Sylvie Claeysen

Michèle Sebben

Carine Becamel

Joël Bockaert
email: bockaert@ccipe.montp.inserm.fr

Aline Dumuis

Novel brain-specific 5-HT 4 receptor splice variants show marked constitutive activity: role of the C-terminal intracellular domain

G-protein coupled receptors; 5-HT 4 R, 5-Hydroxytryptamine 4 receptor; RT-PCR, reverse transcriptase polymerase

INTRODUCTION

The physiological functions of 5-HT both in the central nervous system and the periphery of vertebrates are mediated by a large number of GPCRs. Among the 13 known genes coding for 5-HT GPCR only three (5-HT 4 R, 5-HT 6 R and 5-HT 7 R) are positively coupled to Gs [START_REF] Hoyer | 5-HT Receptor classification and nomenclature: towards a harmonization with human genome[END_REF] . 5-HT 4 receptors are expressed in a wide variety of tissues of vertebrates including brain, oesophagus, ileum, colon, adreno-cortical cells, urinary bladder, human and porcine heart [START_REF] Bockaert | Handbook of experimental pharmacology: Serotoninergic neurons and 5-HT receptors in the CNS[END_REF][START_REF] Eglen | Central 5-HT 4 receptors[END_REF]. Benzamide 5-HT 4 R agonists, such as metoclopramide and cisapride, are therapeutically used as prokinetic drugs. A role for 5-HT 4 receptors in learning and memory has been demonstrated in brain [START_REF] Eglen | Central 5-HT 4 receptors[END_REF][START_REF] Letty | 5-HT 4 receptors improve social olfactory memory in the rat[END_REF][START_REF] Marchetti-Gauthier | BIMU1 increases associative memory in rats by activating 5-HT 4 receptors[END_REF].

Although the pharmacology of 5-HT 4 receptors is similar in the different tissues studied, some still unexplained differences remain. For example, benzamides are super agonists (efficacy greater than that of 5-HT) in colliculi neurons, the preparation on which 5-HT 4 receptors were first described, whereas in many systems they are partial agonists or even antagonists [START_REF] Bockaert | Handbook of experimental pharmacology: Serotoninergic neurons and 5-HT receptors in the CNS[END_REF][START_REF] Dumuis | A non classical 5hydroxytryptamine receptor positively coupled with adenylate cyclase in the central nervous system[END_REF]. A difference in structure may explain some of the observed differences and in particular the super agonism of benzamides in colliculi neurons. This is the reason why we have persevered in our cloning effort of 5-HT 4 receptors especially in brain tissues and further analyzed the coupling efficiency of variants cloned in different species. Gerald et al [START_REF] Gerald | The 5-HT 4 receptor: molecular cloning and pharmacological characterization of two splice variants[END_REF] first reported the cloning of two rat variants, (r5-HT 4S R and r5-HT 4L R now called r5-HT 4(a) R and r5-HT 4(b) R) which differ in their C-terminal tail after the splicing site at L358. We and others have cloned the human and mouse m5-HT 4(a) and m5-HT 4(b) receptor homologues [START_REF] Blondel | Molecular and functional characterization of a 5-HT 4 receptor cloned from human atrium[END_REF]Claeysen et al., 1997a;[START_REF] Claeysen | Cloning, expression and pharmacology of the mouse 5-HT 4L receptor[END_REF]. Blondel et al., however, (Blondel et al., 1998) reported the existence of two different human variants (h5-HT 4(c) R and (d) R) also displaying divergent sequences after L358. In this study, we have cloned two additional splice variants in mouse (m5-HT 4(e) R and m5-HT 4(f) R).

The (e) form has also been cloned in rat and human brain (r and h5-HT 4(e) R). All these variants share the same sequence up to L358, followed by specific C-termini. Splice variants of GPCRs generally differ in their intracellular domains in particular, within the third intracellular loop and the C-terminal tail. These two regions are involved in the fine tuning of the coupling of GPCRs to G proteins [START_REF] Journot | The PACAP receptor: an example of functional diversity among G protein-coupled receptors generated by alternative splicing[END_REF]. The role of C-terminus in maintaining the receptor in an inactive conformation (R) in the absence of agonist has been highlighted by recent studies [START_REF] Mary | A cluster of basic residues in the carboxyl-terminal tail of the short metabotropic glutamate receptor 1 variants impairs their coupling to phospholipase C[END_REF][Mary, #1556;;[START_REF] Hasegawa | Two isoforms of the prostaglandin E receptor EP3 subtype different in agonist-independent constitutive activity[END_REF]Hasegawa, #1654;;[START_REF] Weiss | Effects of carboxyl-terminal truncation on the stability and G protein-coupling activity of bovine rhodopsin[END_REF] [START_REF] Matus-Leibovitch | Truncation of the thyrotropin-releasing hormone receptor carboxyl tail causes constitutuve activity and leads to impaired responsiveness in Xenopus oocytes and AtT20 cells[END_REF]#1651;[START_REF] Parker | Truncation of the extended carboxyl-terminal domain increases the expression and regulatory activity of the avian b-adrenergic receptor[END_REF]Parker, #1652]]. These data support the hypothesis of a GPCR structure likely selected during evolution to constrain allosteric transition from the inactive (R) to active (R*) forms in the absence of agonist [START_REF] Cotecchia | Regions of the a1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function[END_REF][START_REF] Cotecchia | Discrete amino acid sequences of the alpha 1-adrenergic receptor determine the selectivity of coupling to phosphatidylinositol hydrolysis[END_REF][START_REF] Kenakin | Agonist-receptor efficacy I: mechanisms of efficacy and receptor promiscuity[END_REF][START_REF] Kjelsberg | Constitutive activation of the a 1B -adrenergic receptor by all amino acid substitutions at a single site[END_REF]. This transition is governed by the allosteric transition constant J, which is an index of the magnitude of receptor constraint [START_REF] Samama | A mutation-induced activated state of the b 2 -adrenergic receptor. Extending the ternary complex model[END_REF].

The lower the constant, the higher the signal to noise ratio.

The aptitude for a given receptor to convert from R to R*, in the absence of agonist, is also an important physio-pathological parameter to take into account because of: (a) the increasing number of pathologies associated with constitutively active receptors (under the R* form in the absence of agonist) [START_REF] Spiegel | Abnormalities in G protein-coupled signal transduction pathways in human disease[END_REF] and (b) the potential therapeutic use of inverse agonists. Not all receptors have the same propensity to isomerize from R to R* in the absence of ligand. TSHR is one of the GPCRs which has the highest propensity to isomerize. This is probably the reason why mutations of residues localized throughout the TSHR structure, lead to the constitutively active states found in thyroïd hyperplasia [START_REF] Van Sande | Somatic and germline mutations of the TSH receptor gene in thyroid diseases[END_REF]. Here, we report that 5-HT 4 receptors like TSHRs show marked constitutive activity when expressed at low physiological densties. At a similar receptor density, the newly cloned 5-HT 4 receptor splice variants (m5-HT 4(e) R and (f) R) have, in their native conformation, an agonist-independent activity level above the mutated b 2 -AR (CAM b 2 -AR).

MATERIALS AND METHODS

Isolation and sequencing of the mouse 5-HT 4(a), (e) and (f) receptors cDNA by RT-PCR:

Total RNA was isolated from whole adult Swiss mouse brain. Poly(A) + RNA was extracted with Dynabeads Oligo(dT) 25 (Dynal) and reverse transcribed using Superscript II RNase H Reverse Transcriptase (Life Technologies) and random nonamers. RT-PCR was performed with the following oligonucleotides, ON1 [START_REF] Claeysen | Cloning, expression and pharmacology of the mouse 5-HT 4L receptor[END_REF] (accession number Y09585, position 1-24): 5' TAATGGACAAACTTGATGCTAATG 3', and ON2 designed on International Patent WO 94/14957, (Sequence 1, position 1302-1325): 5' TAGTAACCTGTTCATGCAGACACA 3'. ON1 is located in the 5' translated region of m5-HT 4(b) receptor and ON2 is located in the 3' untranslated part of the r5-HT 4S R cDNA. PCR was performed with the Taq DNA Polymerase (Eurobio): 94°C/3 min followed by 30 cycles of denaturation (94°C/30 sec), annealing (55°C/30 sec) and elongation (72°C/1 min). These conditions yielded a large band of near 1228 bp which was subcloned into pCR®2.1 vector using the Original TA Cloning® Kit (Invitrogen) and plasmid DNA was prepared for insert sequencing. Nucleotidic sequence was performed using Sanger dideoxy-nucleotide chaintermination method on denatured double-stranded plasmid templates using T7 DNA polymerase (Pharmacia kit). Three different clones were isolated: the m5-HT 4(a) R cDNA which is the equivalent of the previously cloned r5-HT 4S R in rat [START_REF] Gerald | The 5-HT 4 receptor: molecular cloning and pharmacological characterization of two splice variants[END_REF] and two other cDNA sequences presenting additional cassettes at position 1079 that we named m5-HT 4(e) R and m5-HT 4(f) R, taking into account the other published 5-HT 4 receptors and the order of cloning. To confirm the sequence of the 5' part of the receptor around the ATG codon, we performed a PCR reaction using the following primers: ON3 (Patent, Sequence 1, position 21-41) 5' GGTTGGAAGGAGGAGGATGCT 3', in the 5' non coding sequence, and ON4 (Patent, Sequence 1, position 699-721) 5' GAGGAGAAACGGGATGTAGAAGG 3', in the middle of the receptor sequence. The PCR conditions with the Taq DNA Polymerase (Eurobio) were the same as above. Fragments of the expected size (701 bp) were subcloned using the Original TA Cloning® Kit (Invitrogen). To define the exact sequence of all the 5-HT 4 receptor splice variant cDNAs, the sequences were performed for each cDNA, on three clones obtained from three independent PCR reactions.

