7 in the active g+ conformation and W6.48 is pointing towards TM5 in the active t conformation. The conformation of F6.52 was also modeled in t to avoid the steric clash with W6.48 (Shi et al., 2002). T3.36 in g+ participates, in addition to the known aspartic acid D3.32 in TM3, in the binding of the protonated amine of serotonin; similar as described for histamine binding to the H1 receptor [START_REF] Jongejan | Linking agonist binding to histamine H1 receptor activation[END_REF]. The indole ring of 5-HT expands toward TMs 5 and 6, to hydrogen bond the key S5.43 and N6.55 side chains (Fig. 5A), as proposed by site-directed mutagenesis [START_REF] Mialet | Exploration of the ligand binding site of the human 5-HT(4) receptor by site-directed mutagenesis and molecular modeling[END_REF], without forming a direct interaction with W6.48. This mode of binding explains the fact that T3.36A slightly influences both the binding affinity for 5-HT as well as R*-5-HT (Emax and EC50), and that W6.48A does not modify the binding affinity nor Emax. Thus, the W6.48 toggle switch is not necessary to reach the R*-5-HT, while the T3.36 switch is only partially necessary. R*-5-HT is probably attained through other routes of activation involving TMs 5 and 6 (see Discussion).

The R*-benzamide state is reached following a direct interaction of benzamides with T3.36, triggering its toggle switch from the g-to the g+ conformation.

Notably, the T3.36A mutation modified the process of receptor activation in an agonist-specific dependent manner (Table 1; Fig. 3B). At similar receptor expression density, the Emax of the R*-BIMU8 state was weakly decreased by this mutation, from 97% to 78%; while the EC50 was unchanged (Fig. 3; Table 1). In contrast, activation by 2-methoxy-4-amino-5-chloro benzamides (Szacopride, cisapride) and related aryl ketone (RS 67333) was almost eliminated (Fig. 3B; Table 1).

Importantly, cell surface expression of this T3.36A mutant was similar to that of the WT (Fig. 1A and legend), and the lack of stimulation was not due to a loss of binding affinity of these ligands for the 5-HT4R-T3.36A mutant (Table 1). Since the benzamides are partial agonists on the WT receptor, it was possible that a weaker coupling efficiency due to mutation could have a higher influence on the Emax of benzamides compared to that of WT. Therefore, we studied the influence of receptor density on the stimulation of 5-HT4R by 5-HT, S-zacopride and BIMU8 in WT and T3.36A mutant (Fig. 4).

We have previously shown that, in COS-7 cells [START_REF] Claeysen | Pharmacological properties of 5-Hydroxytryptamine(4) receptor antagonists on constitutively active wild-type and mutated receptors[END_REF], the 5-HT4R activation can be analysed by the two-state model proposed by [START_REF] Leff | The two-state model of receptor activation[END_REF]. In particular the Emax increased as the function of receptor density, but the EC50 remains identical. This was again observed here when the dose-activation curves were determined on WT 5-HT4R (Fig. 4, left panels). The 5-HT and BIMU8

dose-response curves determined on the T3.36A mutant also showed an increase in Emax without change in EC50 when the receptor density increased (Figs. 4D and4F). In contrast, the benzamide Szacopride was totally unable to stimulate the T3.36A mutant whatever the receptor density (Fig. 4E;

Table 1).

In order to propose a structural hypothesis of the small effect of the T3.36A mutation on activity of the R*-BIMU8 state and the specific elimination of the R*-benzamide state we propose a model in Figs. 5B and 5C describing the complexes between these ligands and the 5-HT4R. In addition to other pharmacophoric elements (see legend to Fig. 5), S-zacopride and BIMU8 contain key carbonyl groups, which point towards TM3 (Fig. 5B) and towards TM6 (Fig. 5C), respectively. Thus, the carbonylic oxygen of S-zacopride forms a specific hydrogen bond with the active g+ rotamer of T3.36 (Fig. 5B), whereas the carbonylic oxygen of BIMU8 forms the hydrogen bond interaction with the active t rotamer of W6.48 (Fig. 5C). Accordingly, the T3.36A mutation almost abolished the benzamide and aryl-ketone induced 5-HT4R stimulation (Fig. 3; Table 1). We propose that benzamides and aryl ketones stabilize a R* (R*-benzamides) by triggering the direct conformational transition of T3.36 (from the inactive g-to the g+ conformation) by forming a specific hydrogen bond with T3.36.

