WO1994016067A1

WO1994016067A1 - Serotoninergic receptors (5ht6 and 5ht7) and use thereof

Info

Publication number: WO1994016067A1
Application number: PCT/FR1993/001282
Authority: WO
Inventors: Martial Auguste Simon Ruat; Jorge-Luis Diaz; Regorius Leurs; Joël TARDIVEL-LACOMBE; Elisabeth Marie Traiffort; Jean-Charles Schwartz; Jean-Michel Arrang
Original assignee: Institut National De La Sante Et De La Recherche Medicale
Priority date: 1992-12-30
Filing date: 1993-12-21
Publication date: 1994-07-21
Also published as: FR2699922B1; FR2699922A1

Abstract

Polypeptides having a serotoninergic receptor activity, medicaments based on said polypeptides, nucleotidic fragments coding for them, probes for detecting said fragments, vectors and hosts expressing said polypeptides, and application to the screening of medicaments. New polypeptides having an activity of new serotoninergic receptors 5HT6 or 5HT7 and aminoacid fragments coding for said polypeptides. Cellular hosts transformed by vectors in which these fragments have been inserted express these polypeptides. Applications to said polypeptides and fragments for the production of antibodies, the production of medicaments, nucleotidic probes for diagnosis and detection, screening of medicaments intended particularly for treatment of neurologic or gastro-enterologic disorders and for the identification of tissues or cells.

Description

SEROTONINERGIC RECEPTORS (5HT6 AND 5HT7) AND USES THEREOF

The present invention relates to polypeptides having serotonergic receptor activity.

It also relates to nucleotide sequences, and in particular DNA, coding for these polypeptides as well as to vectors containing said sequences and to cells transformed to express said sequences and in particular the genes coding for receptors containing, or constituted by, said polypeptides, these cells making it possible to study the activity of agonist or antagonist of said receptors.

The invention also relates to nucleotide probes capable of binding by hybridization to said sequences or said genes coding for said polypeptides and intended for the diagnosis of conditions resulting in a qualitative or quantitative modification of the formation or of the activity of said receptors, in humans or animals, and in particular of affections neurological, psychiatric, qastroenterological or neuroendocrine.

The invention also relates to monoclonal or polyclonal antibodies, human or not, directed against said polypeptides.

It also relates to means for screening for drug candidates, making it possible in particular to study the affinity of candidate substances for said polypeptides.

Finally, it relates to medicaments, in particular for the treatment of psychiatric, neurological, qastroenterological or neuroendocrine affections, and containing substances having a high affinity for said polypeptides with serotonergic receptor activity or substances having activity on cells. transfected by said DNA sequences coding for said polypeptides.

It also relates to drugs active on one, some, or all of the serotonergic receptors already identified and / or those described in the present invention and in particular to drugs agonists or antagonists of serotonin.

It was previously accepted that the various effects of serotonin resulted from its interaction with different classes of receptors commonly known as 5HT1a, 5HT1b, 5HT1c, 5HT1d, 5HT1e, 5HT2, 5HT3 and 5HT4 receptors. Among them, the receptor sequences 5HT1a, 5HT1b, 5HT1c, 5HT1d, 5HT1e, 5HT2 and 5HT3 are known (Zifa and Fillion, Pharmacol. Reviews, 44, 401-457, 1992; McAllister et al., Proc. Natl. Acad. Sci., 89, 5517-5521, 1992). With the exception of the 5HT3 receptor, these receptors belong to the family of receptors comprising seven transmembrane domains coupled to a G protein and having a certain degree of homology with receptors of the rhodopsin family which are expressed by photoreceptors of the retina.

These receptors have a particular pharmacological profile and are coupled to different intracellular messengers. The 5HT1a, 5HT1b, 5HT1d, 5HT1 receptors are negatively coupled to adenylate cyclase while the 5HT1c and 5HT2 receptors are positively coupled to phospholipase C leading to the hydrolysis of phosphatidyl inositols. The 5HT4 receptor, the protein sequence of which is not yet known, would be positively coupled to adenylate cyclase.

The existence of serotonergic receptors distinct from those mentioned above, positively coupled to adenylate cyclase and distinct from the 5HT4 receptor, had been suspected by different authors on indirect observations relating to complex biological preparations, but they had never have been isolated, neither identified as to their structure, nor entirely defined by their pharmacological properties.

The invention of the above new polypeptides has made it possible to discover new serotonergic receptors which will be designated, below, by 5HT6 and 5HT7.