Isolation and sequencing of the rat 5-HT 4(e) receptor cDNA by RT-PCR:

cDNA encoding rat brain 5-HT 4(e) receptor, a novel rat variant, has been cloned by RT-PCR on total rat brain poly(A) + RNA (Clontech) following the same procedures as described above. Primers used were: ON5 [START_REF] Gerald | The 5-HT 4 receptor: molecular cloning and pharmacological characterization of two splice variants[END_REF] (accession number U20906, position 19-42) and ON2.

Isolation and sequencing of the human 5-HT 4(e) receptor cDNA by RT-PCR: cDNA encoding human brain 5-HT 4(e) receptor, a novel human variant, has been cloned by RT-PCR on total human brain poly(A) + RNA (Clontech) following the same procedures as described above.

Primers used were: ON6 (Claeysen et al., 1997a) (accession number Y0586, position 1-24) and ON2.

Tissue localization studies:

Brain areas and organs were dissected from adult Swiss mice. cDNA was obtained as described above from poly(A) + RNA. Primers designed to carry out PCR amplification were: one sense primer in the common part of the variant sequences, ON7 (Patent WO 94/14957, Sequence 1, position 864-880): 5'-CCAAGGCAGCCAAGACT-5' and two antisense primers, ON2 for the amplification of the Ctermini of the (a), (e) and (f) forms and ON8 (Patent WO 94/14957, Sequence 3, position 1322-1340) 5' TTGCCTCATGCTCTTGGAA 3', for the amplification of the C-terminus of the (b) form. For the amplification of b-actin as control, we used ON9 (J00691, position 2252-2271) 5' GACTCCGGAGACGGGGTCAC 3' and ON10 (J00691, position 2482-2501) 5' CGCATCCTCTTCCTCCCTGG 3'. The PCR conditions were those described above. The template cDNA used per tube was retro-transcripted from 250 or 500 ng of poly(A) + RNA. The expected sizes of the PCR fragments were 463 bp for m5-HT 4(a) R, 503 bp for m5-HT 4(f) R, 527 bp for m5-HT 4(e) R (primer couple ON7/ON2), 528 bp for m5-HT 4(b) R (primer couple ON7/ON8) and 450 bp for b-actin (primer couple ON9/ON10). PCR products were run on 3% MetaPhor® agarose (FMC®)/ethidium bromide gels and photographed. Positive and negative controls (with or without reverse transcriptase) were performed on the b-actin amplification (data not shown). Southern blotting was done after transfer of the DNA to charged nylon membranes (Qiabond, Qiagen). The hybridization probe was an RT-PCR fragment obtained in the same PCR conditions with r5-HT 4(a) R cDNA as template (a generous gift from Dr. Christophe Gerald) and the two primers ON7 and ON2. The probe was labeled using the Prime-It II Random Primer Labeling Kit (Stratagene). All the receptors were subcloned in the eucaryotic expression vector pRK5, a CMV based expression vector [START_REF] Schall | Molecular cloning and expression of a receptor for human tumor necrosis factor[END_REF].

Construction of truncated 5-HT 4(a) receptor cDNA:

Constructs were obtained by inserting a stop codon after residues 327, 346 or 359 in the 5-HT 4(a) R cDNA sequence, using the QuikChange™ Site-Directed Mutagenesis Kit (Stratagene). Sense primers used: ∆327: 5'-CATCCTCTGAAGTGATGATGAGCG-3' ∆346: 5'-TCCCCTGTTAACCCACAACCATTA-3' ∆359: 5'-CATGTACTAAGGTAGACAGTTTTGCAC -3' Construction of mutated m5-HT 4(e) receptor cDNA:

The mutant A258L was obtained by exchanging the endogenous A258 to L in the m5-HT 4(e) R cDNA sequence, using the QuikChange™ Site-Directed Mutagenesis Kit (STRATAGENE). Sense primer used: 5'-AGACCAAGGCACTCAAGACTTTATGTGT-3'

Construction of epitope-tagged m5-HT 4 R splice variants:

The cDNA of all the 5-HT 4 R variants cloned was subcloned into pRK5, a CMV based expression vector [START_REF] Schall | Molecular cloning and expression of a receptor for human tumor necrosis factor[END_REF]. We then tagged the m5-HT 4(a) , (b) , (e) and (f) receptors with a stretch of nucleotides coding for a 10-amino acid epitope (EQKLISEEDL) of the human c-Myc protein [START_REF] Ramsay | The protein encoded by the human proto-oncogene cmyc[END_REF] in the N-terminal extremity using the QuikChange™ Site-Directed Mutagenesis Kit (Stratagene). Sense primer used: 5'GATTGAATTCTAATGGAACAAAAGCTTATTTCTGAAGAAGACTTGGACAAACTTGATGC-3' (in bold letters: start codon, underlined: cMyc epitope).

Cell culture and transfection:

The cDNAs, subcloned into pRK5, were introduced into LLC-PK1 or in COS-7 cells by electroporation as described by [START_REF] Claeysen | Cloning, expression and pharmacology of the mouse 5-HT 4L receptor[END_REF]. Briefly, cells were trypsinized, centrifuged, resuspended in EP 1X (50 mM K 2 HPO 4 /20 mM CH 3 CO 2 K/20 mM KOH/26.7 mM MgSO 4 /pH 7.4) with 25 to 2000 ng of receptor cDNA. The total amount of cDNA was kept constant at 15 µg/ transfection using wild type pRK5 vector. After 15 min at room temperature, 300 µl of cell suspension (10 7 cells) were transferred to a 0.4 cm electroporation cuvette (Bio-Rad) and pulsed using a Gene pulser apparatus (setting 1000 µfarads, 270V). Cells were diluted in DMEM (10 6 cells/ml) containing 10% dialyzed and decomplemented fetal bovine serum (dFBS) and plated on 10 cm-Falcon Petri dishes or into 12-well clusters at the desired density. Using epitope tagged 5-HT 4 receptors, we estimated the percent of cells that expressed the receptors.

Immunocytochemical fluorescence labeling of epitope-tagged 5-HT 4 receptors in cells:

5-HT 4 receptors on intact cells were visualized using the anti c-Myc primary antibody and a rhodamine conjugated secondary anti-mouse antibody. Cells were grown on coverslips and fixed 24 hours after transient transfection in 4% paraformaldehyde diluted in PBS (pH 7.4) for 30 min at room temperature, washed three times in a glycine buffer (0.1M, pH 7.4) and incubated for 2h at 37°C with monoclonal antibody 9E10, ascite fluid was diluted 1:250 in PBS-gelatine 0.2%. The cells were then washed and incubated for 1h with a rhodamine conjugated secondary anti-mouse antibody 1:40 in PBS-gelatine 0.2%. Coverslips were washed and mounted on glass slides using gel mount (Biomeda Corp., Foster City, CA.). Cells were viewed on Zeiss Axioplan 2 microscope.

Determination of cAMP production in intact cells:

Six hours after transfection, cells were incubated overnight in DMEM without dFBS with 2 µCi [ 3 H]adenine/ml to label the ATP pool and cAMP accumulation was measured as described by [START_REF] Dumuis | A non classical 5hydroxytryptamine receptor positively coupled with adenylate cyclase in the central nervous system[END_REF].

Membrane preparation and radioligand binding assay:

Membranes were prepared from transiently transfected cells plated on 15 cm-dishes and grown in DMEM with 10% dFBS for 6 hours and 20 hours in DMEM without dFBS as previously described [START_REF] Ansanay | Pharmacological comparison between [ 3 H]-GR113808 binding sites and functional 5-HT 4 receptors in neurons[END_REF]. The cells were washed twice in PBS, scraped with a rubber policeman, harvested in PBS and centrifuged at 4°C (900 x g for 4 min). The pellet was resuspended in buffer containing 10 mM Hepes (pH 7.4), 5 mM EGTA, 1 mM EDTA and 0.32 M sucrose and homogenized 10 times with a glass-Teflon potter at 4°C. The homogenate was centrifuged at 20,000 x g for 20 min, the membrane pellet was resuspended in 50 mM Hepes (pH 7.4) (5 mg of protein in 1 ml solution) and stored at -80°C until used.