The R*-BIMU8 state is primarily reached following a direct interaction of benzimidazolones with W6.48, and triggering its toggle switch from the g+ to t conformation.

The computational simulation presented above suggests that BIMU8 may trigger the process of 5-HT4R activation (stabilization of the R*-BIMU8) mainly via the conformational transition of W6.48 from the g+ to the t conformation by a specific hydrogen bond interaction. To test this hypothesis, we analysed the ability of BIMU8 to stimulate the W6.48A mutant. To our surprise the KD value for BIMU8 was unchanged (Table ), whereas this mutation reduced the EC50 by 43-fold (Fig. 6B) and the Emax by a factor of 2, compared to 5-HT stimulation (Fig. 6A). This reduction of the BIMU8stimulation was independent of the receptor density (Fig. 6B). The effects of T3.36A and W6.48A mutations in BIMU8 function suggest that the T3.36 toggle switch plays a minor role in the stabilization of R*-BIMU8 (Emax decreased from 97% to 78%); leaving the W6.48 toggle switch as the main mechanism of 5-HT4R activation by BIMU8 (Emax decreased from 97% to 53%). We tried to generate the double T3.36A-W6.48A mutant receptor to fully impair the R*-BIMU8 state.

Unfortunately, this double mutant receptor was poorly expressed at the cell surface and no conclusion can be drawn.

Molecular modelisation showed that in the absence of W6.48 (W6.48A mutant), the aromatic ring of BIMU8 can occupy the position of the active t conformation of W6.48 in the WT receptor (see Fig. 6C). This position of the aromatic ring of BIMU8 in the receptor is very favourable due to the presence of the F5.47 aromatic side chain. In this new mode of binding, the carbonylic oxygen of BIMU8 forms a hydrogen bond interaction with the active g+ rotamer of T3.36 (Fig. 6C). Likely, in the W6.48A mutant receptor, the new R*-BIMU8 state is stabilized by this hydrogen bond, a mechanism of triggering a R* state similar to that of R*-benzamides (compare Figs. 5B and6C). This mode of binding explains why BIMU8 has similar binding affinity for WT and W6.48A mutant 5-HT4R. Nevertheless, the lack of expression of this double T3.36A-W6.48A mutant receptor impedes to experimentally probe this hypothesis.

DISCUSSION

One of the most important progresses in family A GPCR molecular pharmacology has been to understand, based on biophysical, biochemical and crystallography experiments, how a multitude of chemically different agonists, which do not share a common binding mode, can trigger receptor activation. A few conserved molecular activation mechanisms implying the rearrangement of the side chains of a limited number of residues have been associated with receptor activation [START_REF] Kobilka | G protein coupled receptor structure and activation[END_REF][START_REF] Smit | Pharmacogenomic and structural analysis of constitutive g protein-coupled receptor activity[END_REF]. A key question is to determine which different rearrangements of a given GPCR lead to the different R*s. Are they different for basal constitutively active R* and agonist-induced R*s? Do chemically different agonists induce different rearrangement? Few examples indicate this could be the case. In the b2-adrenergic receptor, salbutamol although being an agonist, does not trigger the TM6 toggle switch whereas the cathecol derivatives do [START_REF] Swaminath | Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states[END_REF].

Here, we show that the R*basal of the WT 5-HT4 receptor is likely to be the result of a simultaneous double toggle switch. One is the conformational change of W6.48 from the inactive g+ (pointing towards TM7) to the active t (TM5) conformation, which is accompanied by the conformational transition of T3.36 from the inactive g-(TM6) to the active g+ (TM7) conformation.

Both coordinated switches are necessary to go from the silent Rg to the R*basal. Indeed, mutation of either W6.48 or T3.36 to alanine kept the WT 5-HT4 receptor to the Rg state whatever the receptor density. This Rg state was, of course, insensitive to inverse agonists. When the receptor is under the Rg state, the levels of cAMP accumulation were close to those observed in the absence of receptors (the mock-transfected cells). The Rg state is not often described in GPCR pharmacology [START_REF] Joubert | A 5-HT4 receptor transmembrane network implicated in the activity of inverse agonists but not agonists[END_REF]. The Rg state of rhodopsin is stabilized by the full inverse agonist cis-retinal, the structure of which is distinct from the ligand-free opsin state R [START_REF] Lin | Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state[END_REF][START_REF] Park | Crystal structure of the ligand-free G-protein-coupled receptor opsin[END_REF].