The subject of the invention is new polypeptides having a serotonergic receptor activity (5HT6 or 5HT7) chosen from the group of polypeptides formed by the sets 1 and 2 defined below:

- Set 1 of the polypeptides constituted by or containing the sequence of 448 amino acids of rat, relating to the receptor 5HT6, defined in the sequence identifier SEQ ID No. 1 of the annexed sequence list or peptide fragments of this sequence, said fragments being such that

- either they contain the site or sites contained in said sequence of 448 amino acids and the presence of which is necessary for the fragment to be capable of binding the serotonin, its agonists or its antagonists,

either they are recognized by antibodies which also recognize said sequence of 448 amino acids but which do not recognize any of the other serotonergic receptors described above, including the 5HT7 receptor,

- Either they are capable of inducing antibodies recognizing said sequence of 448 amino acids but not recognizing any of the serotoninergic receptors described above, including the 5HT7 receptor;

or analoque polypeptides or fragments corresponding to other species, including the human species, and relating to the serotonergic receptor homologous to the 5HT6 receptor.

- Set 2 of the polypeptides constituted by or containing the sequence of 127 rat amino acids, relating to the 5HT7 receptor, defined in the sequence identifier SEQ ID No 2 of the annexed sequence list or peptide fragments of these polypeptides, said fragments being such that

- Either they contain the site (s) contained in said 127 amino acid sequence or any neighboring site on a polypeptide encompassing said 127 amino acid sequence and encoded by a gene expressing the 5HT7 receptor, the presence of which is necessary for the fragment is capable of binding the serotonin to its agonists or antagonists,

either they are recognized by antibodies which also recognize said sequence of 127 amino acids and / or a polypeptide encoded by a gene expressing the 5HT7 receptor but which do not recognize any of the other serotonergic receptors described above, including the 5HT6 receptor,

- Either they are capable of inducing antibodies recognizing said sequence of 127 amino acids and / or a polypeptide encoded by a gene expressing the 5HT7 receptor but not recognizing any of the serotonergergic receptors described above, including the 5HT6 receptor;

or analoque polypeptides or fragments corresponding to other species, including the human species, and relating to the serotonergic receptor homologous to the 5HT7 receptor.

In the following description, 5HT6 and 5HT7 define not only the rat receptors but the homologous serotonergic receptors, human or non-human.

Preferably, the polypeptides of set 1 contain the sites forming part of said sequence of 448 amino acids, the presence of which is necessary, when the polypeptides are presented on a cell membrane in the presence of tritiated serotonin and in the presence of an agonist or a serotonin antagonist, so that the apparent affinity of said antagonists or agonists corresponds to that shown in Table 1 below.

We can see in particular that the affinity of serotonin for the 5HT6 receptor polypeptides the invention is superior to that of LSD.

It can also be seen in table 1 that the affinity of the receptor polypeptide expressed in the cells is greater for spiperone than for mianserin.

The polypeptides according to the invention can be used by being isolated, or fixed on supports, in a completely conventional manner. or also joined to amino acid sequences unrelated to the 5HT6 or 5HT7 receptors. They can be glycosylated or not and include disulfide bridges.

The present invention also relates to compositions having a significant content of such polypeptides, for example contents obtained by expression in a recombinant host, said polypeptides possibly being present in the form of cell membranes enriched in polypeptides.

The present invention also relates to the nucleic acids containing or consisting of the nucleotide sequences coding for any of the polypeptides according to the invention.

The nucleic acids according to the invention may comprise or consist of the complete sequence, that is to say the gene, coding for the 5HT6 or 5HT7 receptors in humans or in animals. Nucleic acids according to the invention are in particular nucleic acids comprising or consisting of the sequences SEQ ID No. 1 or No. 2 or fragments of these sequences placed in the reading frame, as well as the homologous sequences, in particular in humans . Among the nucleic acids according to the invention, a particularly interesting nucleic acid is that consisting by the sequence extending from the nucleotide at position 135 to that at position 1 478 in the identifier SEQ ID nº 1 or by its counterparts in humans or in animals. The nucleic acids also include the fragments coding for the shorter polypeptides defined above.

In addition to the variations in nucleotide sequences that can be observed when passing from the DNA sequences of rats represented on identifiers 1 and 2 to homologous sequences in humans or other animal species, the acids nucleic acids according to the invention may include the variations specific to the degeneration of the code as well as localized mutations insofar as the variant nucleic acids hybridize with the probes capable of hybridizing, in a specific manner, with the sequences ID No. 1 or ID # 2.

The nucleic acids according to the invention can be obtained by conventional chemical synthesis of nucleic acid, by replication in vectors which contain them or by any other means. and in particular multiplication by the so-called PCR method from a small amount of nucleic acid according to the invention.