To perform radioligand binding studies with [ 3 H]GR 113808 (specific activity: 83 Ci/mmol) 100 µl of membrane suspension, prepared as described above, were diluted 5 times with 50 mM HEPES (pH 7.4) (2 to 20 µg of protein) containing 10 mM pargyline and 0.01% ascorbic acid and were incubated at 20°C for 30 min with 100 µl [ 3 H]GR 113808 and 50 µl of buffer or competing drugs. For saturation analysis assays, various concentrations of [ 3 H]GR 113808 (0.001 to 1 nM) were used. For competition binding experiments, the [ 3 H]-GR 113808 concentrations was 0.15 nM. Receptor expression was determined using a saturating concentration of[ 3 H]GR 113808 (0.3-0.5 nM). 5-HT (0.5 µM) was used to determine non specific binding.

For determination of b 2 -adrenergic and CAM b 2 -adrenergic receptor expression in COS-7 cells, membranes preparation and binding studies were performed as described previously [START_REF] Chidiac | Inverse agonist activity of badrenergic antagonists[END_REF]. The specific radioligand [ 125 I]cyanopindolol was used at saturating concentrations (300-500 pM; Kd = 21 nM) and propranolol at 10 µM was added to determine non-specific binding. For binding experiments, membranes were incubated in a buffer containing 7 mM Tris-HCl, pH 7.4, 12.5 mM MgCl 2 , 2 mM EDTA, and proteins (5 to 20 µg) for 90 min at 20°C.

In both assays, the incubation was terminated by vacuum filtration over Whatman GF/B filters presoaked with 0.1% polyethylenimine using a Brendel 48 well cell harvester; the filters were then washed with 3 x 4 ml ice-cold buffer (10 mM HEPES). Filtration was performed in less than 10 seconds. Radioactivity was counted using a complete phase combining system for liquid scintillation counting (Amersham, Les Ullis, France). Protein concentration in the samples was determined using the Bio-Rad protein assay .

Drugs

RESULTS

Primary structure of the different 5-HT 4 R splice variants:

We have published the isolation of m5-HT 4(b) R, previously named long form [START_REF] Claeysen | Cloning, expression and pharmacology of the mouse 5-HT 4L receptor[END_REF] which has also been isolated in rat and human [START_REF] Gerald | The 5-HT 4 receptor: molecular cloning and pharmacological characterization of two splice variants[END_REF][START_REF] Van Den Wyngaert | Cloning and expression of a human serotonin 5-HT 4 receptor cDNA[END_REF].

We performed RT-PCR experiments with poly(A) + RNA prepared from mouse brain using primers designed to clone the variant (a) (described in the experimental procedure section). We obtained three different full length cDNA fragments. One of them corresponded to variant (a) previously cloned in rat and human [START_REF] Blondel | Molecular and functional characterization of a 5-HT 4 receptor cloned from human atrium[END_REF]Claeysen et al., 1997a;[START_REF] Gerald | The 5-HT 4 receptor: molecular cloning and pharmacological characterization of two splice variants[END_REF], whereas two new clones generated from the same RT-PCR corresponded to new 5-HT 4 receptor cDNAs, coding for variants significantly shorter than the (a) and (b) forms. Blondel et al. have reported the existence of two unrelated h5-HT 4 R splice variants (c) and (d) [START_REF] Blondel | Cloning, expression, and pharmacology of four human 5-hydroxytryptamine 4 receptor isoforms produced by alternative splicing in the carboxyl terminus[END_REF] (Fig. 1). Taking into account the already published human variants, we named the present new variants m5-HT 4(e) R and m5-HT 4(f) R (Fig. 1). We also cloned a r5-HT 4(e) receptor, identical, to the m5-HT 4(e) R in length and amino acid sequence after the splicing site by performing RT-PCR experiments with poly(A) + RNA prepared from rat brain. In human, we have also isolated, from brain, a new splice variant very close but not identical in sequence to the (e) form. When comparing its nucleotidic and amino acid sequences to those of rat and mouse (e) forms, we can suggest that this new human variant produced by alternative splicing is the human orthologue of the mouse and rat (e) forms. We therefore called it h5-HT 4(e) R.Whatever the species, mouse, human or rat, the different splice variants have an identical sequence up to L358, whereas the length as well as the composition of the C-terminal end, is specific for each splice variant (Fig. 1). Note the specific characteristics of the last four residues of the Ctermini of the new clones described here. The r5-HT 4(e) , m5-HT 4(e) , (f) as well as of the h5-HT 4(e) receptors contain two PV repeats.

Novel mouse 5-HT 4 R splice variants are brain-specific:

Using pairs of primers, designed as described in the experimental procedure section, the expression of the different m5-HT 4 R transcripts (Fig. 2A) were assayed in brain and in the peripheral tissues.

Note that our RT-PCRs performed either with 250 or 500 ng of poly(A + ) RNA are non-saturating, ie.

the ratio between the different bands amplified remain the same whatever the quantity used (Fig. 2B).

The most interesting observation was the total absence of m5-HT 4(e) R and (f) R mRNA expression in peripheral tissues. In brain, we detected a high level of the long form (a) and (b) mRNAs in all of the structures studied (Fig. 2C). The two new variants (e) and (f) were expressed in colliculi, striatum and hippocampus. In the cerebellum and the cortex the (e) form was not detected. We observed a relatively high level of the m5-HT 4(a) R and (b) R mRNA in heart and kidney but also in bladder, ileum and colon. Oesophagus and adrenal gland showed very low levels of signal and liver did not express detectable amount of any of the four splice variants. As for m5-HT 4(e) R, (f) R, the r5-HT 4(e) R and h5-HT 4(e) R mRNAs have been detected only in brain (data not shown).

5-HT 4(a) receptors have a high constitutive activity:

Since we previously observed that native m5-HT 4(a) R and h5-HT 4(b) R expressed high basal constitutive activities in the absence of agonist (Claeysen et al., 1997a;Claeysen et al., 1997b), we decided to investigate the capacity of 5-HT 4 receptors to convert from R to R* by studying their constitutive activity as a function of receptor density. In COS-7 cells, in the absence of 5-HT, a twofold increase in cAMP production as compared with mock transfected cells, was obtained for a 5-HT 4(a) receptor density (150/200 fmol/mg protein) comparable to those found in brain tissues [START_REF] Waeber | Regional distribution and ontogeny of 5-HT 4 binding sites in rodent brain[END_REF] (Fig. 3A). Because comparison of receptor density is rather complex we have determined the percent of cells expressing the 5-HT 4 receptors. Cells were transiently transfected with the epitope-tagged receptor cDNA and visualized using the anti c-Myc primary antibody. We found that 57 ± 16% (n=6) of the cells expressed the transfected epitope-tagged 5-HT 4 receptor isoforms, whatever the splice variant considered.

The constitutive activity of 5-HT 4(a) R was almost linearly proportional to the receptor density and independent of the species origin of the receptor (Fig. 3A). Such a constitutive activity, was not specific to COS-7 cells since similar results were obtained in LLC-PK1 cells (Fig. 3B). In this cell lane, a two fold increase in cAMP production was observed for a receptor density of 100 fmol/mg proteins. Fig. 3A shows that native 5-HT 4(a) receptors had a much higher constitutive activity than the native hb 2 -AR when expressed in COS-7 cells.

Although our experiments were performed with dialyzed serum, it was important to check whether some contamination by 5-HT, known to stick to plastic surfaces was responsible for the observed intrinsic activity. We therefore used highly potent selective 5-HT 4 R antagonists, some (like RS 100235) were neutral antagonists and had no effect on basal 5-HT 4(a) receptor-induced constitutive activity (Fig. 4A). However other antagonists like GR 125487, behaved as inverse agonists because they reduced this basal constitutive activity [START_REF] Costa | Antagonists with negative intrinsic activity at d opioid receptors coupled to GTP-binding proteins[END_REF]. In addition, both antagonists used in Fig. 4A were highly potent in inhibiting the 5-HT-induced cAMP production in transfected COS-7 cells expressing 5-HT 4(a) receptors (Fig. 4B). These experiments indicated that the observed constitutive activity was not due to 5-HT contamination.