We have also shown that T3.36, in addition to modulate the Rg to R*basal, plays a minor role in the stabilization of R*-5-HT and R*-BIMU8 and is fully required in the stabilization of the R*benzamides (S-zacopride, cisapride and RS 67333). Benzamides were completely inactive on the T3.36A mutant, whatever the receptor density, excluding a simple reduction in the coupling efficiency affecting preferentially the response to partial agonists such as benzamides. Thus, as a hypothesis, we can conclude that S-zacopride, cisapride, and RS 67333 trigger 5-HT4R activation via stabilization of the rotamer toggle switch of T3.36, from the inactive g-to the active g+ conformation. Here, we show for the first time that the 3.36 position is directly implicated in agonist-induced activation and is responsible for the stabilization of an active state.

The 5-HT4R is also activated by benzimidazolone ligands such as BIMU8. We propose that the R*-BIMU8 state is primarily reached following a direct interaction between the carbonylic oxygen of benzimidazolones and W6.48, triggering its toggle switch from the inactive g+ to the active t conformation. A similar mode of interaction between the W6.48 residue and the CSP-2503 agonist has been proposed during activation of the 5-HT1A receptors [START_REF] Lopez-Rodriguez | Synthesis and structure-activity relationships of a new model of arylpiperazines. 8. Computational simulation of ligandreceptor interaction of 5-HT(1A)R agonists with selectivity over alpha1-adrenoceptors[END_REF].

It was surprising that the W6.48A as well as the T3.36A mutant were fully activated by 5-HT. This clearly indicates that the conformational mechanisms occurring from Rg to R*basal are different from those occurring from Rg to R*-5-HT. A two state model was not sufficient to explain those results.

The 5-HT4Rs as well as rhodopsin are, at least, in equilibrium between three states, i.e. the Rg, the R*basal and the R*-5-HT states. The R*-5-HT state can be reached either from the Rg or from the R*basal state. Thus, the relative stimulation by 5-HT was higher in cells expressing the receptor under the Rg state (W6.48A and T3.36A mutants) than under the R*basal state (WT receptors) (Fig. 1). A similar observation can be done considering the WT and the T3.36A mutant and the stimulation by BIMU8. The relative stimulation by BIMU8 was higher in cells expressing the T3.36A mutant (under the Rg state) than the WT receptors (under the R*basal state) (Fig. 3).

To date, there is not a clear idea as to the rearrangements occurring upon 5-HT stimulation.

However, it has been shown for the β2-adrenergic receptor, using fluorescence spectroscopy to monitor agonist-induced conformational changes, that relocation of the extracellular side of TM5 is necessary to facilitate binding of the catechol hydroxyls to the Ser residues at positions 5.42 and 5.46 [START_REF] Swaminath | Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states[END_REF]. In addition, TM6 performs an inward movement of the extracellular part toward TM3 (the global toggle switch of Schwartz et al.) that is stabilized by the interaction with agonists [START_REF] Schwartz | Molecular mechanism of 7TM receptor activation--a global toggle switch model[END_REF]. By homology with these proposals, we suggest that 5-HT would stabilize R*-5-HT by the interaction between the 5-OH and NH moieties with the S5.43 and N6.55 side chains, respectively, triggering the proposed movements of TMs 5 and 6.

The mechanisms by which binding of these chemical signals at the extracellular domain of the receptor trigger a set of conformational rearrangements of the TM segments near the G-protein binding domain are not fully understood. Nevertheless, comparison of the structure of inactive rhodopsin [START_REF] Li | Structure of bovine rhodopsin in a trigonal crystal form[END_REF] with the crystal structure of the ligand-free opsin [START_REF] Park | Crystal structure of the ligand-free G-protein-coupled receptor opsin[END_REF] has provided additional insights into these processes. Specifically, we propose that the T3.36/W6.48 rotamer toggle switch disrupts a conserved hydrogen bond network linking W6.48 and D2.50 [START_REF] Li | Structure of bovine rhodopsin in a trigonal crystal form[END_REF][START_REF] Pardo | The role of internal water molecules in the structure and function of the rhodopsin family of G protein-coupled receptors[END_REF][START_REF] Rosenbaum | GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function[END_REF], triggering the conformational transition of N7.49 towards D2.50 [START_REF] Urizar | An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor[END_REF], and ultimately leading to the extended conformation, pointing towards the protein core, of R3.50 [START_REF] Park | Crystal structure of the ligand-free G-protein-coupled receptor opsin[END_REF]. On the other hand, the agonist-induced inward movement of the extracellular part of TM6 toward TM3 [START_REF] Schwartz | Molecular mechanism of 7TM receptor activation--a global toggle switch model[END_REF] might cause the intracellular movement of TM6 toward TM5, facilitating the observed ionic interaction between E6.30 and K5.66 [START_REF] Park | Crystal structure of the ligand-free G-protein-coupled receptor opsin[END_REF]. Finally, the relocation of the extracellular side of TM5 to facilitate binding of biogenic amine agonists [START_REF] Swaminath | Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states[END_REF] induces the intracellular movement of TM5, enabling the interaction of Y5.58 with the extended conformation of R3.50 [START_REF] Park | Crystal structure of the ligand-free G-protein-coupled receptor opsin[END_REF].