The invention also relates to recombinant vectors, which can be used for cloning or for the expression of nucleic acids, and containing a nucleic acid according to the invention, such as plasmids. phages or viruses containing one or more sequences according to the invention at a site which is not essential for replication. The vectors according to the invention may contain. if necessary, the elements necessary for expression in a cellular host such as promoter, initiation codon. finished. signal sequence of the anchor sequence or any other useful control element.

A subject of the invention is also the cellular hosts transformed by a recombinant vector according to the invention and capable of expressing the coding sequence according to the invention for the synthesis of a polypeptide according to the invention. These hosts may be prokaryotes, such as for example E. coli, or eukaryotes, for example yeast cells such as S. cerevisiae or mammalian cells, for example CHO cells.

The subject of the invention is also the monoclonal or polyclonal antibodies directed against a polypeptide according to the invention. These monoclonal or polyclonal antibodies are obtained by induction from a polypeptide according to the invention and are specific for polypeptides according to the invention, while being capable, or not, of recognizing other serotonin receptors.

Obtaining these antibodies can be carried out in a conventional manner, either by injection of a polypeptide according to the invention into an organism and collection of the serum with optionally purification of the specific antibodies, or for the monoclonal antibodies, by the conventional techniques of cell fusion forming hybridomas.

The antibodies according to the invention are useful for diagnosis, by antibody-antigen reaction demonstrated using a standard revelation technique, for diagnosing pathological proliferation of cells expressing the 5HT6 or 5HT7 receptor.

The subject of the invention is also the nucleotide probes hybridizing with one of the nucleic acids according to the invention or with their complementary sequences or Corresponding RNAs, being specific for the 5HT6 receptor or for the 5HT7 receptor, and which do not hybridize with the nucleic sequences or the corresponding RNAs of the other serotonin receptors described above.

In a manner known per se, it is preferred that the probes comprise at least 10, and preferably at least 15 to 17 nucleic acids.

The probes according to the invention are useful for the diagnosis of neurological, psychiatric, gastroenterological or neuroendocrine disorders linked to qualitative or quantitative abnormalities of the 5HT6 or 5HT7 receptors, including for the search for genetic anomalies at this level. .

The probes are used in a conventional manner by being brought into contact, under the usual conditions, with preparations presenting nucleic acids of cells suspected of exhibiting an abnormality defined above.

In particular, the probes can be used to detect an expression if one or the other of the 5HT6 or 5HT7 receptors by revealing the presence of its messenger RNA.

Advantageously for expression in eukaryotic cells, one can use the endogenous promoter of serotonergic receptors or usual viral promoters, for example SV40 virus or Rous sarcoma virus.

For expression in bacteria, in particular E. Coli, the gene can be inserted under the control of a usual promoter, for example promoter of the lactose operon or of the tryptophan operon.

The invention also relates to a method of screening of drugs intended for the treatment of neurological conditions, in particular Parkinson's disease, Alzheimer's disease, sleep disorders, migraine, and psychiatric such as mood disorders, depressions, psychotic states, or neuroendocrine, and in particular dysfunctions of the hypothalamic-pituitary axis, or qastroenterological, in particular functional disorders of the intestine, motor or secretory disorders.

The screening of a medicament can in particular use the detection of the capacity of a serotonergergic molecule to behave as a ligand with respect to a polypeptide according to the invention, and consists, for example, in bringing into contact, in a suitable medium, the molecule serotonergergic with the membranes of a cellular host transformed by a vector comprising an insert coding poulle polypeptide and capable of expressing said polypeptide so as to present on the membrane surface one or more specific sites of this polypeptide, in such a way that the specific binding of the candidate ligand can be evaluated or indirectly estimated, for example by detecting the possible ligand-polypeptide complex. This method can also include the measurement of a response induced by the binding with the cell, such as for example the production of cyclic AMP so as to make it possible to differentiate the serotonergic molecules which behave as antagonists and those which behave as aqonists , and even to define the relative character of the latter.

For this screening purpose, a method can also be used to determine the affinity of a polypeptide. of the invention for specific ligands, in which a cell host transformed in accordance with the invention is cultivated to express and present in its membrane the 5HT6 or 5HT7 receptor or a corresponding polypeptide, after which the cell culture is brought into contact with the determined ligand and it is checked whether a specific bond is formed between the cellular hosts and the ligands, in particular by radiolocation tests and preferably, measuring the rate of production or release of a suitable indicator such as AMP cyclic, or intracellular ions, these latter tests make it possible to distinguish and define the agonists and their partial character as well as the antagonists.