Mouse 5-HT 4 receptor splice variants exhibit different constitutive activities:

It was of particular interest to check if the difference in the structure of the C-terminus of the different m5-HT 4 receptor splice variants influence their propensity to induce high basal constitutive activity

We actually found that although the (a) and (b) forms gave a similar constitutive activity when studied as the function of receptor density, the shortest forms (e) and (f) presented a two times higher basal constitutive activity (Fig. 5). In comparison, in this experiment, hb 2 -AR do not modify cAMP production when expressed at this density (Fig. 5)

A C-terminal domain of 5-HT 4 receptors inhibits constitutive activity:

Since we found that the shortest splice variants had the highest constitutive activity (Fig. 5) we wondered if the C-terminal domain was important for constraining 5-HT 4 receptors in an inactive state. This turned out to be true since progressive deletions of the C-terminal part of the 5-HT 4(a)

receptor (Fig. 6A) resulted in a progressive increase in constitutive activity for a similar density of wild type and truncated receptors (Fig. 6B). The truncated 5-HT 4 ∆359 receptor identical in length and sequence to the 5-HT 4(f) R variant without its PVPV sequence, represents the common region of the four splice variants. This truncated receptor displayed a similar constitutive activity to that of 5-HT 4(f) R, whereas a larger deletion mutant (deletion of 13 residues: 5-HT 4 ∆346 receptor) dramatically activated the receptor. A larger deletion of the C-terminal (∆327) did not further increase the constitutive activity compared to ∆346. Both deletions (∆346 and ∆327) induced a 5-fold activation of basal cAMP production for receptor densities below 200 fmol/mg protein.

The C-terminal part of the third intracellular loop (i3) constrains the 5-HT 4 receptor in its inactive form:

A comparison between the sequences of the C-terminal part of i3 of m5-HT 4 receptors on one hand and of the hb2-AR on the other hand indicated that among the last 7 residues, 4 of them were identical.

One of them, alanine in 5-HT 4 receptor, leucine in b 2 -AR at position -3 upstream of TM VI, was interesting because the mutation of leucine to alanine was one of the four mutations in i3 of b 2 -AR that produced the CAM hb 2 -AR [START_REF] Samama | A mutation-induced activated state of the b 2 -adrenergic receptor. Extending the ternary complex model[END_REF] (Fig. 7A). It is also this mutation (leucine to alanine) which is the most potent mutation in inducing the constitutive activity of CAM hb2-AR (S.

Cotecchia, personal communication). Therefore, one possible explanation for the highest constitutive activity of native 5-HT 4 receptors as compared to native b 2 -AR, was the presence, at this homologous position, of an alanine (A258) in the wild type 5-HT 4 receptor (Fig. 7A) instead of a leucine in b 2 -AR. We therefore mutated this A258 to L in the 5-HT 4(e) receptor. Instead of reducing the constitutive activity of the receptor we obtained a dramatic increase (Fig. 7B). For comparison we note that the constitutive activity of the native 5-HT 4(e) receptors was as high as the constitutive activity of CAM hb 2 -AR .

Basal and 5-HT stimulated cAMP productionin cells transfected with 5-HT 4(a) , (e) receptors and 5-HT 4 mutated receptors:

The short C-terminal sequence 5-HT 4(e) receptors had a higher basal constitutive activity than the long C-terminal terminal sequence 5-HT 4(a) receptors as already described in fig. 5. However, they had identical activity in the presence of 5-HT (Fig. 8A). In this experiment, we expressed about 1000 fmol/mg protein of 5-HT 4(a) and (e) receptors. The identical 5-HT-stimulated activity observed with these two 5-HT 4 variants was not due to a limiting step in cAMP formation since increasing the number of expressed receptors from 1000 to 4000 fmol/mg protein increased the 5-HT stimulated activity proportionally (data not shown). Fig. 8B illustrates the relative efficacity of 5-HT to stimulate over the basal constitutive activity of the wild types and the mutated receptors. The higher the basal constitutive activity, the lower the relative efficacy of 5-HT. Note that the 5-HT-maximum response was slighly lower in severely truncated receptors (Fig. 8 A). A decrease in the mutated receptor stability during incubation or an increase in desensitization can be proposed as an explanation [START_REF] Gether | Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility[END_REF][START_REF] Pei | A constitutively active mutant beta 2-adrenergic receptor is constitutively desensitized and phosphorylated[END_REF].

Substitution of A258 by L within the third intracellular loop i3 and C-terminal truncations (∆346 and ∆327) of m5-HT 4(e) receptors increase the affinity of 5-HT and its potency in stimulating cAMP:

The extended ternary complex [START_REF] Samama | A mutation-induced activated state of the b 2 -adrenergic receptor. Extending the ternary complex model[END_REF] as well as the cubic ternary complex [START_REF] Kenakin | The evolution of drug-receptor models: the cubic ternary complex model for seven transmembrane domain receptors[END_REF] introduced for the modeling of the G protein receptor isomerization R to R* and their interaction with G proteins, indicated that the observed affinity of agonist increased as the function of the isomerization constant J. Indeed, we observed that the affinity of 5-HT for both the mutated 5-HT 4(e) R A258L and C-terminal deleted (∆346 and ∆327) 5-HT 4 receptors expressed at similar densities, increased by almost two logs 10 when compared with that of the native receptor. 5-HT-binding was measured by competition. The Kd values for 5-HT were 47 ± 12 nM for the wild type 5-HT 4(e) R, 0.53 ± 0.6 nM and 1.34 ± 0.15 nM for the truncated 5-HT 4 R ∆346 and 5-HT 4 R ∆327 respectively and 0.85 ± 0.10 nM for the mutated 5-HT 4(e) R A258L (Fig. 9A). The difference in affinity observed was certainly not due to a difference in the coupling to G proteins (different proportion of the high affinity state), since a high GppNHp concentration (10 µM) did not modify this difference (data not shown). In contrast to these results, we found no difference between the affinity of 5-HT for the different natural splice variants. The EC 50 of 5-HT to stimulate cAMP were about 10 times lower when determined in mutant receptor transfected cells rather than in native receptors transfected cells (Fig. 9B) The EC 50 values were 8 ± 2 nM, 0.3 ± 0.10 nM, 0.7 ± 0.10 nM and 0.9 ± 0.15 nM for 5-HT 4(e) R, 5-HT 4 R ∆346, 5-HT 4 R ∆327 and 5-HT 4(e) R A258L, respectively.

DISCUSSION

We report the cloning of new 5-HT 4 R splice variants (m5-HT 4(e) R and (f) R; r5-HT 4(e) R and h5-HT 4(e) R) which possess original C-terminal domains compared to already cloned 5-HT 4 R splice variants [START_REF] Blondel | Cloning, expression, and pharmacology of four human 5-hydroxytryptamine 4 receptor isoforms produced by alternative splicing in the carboxyl terminus[END_REF][START_REF] Blondel | Molecular and functional characterization of a 5-HT 4 receptor cloned from human atrium[END_REF]Claeysen et al., 1997a;[START_REF] Claeysen | Cloning, expression and pharmacology of the mouse 5-HT 4L receptor[END_REF][START_REF] Gerald | The 5-HT 4 receptor: molecular cloning and pharmacological characterization of two splice variants[END_REF][START_REF] Van Den Wyngaert | Cloning and expression of a human serotonin 5-HT 4 receptor cDNA[END_REF]. They have in common an intriguing and unique PVPV C-terminal sequence that is also found in the C-termini of some EPH/ELK family of receptor tyrosine kinases [START_REF] Wicks | Molecular cloning of HEK, the gene encoding a receptor tyrosine kinase expressed by human lymphoid tumor cell lines[END_REF]. We can only speculate on the possible roles for this sequence but one could be the interaction of these splice variants with proteins similar to PDZ-domaincontaining proteins. Because of the roles of 5-HT 4 receptors in learning and memory [START_REF] Ansanay | A cyclic AMP-dependent, longlasting inhibition of a K + current in mammalian neurons[END_REF][START_REF] Eglen | Central 5-HT 4 receptors[END_REF][START_REF] Letty | 5-HT 4 receptors improve social olfactory memory in the rat[END_REF][START_REF] Marchetti-Gauthier | BIMU1 increases associative memory in rats by activating 5-HT 4 receptors[END_REF] it is interesting to note that the newly cloned PVPV-containing receptors are only expressed in brain tissues.

In view of the large number of 5-HT 4 receptor variants only differing in their C-termini domains it was of particular interest to analyze the role of these domains in G protein coupling. Over the past ten years our view on how GPCRs are activated has been re-assessed based on the pioneering works of Costa and Lefkowitz's laboratories [START_REF] Costa | Antagonists with negative intrinsic activity at d opioid receptors coupled to GTP-binding proteins[END_REF][START_REF] Cotecchia | Regions of the a1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function[END_REF]. They demonstrated that GPCRs can isomerize from an inactive form R to an active form R* in the absence of agonist. GPCRs are no longer considered as shifting from an inactive to an active conformation when interacting with the agonist but it is now proposed that the agonist is only stabilizing the R* form. Thus, GPCRs can be considered as allosteric molecules which already includes ionotropic receptors such as nicotinic receptors [START_REF] Changeux | Allosteric receptors after 30 years[END_REF][START_REF] Monod | On the nature of allosteric transitions: a plausible model[END_REF]. Among the numerous theoretical consequences of this view is the idea that the structure of the receptor has been selected to constrain the receptor under the R form in the absence of agonist. The higher the constraint (low isomerization J constant), the higher the signal to noise ratio in the presence of agonist.