We have previously established, using circular dichroism difference spectra of purified 5-HT4R, that free (or neutral antagonist-occupied), agonist-occupied (partial or full agonist) and silent (inverseagonist-occupied) receptors involved different arrangements of the e2 loop [START_REF] Baneres | Molecular characterization of a purified 5-HT4 receptor: a structural basis for drug efficacy[END_REF].

Here, in a cellular context, we provide arguments in favour of different conformational arrangements during stabilization of different 5-HT4 R*s: the R*basal, the R*-5-HT, the R*-benzamides and the R*-BIMU8. (B) Basal level and maximum 5-HT (10 -5 M) stimulation of cAMP formation were measured after 10 min incubation. Each value was estimated as the percentage of maximum cAMP production (8.9 ± 0.7 pmol/100 000 cells) in the WT induced by 5-HT (10 -5 M).

FOOTNOTES

(C) Statistical preferences of threonine (T), serine (S), and cysteine (C) side chain rotamer distribution (t: trans, g+: gauche+ or g-: gauche-; see Methods) when located in a a-helical structure (adapted from Ballesteros et al. [START_REF] Ballesteros | Serine and threonine residues bend alpha-helices in the chi(1) = g(-) conformation[END_REF]). Active conformations in the 5-HT4R are shown in bold and asterisked. In these models the protonated moiety of the ligands interact with D3.32; the 5-OH and NH moieties of 5-HT hydrogen bond the key S5.43 and N6.55 side chains (A) as proposed by site-directed mutagenesis [START_REF] Mialet | Exploration of the ligand binding site of the human 5-HT(4) receptor by site-directed mutagenesis and molecular modeling[END_REF]; the -C(CH3)2 and -Cl moieties of BIMU8 and S-zacopride, respectively, are located in a small hydrophobic cavity between TMs 3-5 (B, C); and the -NH2 group of S-zacopride hydrogen bonds S5.43 (B). BIMU8 and S-zacopride form an intramolecular hydrogen bond between the -NH amide and a carbonyl or methoxy group, respectively. BIMU8 elicits 5-HT4R activation by forming a hydrogen bond interaction with W6.48 in the active t conformation (C), while S-zacopride triggers activation via the interaction with T3.36 in the active g+ conformation (B). The atoms implicated in these interactions are pointed out: oxygen of the drugs is asterisked and T3.36 or W6.48 hydrogen is labelled with a filled square. (C) Computational models of the complex between BIMU8 and W6.48A mutant receptor. In the absence of W6.48, the aromatic ring of BIMU8 occupies the position of the active t conformation of W6.48 in the WT receptor (see Fig. 5C); and the carbonylic oxygen hydrogen bonds the active g+ rotamer of T3.36. The atoms implicated in this interaction are pointed out: oxygen of the drug is asterisked and T3.36 hydrogen is labelled with a filled square.

TABLE 1

Pharmacological properties of a series of 5-HT4R ligands on WT 5HT4R, T3.36A and W6.48A mutant receptors.

All the data are expressed as means ± SEM of at least four separate experiments.

(a) Binding assays were performed by using [ 3 H]GR 113808 as the specific 5-HT4R radioligand in COS-7 cells membranes expressing either WT 5-HT4R, 5-HT4-T3.36A or 5-HT4-W6.48A. Non-specific binding was determined with BIMU8 (10 µM). KD values were determined from Cheng-Prussoff equation.

(b) The agonist activity or potency (EC50) was accessed as the concentration that gave 50% increase in the response to cAMP formation.