This process also makes it possible to determine the polypeptide fragments according to the invention, for example those comprising a part of the sequence of 448 amino acids SEQ ID No 1, and which include the operational sites allowing the binding and / or the induction of cellular responses. mentioned above.

It is also possible to identify cells or tissues expressing spontaneously at their membrane surface a polypeptide according to the invention, these cells or tissues then being usable for serving in the development or screening of drugs or ligands interacting with the polypeptides according to l 'invention.

The invention also relates to medicaments containing, in a dose intended to be administered, a therapeutically effective amount of an active substance on a polypeptide according to the invention having a 5HT6 or 5HT7 receptor activity in a pharmaceutically acceptable vehicle. The invention also relates to medicaments, containing, in a pharmaceutically acceptable vehicle, an effective amount of a substance active on the other serotonergic receptors but not active on at least one of the 5HT6 and 5HT7 receptors.

Other advantages and characteristics of the invention will appear on reading the following description, given by way of nonlimiting example.

In this description, the sequence identifier SEQ ID No. 1 represents the sequence of a DNA fragment of 1661 bases, of which 448 codons, identified on the sequence, code for a polypeptide of 448 amino acids constituting a receptor of the serotonin designated by 5HT6.

The sequence No. 2 represents a DNA fragment, of which 127 codons code for a polypeptide of 127 amino acids constituting a fraoment of the 5HT7 receptor for serotonin.

The above sequences correspond to nucleic acids in rats.

The corresponding nucleic sequences in other mammals and in humans can be obtained by hybridization from a library or from a cDNA library with a probe consisting of all or part of the sequences represented, then sequencing of a quite classic way of the fragments thus recognized.

Cloning of the serotonin 5HT6 and 5HT7 receptors

1. Cloning of the 5HT7 receptor

A rat brain cDNA library was screened under low streak conditions with a DNA probe labeled with ³² P and corresponding to the nucleotide sequence coding for the rat substance P receptor. In these conditions, a clone (λSP45) hybridizes strongly with this probe. The nucleotide sequence of the DNA fragment (1124 bp) thus isolated shows that this cDNA comprises a sequence coding for a peptide of 127 amino acids coding for the terminal MB6, MB7 and NH _{2 regions} of a protein and having homologies with the family of rhodopsin homologous receptors.

2. Cloning of the 5HT6 receptor

A DNA fragment of this clone (nucieotide 8 to nucieotide 539) is then labeled with ³² P, then used to screen a cDNA library of rat hypotha lamus (Strat aqene) under stri ngence conditions. l evée. Twelve clones are then isolated and analyzed by restriction analysis by Southern techniques and the clones having the longest inserts are selected. One of them, called λHPT3, comprises the nucleotide sequence 404 to 1 661 of the sequence of FIG. 1. A DNA probe labeled with ³² P, located between nucleotides 404 and 607 is used to screen a rat genomic band , partially cut with the HaelII enzyme. A clone strongly hybridized with the probe and a positive fragment, EcoRI-Hind III, of 6.5 kb is subcloned into plasmid (PGEM 4Z). A nucleotide sequence going from nucieotide 1 to nucieotide 682 of FIG. 1 is thus isolated and sequenced. Nucleotide primers (primer 1: 5'-GGAGATCTCCATTGTGCAGTCTCAGCCAATGA-3 ', located downstream of the stop codon TGA of FIG. 1, and primer 2: 5'-AAA AGC TTG GCG GGG TCG CCG GCT CCAT-3', located upstream of the first methionine) are then synthesized (PCR-Mate .391A DNA synthesizer, Applied Biosystems). They are then used in the RT-PCR technique as follows. We synthesize a single strand cDNA using AMV reverse transcriptase (20U, Boehringer) and poly (A) ⁺ mRNA (2 pci) from rat hypothalamus and primer 1 described above. This matrix is then amplified (Kawazaki ES et al. 89-97, "PCR Technclogy"; ed. Erlich HA Stockholm Press, 1989) using primer 2 (75 mM each) for 35 cycles as follows (94.5 ° C 1 minute 30; 56 ° C, 1 minute 30 and 72ºC, 3 minutes) with 5U of DNA polymerase Tag (Perkin Eliner Cetus).

A DNA band of expected size 1.5 kb is extracted after electrophoresis on agarose qel and digested with HindIII and Bgl II, then subcloned in a plasmid pGEM 4Z. The sequencing of several plasmids shows that the sequence obtained is identical to the sequence delimited by primers 1 and 2 of the indicator SEQ ID No. 1.

3. Expression of the 5HT6 receptor in cells

CHO.