The evaluation of the capacity of a native receptor to spontaneously isomerize from R to R* is certainly a very important pharmacological characteristic, because this may explain part of its physiological and possible pathological behavior as well as the effect of drugs classified as inverse agonists (able to reverse the isomerization because of their higher affinity for R than for R*) on this receptor.

Native 5-HT 4(a) receptors of different species (mouse, rat, human) have a much higher capacity to convert from R to R* than most of the other GPCRs studied so far. Indeed, when expressed at low density (100 to 200 fmol/mg), 5-HT 4(a) receptors produced a two-fold increase in the cAMP production compared to mock transfected cells. In comparison, hb 2 -AR do not modify cAMP production when expressed at this density (Fig. 3 and [START_REF] Samama | A mutation-induced activated state of the b 2 -adrenergic receptor. Extending the ternary complex model[END_REF]). Indeed in COS-7 cells, less than a two fold increase in cAMP production, was obtained when native b 2 -AR were expressed at a density of 20 pmol/mg protein [START_REF] Samama | A mutation-induced activated state of the b 2 -adrenergic receptor. Extending the ternary complex model[END_REF]. Increasing native 5-HT 4(a) receptor density to 400 fmol/mg protein was sufficient to increase cAMP production by four fold as compared to mock transfected cells. This high capacity to isomerize was not restricted to expression of the receptor in COS-7 cells, since in LLC-PK1 cells, when the receptor density reached a density as low as 150 fmol/mg protein, the cAMP produced was 3 times that found in mock transfected cells.

The C-terminal domains of the intracellular i3 loop of 5-HT 4 R and hb 2 -AR are remarkably similar.

Upstream of the TM VI, 4 of the last 7 residues are identical. One of the divergent position is an alanine residue in 5-HT 4 receptor which is a leucine in hb 2 -AR (localized at residue -3 upstream of TM VI). This position is critical for keeping GPCR in an inactive form [START_REF] Kjelsberg | Constitutive activation of the a 1B -adrenergic receptor by all amino acid substitutions at a single site[END_REF][START_REF] Samama | A mutation-induced activated state of the b 2 -adrenergic receptor. Extending the ternary complex model[END_REF]. Mutation of this position in a 1B -AR [START_REF] Kjelsberg | Constitutive activation of the a 1B -adrenergic receptor by all amino acid substitutions at a single site[END_REF] and TSHRs [START_REF] Parma | Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenoma[END_REF] increases their constitutive activity. In CAM b 2 -AR, in which four residues of the Cterminal part of i3 were mutated [START_REF] Samama | A mutation-induced activated state of the b 2 -adrenergic receptor. Extending the ternary complex model[END_REF], this position is crucial for constitutive activation (S. Cotecchia, personal communication). Since native 5-HT 4 receptors have a much higher constitutive activity than native hb 2 -AR and have, as CAM hb 2 -AR, an alanine at this position we mutated the alanine of 5-HT 4 receptor to leucine to see if the constitutive activity would drop to the level of native hb 2 -AR. This was not the case and, in contrast, a further and dramatic increase in 5-HT 4 receptor constitutive activity was observed. However, this further indicates that the C-terminal part of the i3 loop is also crucial to keep 5-HT 4 receptor in the R inactive state and that the native primary sequence is the one which probably gives the conformation having the lowest constitutive activity.

The highest basal constitutive activity of the short C-teminal sequence splice variant (5-HT 4(e) R , (f) R) compared to the long C-terminal sequence variants (5-HT 4(a) R , (b) R) can be explained by either one of the following hypotheses. In the first one, the shortest splice variants have a greater ability to isomerize from R to R* (and an equal efficacy of their R* form to stimulate G proteins). In the second one, the efficacy of their R* forms to stimulate G proteins are different and their ability to isomerize are identical. It is difficult to completely exclude the second hypothesis. However, the observation that the 5-HT induced maximum responses were identical whatever the splice variant considered argue in favor of a higher capacity of short C-terminal sequence splice variants to isomerize from R to R*.

One important issue is to know whether such a constitutive activity is present or not in neurons or other peripheral tissues (atria, colon, ileum etc..) in which 5-HT 4 receptors are expressed at comparable density [START_REF] Eglen | Central 5-HT 4 receptors[END_REF][START_REF] Waeber | Regional distribution and ontogeny of 5-HT 4 binding sites in rodent brain[END_REF]. Comparison of receptor density is rather complex because even though around 57% of the cells expressed the transfected tagged 5-HT 4 receptors (whatever the splice variant considered), the quantity of cells expressing this receptor in heterogeneous tissues such as brain for example, remains unknown. In addition, the distribution of 5-HT 4 receptors in COS-7 cell and neuronal membranes are completely different. In preliminary experiments, we did not find a reduction of basal cAMP production in primary colliculi neurons expressing 5-HT 4 receptors using the inverse agonist GR 125487 (data not shown). Since the isomerization constant depends on environmental conditions, it is also possible that proteins that interact or chemically modify the C-termini (one may think of a phosphorylation of the serinethreonine rich domain upstream of the splicing site) reduce the J constant in neurons. The search for such putative interacting proteins will certainly be of interest.

There are increasing data to indicate that specific sequences of the C-terminal end of GPCRs modulate the isomerization of these receptors from R to R*. A cluster of basic residues present in the C-terminal tail of the short variants of mGluRs (mGluR1b, c, d), impairs their ability to isomerize from R to R*.

Removing this cluster by mutation reveals their constitutive activity [START_REF] Mary | A cluster of basic residues in the carboxyl-terminal tail of the short metabotropic glutamate receptor 1 variants impairs their coupling to phospholipase C[END_REF]. In addition, the effect of this inhibitory cluster is suppressed by a long C-terminal domain of mGluR1a which, in fact, has a high constitutive activity [START_REF] Mary | A cluster of basic residues in the carboxyl-terminal tail of the short metabotropic glutamate receptor 1 variants impairs their coupling to phospholipase C[END_REF]. Truncation of the last residues of TRHR and prostaglandin E receptors causes constitutive activity [START_REF] Hasegawa | Two isoforms of the prostaglandin E receptor EP3 subtype different in agonist-independent constitutive activity[END_REF][START_REF] Matus-Leibovitch | Truncation of the thyrotropin-releasing hormone receptor carboxyl tail causes constitutuve activity and leads to impaired responsiveness in Xenopus oocytes and AtT20 cells[END_REF]. The last 12 residues of bovine rhodopsin have also been proposed to be involved as negative regulator of GTP exchange [START_REF] Weiss | Effects of carboxyl-terminal truncation on the stability and G protein-coupling activity of bovine rhodopsin[END_REF]. Removal of the extended C-terminal domain of avian b 2 -AR increases its activity [START_REF] Parker | Truncation of the extended carboxyl-terminal domain increases the expression and regulatory activity of the avian b-adrenergic receptor[END_REF].

We found that truncation of the C-terminal domain consisting in the removal of the sequences that are divergent in individual 5-HT 4 receptor splice variants (∆359) gave a constitutive activity similar to those of the shortest splice variants (5-HT 4(e) R and (f) R). Since the 5-HT 4(a) ∆359 receptor is identical to the 5-HT 4(f) receptor with its PVPV sequence removed, the PVPV sequence is not responsible for intrinsic activity of the shortest splice variants (Fig. 6). In contrast truncation of the last 13 residues (12 of which are common in C-terminal tails of all of them) (∆346), resulted in a dramatic release on the receptor constraint, the basal activity increased 10-fold when as low as 500 fmol/mg protein of receptors are expressed. A further truncation (∆327) did not exacerbate this constitutive activity. This indicates that within the 13 residues comprised between ∆346 and ∆359, one set of residues is highly potent in maintaining the receptor under the R form. Interestingly, this sequence contains 6 serine and threonine residues susceptible to be phosphorylated, a post translational modification which may modulate the receptor intrinsic activity. Removal of this inhibitory sequence rendered the receptor so active that it became less sensitive to 5-HT.