(c) The maximal activity of each drug (EMAX) is expressed as percentage of the maximum 5-HT response obtained with the WT at similar receptor density.

ND: non determined. 5-HT 7.7 ± 1.4 10 -8 3.6 ± 0.7 10 -9 100% 1.4 ± 2.3 10 -6 8.6 ± 3.3 10 -8 74 ± 1% 7.4 ± 2.6 10 -8 8.9 ± 3.2 10 -8 100% BIMU8 5.3 ± 1.2 10 -8 1.8 ± 0.7 10 -8 97 ± 4% 2.1 ± 0.1 10 -7 7.7 ± 1.7 10 -8 78 ± 2% 1.1 ± 0.7 10 -7 5.4 ± 1.9 10 -7 53 ± 3% S-zacopride 6.4 ± 0.9 10 -7 1.1 ± 0.2 10 -7 77 ± 1% 7.2 ± 1.5 10 -7 ND ND Cisapride 2.0 ± 0.2 10 -7 4.3 ± 3.1 10 -8 64 ± 1% 7.3 ± 1.4 10 -7 ND ND RS 67333 5.5 ± 0.2 10 -9 2.6 ± 1.5 10 -9 70 ± 2% 1.2 ± 0.3 10 -8 ND ND

  Figure 1. Constitutive activity of the WT 5-HT4R is largely dependent on T3.36.

(D) 5 -

 5 HT4R activation consists of a concerted T3.36/W6.48 rotamer toggle switch. T3.36 undergoes a conformational transition from the inactive g-to the active g+ conformation, in conjunction with the conformational transition of W6.48 from the inactive g+ to the active t conformation.

Figure 2 .

 2 Figure 2. Lack of basal activity and inverse agonist RO 116-1148 effect in T3.36A and W6.48A

Figure 3 .

 3 Figure 3. Influence of T3.36 in the agonist-induced activation of the 5-HT4R.

Figure 4 .

 4 Figure 4. Effect of increasing T3.36A receptor density on agonist activation.

Figure 5 .

 5 Figure 5. Computational models of the complexes between 5-HT4R and BIMU8 or Zacopride.

Figure 6 .

 6 Figure 6. Influence of W6.48 in the BIMU8-induced activation of the 5-HT4R.

  Figure2

L.P.P. and J.S. equally contributed to this work.

Acknowledgements

Binding experiments, cAMP measurement, and ELISA were carried out using facilities of the Pharmacological Screening platform of the Institut de Génomique Fonctionnelle. We are grateful to Angela Turner Madeuf for help in language revision and to Elisabeth Cassier for technical assistance.


K Poncelet

F Kazymyrenko

Hild

A Carpiuc

C Jailin

M Poncelet

K Kazymyrenko
email: kyrylo.kazymyrenko@edf.fr

F Hild

Stable control for mixed-mode concrete fracture

Stable control for mixed-mode concrete fracture

Introduction

The knowledge of the concrete behavior is essential when analyzing the aging and leakage phenomena of production facilities. On the industrial level the concrete fracture characterization is currently performed through standard experimental tests that are not sufficiently rich to determine in an optimized manner all the corresponding characteristics. First there is a general lack of mixed mode loading experiments that produce non trivial crack paths. Moreover, even for some well defined material properties (as for example critical shear stress) the corresponding loading tests are not sufficiently developed, as the results are too dependent on aggregate distribution and specimen sizes. It means that we are still not able to locally reproduce some specific mechanical stresses distribution and repeatedly check it in order to attain necessary accuracy. Therefore an improvement in corresponding measurement technique is still needed.

When studying concrete fracture behavior the main difficulty is related to instable crack propagation. In the past some relevant works in mixed mode fracture were conducted by Nooru-Mohamed [START_REF] Nooru-Mohamed | Mixed-mode fracture of concrete: an experimental approach[END_REF]. More recently the work was extended employing an up-to-date full-field measurement technique [START_REF] Carpiuc | Innovative tests for characterizingmixed-mode fracture of concrete: from pre-defined to interactive and hybrid tests[END_REF].

In this work, we develop a stable crack path control technique that allows "customized" cracking tests creation for the characterization of various materials. Using this technique well instrumented and discriminating mixed-mode crack propagation tests performed on VERCORS concrete samples are presented. Finally we discuss the relevance of these tests for various parameters identification and some possible extension of stable crack path control technique.

Eigen stresses loading history map

When analyzing experimental data some common characteristics are usually determined, as for example, force-displacement curves and crack pattern evolution. While crack pattern has clear physical meaning force-displacement curves interpretation is trickier as they could be numerous. For a simple experiment as three point flexure a single force-displacement curve is generated and it could be easily analyzed even by non specialist. For more complex experiments, for instance, a typical Nooru-Mohamed test, the number of possible force-displacement curves explodes and it becomes much more difficult to conclude which kind of loading was really applied during the test. In order to simplify this analysis we introduce an eigen stresses loading map picture. The idea is to present on a single picture the whole loading map. Once the experi-

rd Conference on Technological Innovations in Nuclear Civil

Engineering TINCE 2016, Paris 5 th to 9 th September ment is completed we perform its finite elements simulation using a non local damage model. On each loading step the stress field of each Gauss point of the structure subjected to damage evolution is recoded. All these data could then be represented on either 2D or 3D eigen stresses maps. We find out that the simplest way is to filter values close to the initial elastic domain and then to plot obtained subset on 2D plane (plane stress or plane strain depending on the experiment). For example for three point beam flexure it clearly indicates that during the whole loading mode I stress component (𝜎 𝑐𝑟𝑖𝑡 = 3.8 𝑀𝑃𝑎) were probed in priority (𝜎 1 , 𝜎 2 ) ≈ (0, 𝜎 𝑐𝑟𝑖𝑡 ).

Stable crack control

Mixed mode crack propagation could be either generated by the non trivial sample geometry [START_REF] Brokenshire | Torsional fracture tests[END_REF], [START_REF] Winkler | Traglast untersuchungen von unbewehrten und bewehrten Betonstrukturen[END_REF] or by non trivial loading itself. As our main goal is to conduct multiple different fracture tests the last approach is more suitable: the easiest Nooru-Mohamed type geometry is chosen with the rectangular notched concrete sample mechanically loaded by two rigid steel slabs (Figure 2).

In general flat crack (2D) propagation path control needs the continuous adjustment of at least 2 degrees of freedom: one for uniaxial charging and the second one for crack orientation changing. For the experiments where the loading is applied to the sample through rigid slabs one additional in-plane rigid body motion degree of freedom is also available.

To the extension of the classical Nooru-Mohamed tensile and shear translations we use in our experiments the loading slab's rotation. The last one serves to apply the supplementary tension-compression gradient loading in front of the crack tip. At our preliminary validation tests the lack of rotation control was clearly identified as an important issue for the crack stability control. The first option for crack control is a manual change of loading composition on some relevant crack positions. The empirical operator decision based strategy is already non trivial, as one should first be able to measure crack tip position during the experiment and second somehow identify these relevant crack positions. Even so in this article we present two tests that are precisely based on the last strategy, we propose to go further and describe more advanced crack control technique allowing precise crack control. The reason for that is that we have recently realized this kind of tests, but the results are too raw to be presented in the current paper. Let's illustrate how the crack orientation could be controlled using Linear Elastic Fracture Mechanics (LEFM) principles. If our system is resorted to just two translational degrees of freedom (𝑈 𝐼 and 𝑈 𝐼𝐼 ) then they are linearly related to the couple of stress intensity factors𝐾 𝐼 , 𝐾 𝐼𝐼 :

I II I I I I I II II II II II K KK U K KK U               Equation 1
At each loading step we need to create a finite elements mesh corresponding to the real crack propagation. Then the four elements of the sensitivity matrix are identified using stress intensity factor calculations for two virtual unitary loading:

K K K K K K K K K K K K                                         Equation 2
The value of stress intensity factors (SIF) are fixed by two previously mentioned criteria: crack initiation at 𝐾 𝐼 , ≤ 𝐾 𝐼𝑐 and crack orientation angle fixed by the maximal normal stress condition:

Equation 3

General stable crack control technique follows the same idea of crack reorientation developed just above, taking into account this new stability condition. For each new experiment a different target crack path is set prior to the test, then two in-plane translation degrees of freedom are used to control the crack orientation and in-plane rotation is used to stabilize the crack ad-vancement. The testing machine, a hexapod [START_REF] Nierenberger | Multiaxial testing of materials using a Stewart platform: Case study of the Nooru-Mohamed test[END_REF], serves to apply three rigid body motions for in-plane loadings, namely, tension, shear and in-plane rotation. High resolution cameras register full displacement field on both sides of the sample in order get exact crack position at each loading time step. With the proposed experimental setup a new set of benchmark tests for different loading histories have been obtained. We presented here two of the advanced tests (Figure 4): during the evolution of the crack path, the orientation of the crack were redirected on multiple occasions in certain key positions of the crack tip.