In conclusion we can propose a model in which (Fig. 10): (1) the 5-HT 4 receptor protein, up to residue 346, is mainly a constitutive active receptor coupled to Gs, (2) the last common sequence in the Cterminal domain (around 13 residues between 346 and 359) is mainly engaged in the constraint of the receptor under its inactive form 3) the specific sequences of 5-HT 4(e) and (f) ) receptors are neutral in the modulation of the isomerization because their constitutive activity did not differ from that of ∆359, All the cloned 5-HT 4 receptors share the same amino acid sequence up to Leu 358 and differ from Leu 358 on (boxed). †: receptors cloned by Gerald et al. [START_REF] Gerald | The 5-HT 4 receptor: molecular cloning and pharmacological characterization of two splice variants[END_REF]. *: receptors cloned by our group (Claeysen et al., 1997a;[START_REF] Claeysen | Cloning, expression and pharmacology of the mouse 5-HT 4L receptor[END_REF]. ‡: receptors previously cloned by Blondel et al. [START_REF] Blondel | Cloning, expression, and pharmacology of four human 5-hydroxytryptamine 4 receptor isoforms produced by alternative splicing in the carboxyl terminus[END_REF]. §: receptors cloned by [START_REF] Van Den Wyngaert | Cloning and expression of a human serotonin 5-HT 4 receptor cDNA[END_REF]. Note that the amino acid sequence after Leu 358 of newly cloned m5-HT 4(e) and (f) , r5-HT 4(e) and h5-HT 4(e) receptors are shaded.

The EMBL Nucleotide Sequence Database accession numbers for mouse 5-HT 4(a) , (b) , (e) and (f) receptors are Y09587, Y09585, Y09588 and AJ011369 respectively. For rat 5-HT (a) , (b) and (e)

receptors the numbers are: U20906, U20907 and AJ011370 respectively and for human 5-HT 4(a) , (b) , (c) , (d) and (e) receptors Y09586, Y10437, Y12506, Y12507 and AJ011371 respectively.

b 2 -

 2 adrenergic receptor and CAM-b 2 -AR cDNAs: The human b 2 -adrenergic receptor cDNA (hb 2 -AR) and the CAM-b 2 -AR cDNA were obtained from Susanna Cotecchia.

  methylsulphonyl-amino)ethyl]4-piperidinyl]methyl-1-methyl-indole-3 carboxylate, maleate) and GR 125487([1-[2-(methylsulphonyl-amino)ethyl]4-piperidinyl]methyl-5fluoro-2-methoxy-1H-indole-3-carboxylate, hydrochloride) were generously donated by Glaxo (U.K.); [ 3 H]GR 113808 was purchased from Amersham (U.K.); 5-HT (serotonin) and isoproterenol were purchased from Sigma Chemical Corp., St. Louis, MO, USA. RS 100235 (1-(8-amino-7-chloro-1,4-benzodioxan-5-yl)-3-[[3,4-dimethoxyphenyl)prop-1-yl]piperidin-4-yl]propan-1-one) generously donated by Roche-Syntex Laboratories, Palo Alto, USA.

( 4 )

 4 Fig. 1: C-terminal amino acid sequences of the 5-HT 4 receptor splice variants.

Fig. 2 :

 2 Fig. 2: Regional distribution of the four mouse 5-HT 4 receptor splice variants. A) Theoretical PCR results. Lane 1: primer couple ON7/ON2 (see Experimental Procedures) amplifying the mouse (a), (e) and (f) forms. Lane 2: primer couple ON7/ON8 (see Experimental Procedures) amplifying the mouse (b) form.B) Distribution of mouse 5-HT 4 R transcripts. RT-PCR analysis performed with 250 and 500 ng of poly(A + )RNA from colliculus (collic.) and cortex of adult Swiss mice using primer couple ON7/ON2

Fig. 3 :

 3 Fig. 3: Basal levels of cAMP in COS-7 and LLC-PK1 cells transiently expressing increasing concentrations of human, rat and mouse 5-HT 4(a) receptor splice variants.A) COS-7 cells were transfected with increasing concentrations of human, rat or mouse 5-HT 4(a) R and hb 2 -AR receptor cDNA (25 ng to 1000 ng cDNA/10 7 cells). The total amount of DNA was kept constant at 15µg/10 7 cells using wild type pRK5 vector. The percent conversions of [ 3 H]-ATP to [ 3 H]-cAMP in mocked transfected cells was 0.12 ± 0.03 (control). Basal levels of cAMP productions at different receptor concentrations, measured after 30 min-incubation, are expressed as a percent of control.B) LLC-PK1 cells were transfected with increasing concentrations of h5-HT 4(a) receptor cDNA/10 6 cells (25 ng to 1000 ng cDNA) or with equivalent concentrations of pRK5 plasmid (mock-transfected cells). The percent conversion of [ 3 H]-ATP to [ 3 H]-cAMP in mock-transfected cells was 0.115 ± 0.021 (control). Basal levels of cAMP production, measured at different receptor concentrations after 30 min-incubation, are expressed as a percent of control.In A and B, 5-HT 4 receptor densities were determined by using a saturating concentration of [ 3 H]GR 113808 (0.4-0.5 nM), hb 2 -AR densities were determined by using a saturating concentration of [ 3 I]CYP (0.5 nM). Each point is the means ± SEM of three independent experiments.

Fig

  Fig. 4: Effect of two potent 5-HT 4 R antagonists: RS 100235 and GR 125487 on basal and 5-HTstimulated 5-HT 4(a) receptor. The effects of RS 100235 and GR 125487 on cAMP production, in the absence (A) and presence (B) of 5-HT (10 -7 M) were evaluated on h5-HT 4(a) receptor splice variant transiently transfected in COS-7 cells expressing 320 ± 45 fmol/mg protein. Levels of cAMP accumulation were measured after 15 min-incubation and expressed as a percent of control. The percentage conversion of [ 3 H]-ATP to [ 3 H]-cAMP in mock-transfected cells was 0.14 ± 0.018 (control). Each value is the means ± SEM of three independent experiments each performed in triplicate.

Fig. 5 :

 5 Fig. 5: Mouse 5-HT 4 receptor splice variants exhibit different basal activities.The activity was measured by increase in cAMP production in COS-7 cells, expressed m5-HT 4(a) ,(b) , (e) , (f) receptor or hb 2 -A receptor as a function of receptor density. COS-7 cells were transfected with increasing concentrations of m5-HT 4(a) , (b) , (e) , (f) and hb 2 -A receptors cDNA (10 ng to 1000 ng cDNA/10 7 cells). Basal levels of cAMP accumulation were measured after 30 min-incubation. The percent conversion of [ 3 H]-ATP to [ 3 H]-cAMP in mocked transfected cells was 0.12 ± 0.03 (control). Basal cAMP production measured at different concentrations of receptor is expressed as a percent of control. 5-HT 4 and hb 2 -A receptors densities were determined as indicated in Fig.3. Data for each of the splice variant represent the means ± SEM for at least three independent experiments each performed in triplicate.

Fig. 6 :A)

 6 Fig. 6: A common sequence in the C-terminal domain of 5-HT 4 receptor temporizes its basal activity. A) Schematic representation of the C-terminal tail of the m5-HT 4(a) and (f) receptor splice variants. The arrows indicate the sites substituted by a stop codon to produce the truncated receptors (5-HT 4 R ∆359, 5-HT 4 R ∆346 and 5-HT 4 R ∆327). Serine-threonine clusters are shaded. B) COS-7 cells were transfected with increasing concentrations of m5-HT 4(a) R, m5-HT 4(f) R, m5-HT 4 R ∆359, m5-HT 4 R ∆346 or m5-HT 4 R ∆327 receptor cDNA/10 7 cells (10 ng to 2000 ng cDNA)The percent conversion of [ 3 H]-ATP to [ 3 H]-cAMP in mocked transfected cells was 0.15 ± 0.03 (control). cAMP productions measured at different concentrations of receptor are expressed as a percent of control. Each point is the means ± SEM of three independent experiments each performed in triplicate.

Fig. 7 :

 7 Fig. 7: Mutations in the C-terminal part of the i3 loop of 5-HT 4(e) and b 2 -A receptors increase constitutive activity.Relationship between receptor density and basal levels of cAMP production measured in COS-7 cells transfected with m5-HT 4(e) or m5-HT 4(e) A258L or hb 2 or CAM hb 2 receptors.A) Amino acid sequences of the C-termini of the intracellular loop i3 in m5-HT 4 R and hb 2 -AR, mutated residues in m5-HT 4(e) R A258L and CAM hb 2 -AR. The mutated residues are shaded.