Experimental techniques

Performing more complex tests with crack patterns that present several reorientations or/and bifurcations is made possible by the evolution of the experimental techniques. Combining the use of multiaxial testing machine with 6-axes load-cells and with Digital Image Correlation (DIC) measurements permits to predict the behavior of materials and structures under complex generalized loading conditions. The development of multiaxial loading machines is rather recent, even though these machines have the advantage of applying boundary conditions (controlled in displacement or force) very close to the real-life cases, and also various loading types on the same specimen geometry. For the present work a Gough-Stewart platform, a hexapod (Figure 3), is used to perform mixed-mode fracture tests on concrete specimens [START_REF] Nierenberger | Multiaxial testing of materials using a Stewart platform: Case study of the Nooru-Mohamed test[END_REF]. The 6-axes loading machine assures both the stable crack propagations and the control of the crack orientation with only three degrees of freedom, nevertheless the extra loading axes are used to ensure zero displacement along the other directions.

An important experimental challenge is generated by the stiffness of the specimen, the difference of orders of magnitude between the size of the specimen (i.e., 200 mm) and the characteristic displacement at failure (i.e., ~20 μm) while the applied loading force goes up to 20 kN. To overcome this challenge and to ensure the required accuracy complex measurements and a specific control of the machine were developed. The 3D control of the machine is assured by a non-contact optical full field measurement technique [START_REF] Le Flohic | A 3D displacement control by digital image correlation for the multiaxial testing of materials with a Stewart platform[END_REF]. Thus a digital image correlation algorithm is used to measure the 3D displacements of the upper-end effector (labeled 4 in Figure 3) and to correct in real time the displacements applied to the specimen.

Another important aspect when performing rich and discriminating mixed-mode crack propagation tests is the use of the Digital Image Correlation (DIC) technique. The displacement fields on each face of the sample, assessed by DIC, give access to the crack pattern, and also to the actual boundary conditions that are crucial for a faithful numerical analysis of the test. Moreover, DIC computations can be performed during the tests to determine the propagation of the crack and adjust the boundary conditions accordingly. This technique was extensively used in this work, and the instrumentation of tests will be presented in the following.

Instrumentation

In order to have the complete description of the 3D mechanical behavior of the specimen, a 6-axes load-cell (labeled 7 in Figure 3) is used to measure both the applied forces and torques. The cracking state is analyzed on each face of the sample using in plane DIC and the outof-plane motions are determined using stereo DIC (Figure 4). The displacement fields on each face of the sample give access to the crack pattern, and also to the actual boundary conditions that are crucial for a faithful numerical analysis of the test. Considering the particular control of the machine, by the 3D optical displacement loop, a second measurement setup composed of 6 displacement sensors (Figure 4) to check the applied boundary conditions is installed.

Experimental tests

The experimental setup previously presented is used to perform mixed-mode fracture tests on concrete specimens. Using a combination of tension, shear and in-plane rotation stable TINCE 2016, Paris 5 th to 9 th September and complex crack patterns are obtained. The shear direction is closely related to the orientation of the crack, the tensile load is responsible for the actual propagation and the rotation increases the stable propagation period by generating a tension/compression stress gradient. Therefore using a multiaxial testing machine coupled with DIC measurements several cases of stable propagation are studied to illustrate phenomena like crack reorientation, crack branching, successive crack initiations, crack coalescence, crack closure and mode III crack propagation.

In the following, the results of two interactive tests are presented in a succinct manner only to give a hint of the capacity of the developed experimental setup, nevertheless their detailed description is given in [START_REF] Carpiuc | Innovative tests for characterizingmixed-mode fracture of concrete: from pre-defined to interactive and hybrid tests[END_REF], all the experimental data and the material characteristics are available. Applied boundary conditions and both tests' descriptions could be equally downloaded from Code_Aster website (www.code-aster.org, test ssnp168).

The tests are performed on 50x200x200 mm mortar specimens. Depending on the test, two notches (5x40 mm) or only one (5x25 mm) are sawed at mid height of the specimen (Figure 5) For the I1 test the loading path is chosen to generate several reorientations and the branching of the crack. It consists of a series of tension, rotation and shear steps (Figure 6a) that leads to initiation, propagation and reorientation of a crack following a zigzagged path (Figure 6b). considered sufficiently propagated the loading is changed to reorient/branch the crack. The evolution of the crack at the end of each propagation step is given in Figure 7. It is also important to note that the test is quasi-static (i.e., the test lasts more than 10 h with a stable crack propagation during the whole time) so only the final unstable crack propagation is a dynamic event. Test I2 addresses phenomena such as successive crack initiation and propagation, crack closure and friction and crack coalescence. The chosen geometry for the I2 test is the one with two notches (Figure 5b). The test can be divided into four loading steps composed of combinations of elementary loading paths like tension, shear and in-plane rotation (Figure 6a). More in detail the test starts by applying positive in-plane rotation combined with a positive shear force (labeled 2 in Figure 6(a) to initiate and propagate a crack upward. Afterward, the system is unloaded and reloaded by coupling a negative in-plane rotation with a positive shear force leading to the closure of the crack and the initiation of a second crack on the opposite notch. After a second unloading, the final step is a proportional tensile -negative shear loading that reorients both cracks and joins them. Each combination of loadings is carefully chosen to obtain the aforementioned phenomena (i.e., successive crack initiation and propagation, closure, friction and coalescence). The evolution of the crack after each propagation step in given in Figure 8. While there seems to be no difference between proportional Nooru-Mohamed test and sequential rotation test, it is clearly seen that zigzag type interactive test (I2) is covering the initial elastic yield surface in the most complete way; the last one is also complementary to the 3 point flexure test.

Toward a new kind of custom-made experiments

Path control technique is not always relevant when studying some specific material property. For example as could be seen from (Figure 9) I2 (sequential rotation test) is not richer than standard Nooru-Mohamed test if one needs just to probe the elastic-domain shape: both have reasonably similar results. Instead of controlling crack path orientation, which is equivalent to stress intensity factor ratio control, another possible way of producing new experiments could be based on the repeated realization of the needed local mechanical loading within the single experiment. All three rigid body motion slab's degrees of freedom are then adjusted in order to reproduce locally (close to the crack tip) some fixed in advance stress distribution just before crack propagation to the next loading step. Thanks to this new approach material property identification accuracy could be refined to obtain necessary precision within a single test.

Conclusion

We propose a new database for concrete fracture analysis of the VERCORS mock-up building. Some most discriminate tests were obtained relying on the developed crack path control technique. By analyzing eigen stresses loading history map we confirm the mixed-mode nature of these tests. Their relevance for different mechanical parameters identification is discussed.

Figure 1 .

 1 Figure 1. Left: two typical initial elastic domains for different concrete types. Right: Eigen stresses loading history (dots) for three point beam flexure in the bitensile quadrant loading domain.

Figure 2 .

 2 Figure 2. Classical Nooru-Mohamed experiment. The loading is applied to the sample trough two rigid steel slabs using two translational degrees of freedom: tension and shear.

Figure 3 .

 3 Figure 3. The 6-axis testing machine. The real setup (left) and the CAD representation (right): (1) base, (2) actuators, (3) moving platform, (4) upper end-effector, (5) room for specimen, (6) optical setup in closed configuration with two cameras, (7) passive hexapod (load cell)

Figure 4 .

 4 Figure 4.Hybridtest principle (left) and the experimental setup (right).

Figure 5 .

 5 Figure 5. The geometry types: single notch (a), and doublenotches (b)

Figure 6 .

 6 Figure 6. (a) The three elementary loading paths: 'tension' (1), 'shear' (2) and 'rotation' (3), (b)The expected crack pathThe crack evolution is controlled by DIC, images are taken every 5 seconds and a DIC computation is performed every 10 images to detect the advancement of the crack. When the crack is

  Vertical displacement fields showing the crack advancement for test I1 after each propagation step

Figure 9 .

 9 Figure 9. Bitensile quadrant zoom of the eigen stresses loading history maps for (from left to right): 3 point flexure, proportional Nooru-Mohamed test, sequential rotation test (I2) and zigzag type test (I1).

3 rd Conference on Technological Innovations in Nuclear Civil Engineering TINCE 2016, Paris 5 th to 9 th September

rd Conference on Technological Innovations in Nuclear Civil Engineering TINCE 2016, Paris 5 th to 9 th September