Fig. 8 :

 8 Fig. 8: Mutations in 5-HT 4 receptor decrease responsiveness to an agonist proportionally to increasing constitutive activity. A) COS-7 cells were incubated for 15 min at 37°C with vehicle (basal) or with 10 -6 M 5-HT for cells expressing wild types or mutated 5-HT 4 R and with vehicle (basal) or with 10 -6 M isoproterenol for those expressing wild type or mutated hb 2 -AR. The expression levels of 5-HT 4(a) , 5-HT 4(e) , 5-HT 4(e) A258L, 5-HT 4 ∆346, 5-HT 4 ∆327, hb 2 -A and CAM hb 2 -A receptors were 940 ± 56, 1060 ± 126, 1180 ± 170, 1090 ± 165, 827 ± 89, 860 ± 35 and 980 ± 105, fmol/ mg protein, respectively.Percent conversion of [ 3 H]-ATP to [ 3 H]-cAMP in mocked transfected cells was 0.12 ± 0.03 (control).Results are expressed as the percent of control. Data are means ± SEM of four experiments. B) Levels of agonist-stimulated cAMP production in the experiment reported in (A) are expressed as the percent over basal cAMP formation.

Fig

  Fig. 9: Competition binding and 5-HT-induced cAMP formation in COS-7 cells expressing wild-type or mutated 5-HT 4 receptors. A) Competition of 5-HT for [ 3 H]GR 113808 binding on membranes derived from COS-7 cells expressing comparable receptor densities (between 827 and 1180 fmol/mg protein as in experiments reported in Fig. 8) of m5-HT 4(e) R, m5-HT 4 R ∆346, m5-HT 4 R ∆327 or m5-HT 4(e) R A258L. The results expressed as a percent of the specific binding in the absence of a competiting agent are from a representative experiment of three to six independent experiments. B) Intracellular cAMP levels were measured at increasing concentrations of 5-HT in the same transfected COS-7 cells as for binding experiments and in experiments reported Fig. 8. The 100%

Fig. 10 :

 10 Fig. 10: Proposed mechanism for modulation of the constitutive activation of 5-HT 4 receptor splice variants by common and specific carboxyl-terminal domains.The 5-HT 4 receptor up to residue 346 which is common to all the four 5-HT 4 R splice variants is mainly a constitutive receptor coupled to Gs. The common sequence in the C-terminal domain between residues 346 and 359 imposes conformational constraints maintaining the receptor under its inactive form. The specific sequences of the (a) and (b) forms reduce its constitutive activation to an even greater extent, either by increasing the inhibitory effect of the C-terminal common sequence or by a direct negative effect on the isomerization. The specific sequences of (e) and (f) splice variants which share the same PVPV C-terminal sequence do not add supplementary constraint to that imposed by the amino acid sequence 346 to 359.

Figure 1 Figure 10

 110 Figure1

  agarose gels, 25µl of a 50 µl reaction were loaded. A photograph of the ethidium bromide stained gels is shown. Lower panel: southern blot of the PCR results, a 2h exposure of the autoradiogram is shown.Collic., colliculus; Hippoc., hippocampus; Striat., striatum; Cereb., cerebellum; Ventri., ventricle; OEsoph., oesophagus; Adr.gl., adrenal gland.

	(Lane 1) or primer couple ON7/ON8 (Lane 2) (see Experimental Procedures). 35 µl of a 50 µl reaction
	were loaded and separated on 3% agarose gels. A photograph of the ethidium bromide stained gels is
	shown.
	C) Distribution of mouse 5-HT 4 R transcripts. RT-PCR analysis performed with 500 ng of
	poly(A + )RNA from various structures of adult Swiss mice using primer couple ON7/ON2 (Lane 1)
	or primer couple ON7/ON8 (Lane 2) (see Experimental Procedures). Upper panel: PCR products
	were separated on 3%

ACKNOWLEDGMENTS

This work was supported by grants from the Fondation pour la Recherche Médicale and Roche Laboratories. We are grateful to Dr. Christophe Gerald (Synaptic Pharmaceutical Corporation, Paramus N.J., USA) for the gift of rat 5-HT 4L R and 5-HT 4S R cDNA, to Pr. Susanna Cotecchia (Institut de Pharmacologie et Toxicologie, Université de Lausanne, Switzerland) for the gift of human b 2 -adrenergic receptor (hb 2 -AR) and CAM-b 2 -AR cDNAs and to Dr. Bernard Mouillac (U. 469 INSERM Montpellier France) for the gift of monoclonal antibody 9E10. For helpful comments and valuable discussions we thank Drs. L. Fagni, J.-P. Pin., L. Prézeau, L. Journot, L. Vitkovic and V. Homburger. Mrs. A.L. Turner-Madeuf and M. Passama are acknowledged for their help in language revision and preparation of figures respectively.

This work was supported by grants from the Fondation pour la Recherche Médicale and Roche Laboratories.


Assoc. Prof Sanda-Carmen Georgescu

Édouard Canot

Jean-Luc Achard

OIL IN WATER INJECTION FROM A SUBMERGED NOZZLE

Keywords: Vortex ring, interfacial tension, boundary element method NOMENCLATURE

When a certain amount of oil is injected upwardly through a vertical nozzle submerged in water, the oil-water interface evolves, for large orifices, from a sessile shape, to a cap profile with a roll-up border.

The numerical simulation of such liquid-liquid interface evolution was made through the Boundary Element Method (BEM), by assuming a potential flow of a viscous fluid. At the end of the computed process, the velocity field behaviour leads to an inward rolling up movement of the cap border, simultaneously with the interface necking near the orifice level. Such velocity field precedes typically a vortex ring formation.

An olive oilwater couple has been selected, because of the appropriate physical properties of those liquids: comparable densities, huge difference of viscosities, and very small interfacial tension.

INTRODUCTION

A vortex ring is produced when the linear momentum is imparted to the fluid with axial symmetry [1]. A familiar example of flow with circular vortex lines is the tobacco smoke ring, formed by a particular puffing technique. The upward injection of a small quantity of coloured liquid into a liquid pool [1, plate 20] gives rise to a vortex ring that travels away from the orifice: within the resulted torus, a roll-up movement is developed in cross-section. Similar phenomenon can be obtained by pushing downward some amount of liquid throughout a submerged nozzle: for such case, the velocity field measured by Particle Image Velocimetry near the viscous vortex ring core has been compared by Dazin et al. [2] to theoretical models derived for thin core vortex rings in an inviscid fluid. The velocity field induced by an inviscid vortex ring has been analysed in [3].

It is possible to produce a vortex ring, or toroidal bubble, by injecting a volume of air from a submerged nozzle [1]. The toroidal bubble rebound in collapsing bubble with jet formation and impact [4], as well as the toroidal bubble entrapped within the liquid during a bursting bubble collapse at a free surface [5], or during a plunge of a vertical liquid jet on the free surface of a pool [6], are also associated to vortex rings.

Drops falling into a liquid pool produce vortex rings that penetrate more deeply into the pool [1,7]. The jet impinging on a cylindrical surface [8], or on a convex surface [9] equally leads to vortex rings formation.

Some complex phenomena also involve vortex ring formation: it is the case of the fuel injection process that can be accompanied by the development of a fountain structure consisting of a spray jet and a vortex ring [10], or the case of unsteady shear flow induced by vortex ring / wall interactions [11].

When a certain amount of oil is injected upwardly through a large nozzle orifice submerged in water, the oil-water interface evolves from a sessile shape, to a cap profile with a roll-up border (Figure 1). At the end of the process, the velocity field behaviour leads to an inward rolling up movement of the cap border, simultaneously with the interface necking near the orifice level. It is expected that after the complete necking and the detachment of the oil drop from the nozzle, the drop interface will break up from a cap shape into a toroidal shape, the torus being formed from the previous cap border roll-up. Such toroidal oil drop corresponds to a vortex ring structure, and can be added to the previous examples.

Within the present paper, the numerical simulation of the interface evolution for oil in water injection was made through the Boundary Element Method (BEM), by assuming a potential flow of a viscous fluid [12]. Our computations are not performed till the toroidal oil drop formation, because of the complex phenomenon related to the interface rupture and strong changes of the geometrical configuration. But it is highlighted that when injecting oil in water from a submerged nozzle, for large orifice sizes, the liquid-liquid interface rolls up and creates the velocity field that can lead to a vortex ring formation. It is showed that the phenomenon is strongly dependent on the capillary forces, on the viscous forces (introduced here only through the normal stress at the interface), and on the gravity, the last one being important especially when increasing the nozzle orifice radius. Within the present study, an olive oil water couple has been selected, because of the appropriate physical properties of those liquids, namely comparable densities, huge difference of viscosities, and very small interfacial tension.

PROBLEM STATEMENT

The free boundary problem is axisymmetric. In a meridian plane rOz, the oil domain  (Figure 1) is bounded by three surfaces: the oil surface o  in cross-section inside the cylindrical nozzle, the vertical solid wall s  of the nozzle, and the oil- water interface  . The unit normal n on the boundary points inwards the oil domain. The azimuthal angle  is defined between the radial unit vector and the tangent unit vector t.

Figure 1. Geometric axisymmetric configuration

The curvilinear abscissa s starts from the Oz axis inside the nozzle, follows radially the surface o  , then follows upwards the solid surface s  , then the interface  , and reaches its maximum value on the Oz axis, at the apex of the oil-water interface.

The oil flow at the exit of the nozzle is assumed to be potential, with a velocity field

   v
. The Laplace equation for the velocity potential  is:

0 2    . ( 1
)
The velocity is defined by its normal component

n v n    
, and tangential component s   . The Euler's equation for the oil motion is written:

    z g z p v t o o 2            1 2  , (2)
where o p is the oil pressure, and o  the oil density.

The difference between the values of the stress tensor on either side if the oil-water interface is a normal force due wholly to the surface tension [1]. The component of the surface force normal to the interface, namely the normal momentum balance at any point of the interface  is written:

                  2 1 1 1 2 R R z p n v z p w n o o   , ( 3
)
where o  is the oil dynamic viscosity,  is the oil- water interfacial tension, 1 R and 2 R are the local principal radii of curvature. The water pressure can be defined as:

    z g p z p w w w 0   
, where

  0 w p is the hydrostatic pressure at the orifice level, at 0  z . Within the balance (3), only the normal viscous stress   n v n o   2
on the oil side of the interface has been considered. The normal viscous stress on the water side of the interface has been neglected, due to the water viscosity, which is negligible with respect to the oil viscosity. The oil pressure can be expressed from (3) as:

    . 2 1 1 0 2 2 2 1 n z g R R p z p o w w o                     (4)
Combining Euler's equation ( 2) and the normal momentum balance (4) in order to reduce the oil pressure term, we obtain the Bernoulli's equation at any point of the interface  : . The Froude number is expressed for the studied problem as:

  . 2 1 1 2 2 2 2 1 2 n z g R R v t o w o                                    (
  2 gR Fr o w      , ( 6
)
while the Reynolds number is:

o o R Re     . ( 7
)
Dimensionless variables will be denoted by an asterisk.

The dimensionless form of the Bernoulli's equation ( 5

), , 2 1 1 1 1 2 2 2 2 1 2                            n Re z Fr R R We v t   (8)
gives the local time derivative of the velocity potential,

    t  . The singularities issued in (8) at 0   r
, are removed by taking into account that on the Oz axis, the axisymmetric curvature

     r R  sin 1 equals the planar curvature      s R  1 1
. The expressions of the dimensionless normal gradient of normal velocity

2 2     n
 on the Oz axis, and outside of it, are written [13,14]:

, 0 at , 2 2 2 2 2 2                     r n s s n     (9) and . 0 at , sin cos 2 2 2 2                                      r n r s s r s n        (10)
The global mechanical energy balance can be expressed only in surface integrals terms [14]:

                         A We A n t d 1 d 2 1 d d                A z Fr t d 2 1 d d 2                 A n n Re

NUMERICAL METHOD

The distorted oil-water interface evolution is an axisymmetric transient free-boundary problem that is modelled here through a Boundary Element Method (BEM). An irrotational flow model is appropriate because of the impulsive character of the process. Within the model, viscous effects are partially included through the normal viscous stress at the interface (expressed in terms of the velocity potential), as it is allowed for potential flows of fluids with constant viscosity [12].

The BEM numerical code (built in Fortran), has been successfully used in the highly distorted gas-liquid interface evolution, within a potential flow of a viscous fluid assumption, both for the collapsing bursting bubbles at a free liquid surface, terminated by microjet breaking and droplets ejection [13][14][15][16], and for drops formation and ejection from a vertically capillary nozzle, by piezoelectric stimulation [17,18]

is assumed to be ) 0 ( ) 0 (         t v z t n 
. For any further moment, the velocity potential

) 0 (    t 
on  is obtained within the BEM.

 The time progression, made with an explicit numerical scheme of 4th order Runge-Kutta type . The temporal interface evolution is determined through a Lagrangian description of a variable number of nodes, unevenly redistributed on the boundary

) (  t  
at each time step, with respect to the adaptation at surface gradients [16].

In the absence of non-linear analytical tests, the evaluation of numerical code accuracy is checked through the global mechanical energy balance (11). Upon azimuthal integration, the mechanical energy balance deals with line integrals of the terms computed through the BEM, thus being easily implemented in the computational procedure.

NUMERICAL RESULTS

Within this paper, the BEM simulations point on the liquid-liquid interface evolution for the case of upwards oil injection through a vertical nozzle submerged in water.

An olive oilwater couple has been selected. . Due to the choice of scales, the Weber number takes always the unit value. The viscous and capillary effects are dominant for very small orifices (say 1  R mm), and the gravity becomes less important. So, the oil-water interface is less distorted, and continues to grow up till the end of the oil injection (for

  o t t
 ), then it detaches after a complete necking of the interface near the orifice. Within this paper, the oil-interface evolution for small orifice sizes is not presented, because it does not correspond to a vortex ring formation.

For large orifice sizes, e.g. for 5  R mm, the oil-water interface evolves from a sessile shape, to a cap profile with a roll-up border: at the end of the computed process, the velocity field behaviour leads to an inward rolling up movement of the cap border, simultaneously with the interface necking near the orifice level. Such velocity field precedes typically a vortex ring formation. Further, numerical results are presented for large orifice radius cases.

When increasing the orifice radius, the Froude number decreases drastically, so effect of the gravity becomes more and more important. Over the whole orifice radius range, the Reynolds number keeps very small values, so the viscous effects always remain important.

The oil-water interface evolution is discussed firstly for an orifice radius , is presented in Figure 3. The variation of the velocity of the interface apex (the node placed on the Oz axis) versus time is presented in Figure 4. The moment

3     o t t
 is marked on the diagram: before that moment, the apex velocity decreases monotonously; when the oil injection is stopped, there is a drop in apex velocity; then, it continues to oscillate due to inertial effects.

In Figure 5 we present the velocity vectors (with their size and direction) in each node of the interface, at the same time moments as in Figure 3.  , the roll-up is amplified by the interface necking that starts near the orifice level. This velocity field is supposed to lead to the oil drop detachment and vortex ring formation (the present computations are preformed till the complete necking and interface rupture). ), the velocity field behaviour near the orifice corresponds to a slight rolling up movement of the cap border, with respect to the previous case (see last frame of Figure 5, for 5 , 7  R mm). In Figures 8 and9, we present superposed frames of the interface evolution, and the velocity vectors in each node of the interface, for the case of an orifice with a radius ), the velocity field behaviour near the orifice corresponds to a stronger rolling up movement of the cap border, with respect to both previous cases (see the last frame of Figure 5, for  is marked on the diagram: the apex velocity decreases before that moment, then, when the oil injection is stopped, there is a drop in apex velocity. After that velocity dropping, the apex velocity continues to decrease, without oscillating like for smaller orifice radius cases. Due to the fact that the gravity force becomes dominant over the capillary and viscous forces for such great orifice radius, after stopping the oil injection, the necking of the interface will continue and the cap border is supposed to continue to roll up simultaneously.

CONCLUSIONS

The interface evolution during the oil injection in water has been modelled through BEM, in a viscous potential flow assumption. For large nozzle orifices, the oil-water interface evolves from a sessile shape, to a cap profile with a roll-up border. It is supposed that the process ends by the complete necking of the interface near the orifice, and a vortex ring formation.

First of all, it must be highlighted that we do not dispose of any experimental or numerical evidence on the oil injection in water from a submerged orifice, not even for other liquid-liquid couples characterised by an interfacial tension. Within the existing experimental and theoretical evidence related to a liquid injection into another liquid, there is not a well delimited interface between the two liquids: usually, the injected liquid is the same (coloured or not) as the surrounding-one.

We found that at the end of the computed process, the velocity field behaviour leads to an inward rolling up movement of the interface cap border, simultaneously with the interface necking near the orifice level. Such velocity field precedes typically a vortex ring formation. It is obviously

  5)So, within this type of potential model, viscous effects can only be partially considered through the boundary condition[12], namely the normal momentum balance (4) at any point of the fluidfluid interface  .We adopt the nozzle orifice radius R as length scale, and the ratio between length and velocity as time scale. According to the choice of scales, the Weber number takes always the unit value 1  We

  axisymmetric surface element. It is highlighted that the right hand term of (11), namely the dissipation of the mechanical energy due to shear viscosity, depends only on the normal component of the velocity, and on the normal second derivative of the velocity potential.



  corresponding normal component, and tangential component of the velocity. Kinetic conditions of Neumann type are defined on the solid surface s At the initial moment, the interface is considered slightly upward oriented (as a non-flat meniscus), and the velocity potential

  [18], allows the connection of two successive steps, to determine the new potential values and interface position at the following instant ) (

  behaviour leads to an inward rolling up movement of the cap border.

  For

Figure

  Figure 5. Velocity vectors in each node of the oilwater interface at different moments for 5 , 7  R mm

Figure 9 .

 9 Figure 9. Velocity vectors in each node of the oilwater interface at different moments for 10  R mm