METHOD

The expression vectors are derived from the plasmid pSV D2 (Giros, B. et al., Nature 342. 923-926, 1989). The vector for expressing the 5HT6 receptor is obtained by replacing the HindIII-BglII restriction fragment of pSVD2 with a HindIII-BglII restriction fragment of the RT-PCR clone obtained above, leading to the plasmid PSV 5HT6. These constructs are cultivated, then purified on a cesium chloride gradient (Sambrook J. et al., Molecular cloning - a laboratory manual. C. Nolan, ed., Cold Spring Harbor Laboratory Press, 1989) and transfected (Graham, FL and al., Virol. 52, 456-467, 1973) to Chinese hamster ovary (CHO-K1) cells deficient in dihydrofolate reductase. The stable transfectants are selected in a culture medium (Dubelcco's Modified Eagle medium) without hypoxanthine and thymidine free.

The expression of the 5HT6 receptor is demonstrated by the binding of serotonin- ³ H to membranes of transfected cells; according to the technique of Peroutka and Snyder (Peroutka SJ and Snyder SL; Mol. Pharmacol., 16, 687-699, 1979). The binding experiments are ef fec killed in a 50 mM Tris-HCl buffer pH = 7.6 (total volume 500 μl) containing CaCl ₂ 4 mM, 0.1% ascorbic acid, pargyline

10 ^-5 M, and serotonin- ³ H has a concentration of 1 to 2 nM.

RESULTS

5HT6 receptors have been expressed in CHO cells which normally do not have binding sites for ³ H serotonin.

In transfected clones of CHO cells, permanently expressing these sites, the K _D values of serotonin are 1 nM for the 5HT6 receptor and the Bmax values (Bmax representing the maximum concentration of 5HT6 receptor) are approximately 1 pmol / mg of membrane protein.

Table 1 clearly indicates that the gene transfected into CHO cells codes for a serotonergic receptor as indicated by the high affinity of serotonin and its derivatives such as 5-methoxytryptamine. However, the pharmacology of this 5HT6 receptor can in no case be confused with that of a 5HT 1A receptor (8-OH-DPAT has an affinity less than the nanomolar for this receptor), 5HT2 (spiperone and ketanserin have affinities of the order of nM) or 5HT4 (ICS 205 930 has an affinity of 0.5 nM on the 5HT3 receptor and 300 nM on the 5HT4 receptor) or another class of receptors which have been cloned to date.

Characterization of 5HT6 receptor gene transcripts in several tissues

Poly (A) ⁺ RNAs are prepared from several brain or peripheral tissues of rats or guinea pigs (Chirgwin et al. Biochemistry 18. 5294-5299, 1979; Aviv et al., Proc. Nat l. Acad. Sci ., 69, 1408-1412, 1972). Samples (8 μg) are denatured, subjected to agarose electrophoresis, transferred to nitiocellulose membranes and hybrids (Traiffort et al. Proc. Natl. Acad. Sci., 89, 2619-2 653, 1992) using a fragment of

1,106 bp obtained by DNA amplification using oligonucleotide primers, then labeled with ³² P by the "nick-translation" technique. This dotted fragment corresponds to the transmembrane domains 1 to 7 and is used at the rate of 14 × 10 ⁶ dpm / ml. The membranes are exposed for 7 days at - 80ºC with amplifier screens. In the rat, two transcripts (3.9 and 3.2 kb) can be observed in the brain tissues studied. The hypothalamus, trunk and hippocampus are the regions which seem to express the lowest densities of transcripts of the 5HT6 receptor. In the guinea pig, two transcripts (3.8 and 3.0 kb) are also observable in most of the brain regions studied. The thalamus and the trunk seem to be the regions which express the highest densities of transcripts of the 5HT6 receptor.

Responses induced by seiotonino in CHO cells transfected by the 5HT6 receptor

Accumulation of cAMP: according to the technique described in Traiffort E. et al., Proc. Natl. Acad. Sci., 89. 2644-2653, 1992), cells grown on plagues of 96 wells are washed, then incubated in the presence of increasing concentrations of serotonin for 10 minutes at 37ºC. CAMP is extracted, then analyzed by radioimmunoassay (New England Nuclear). Serotonin (300 nM) induces significant stimulation of cAMP (1.22 ± 0.06 pmol / well) compared to the basal level in the absence of serotonin (0.07 ± 0.03 pmol / well). This measurement makes it possible to determine the agonist character of a molecule.

TABLE 1 pharmacology of the 5 HT6 receptor expressed in CHO cells

ANNEX 1

LIST OF SEQUENCES

SEQ ID # 1

Sequence length: 1,661 base pairs

Type of sequenced molecule: single-strand cDNA

5HT6 receptor nucleotide sequence.

116 gcagcggcgctcggcacg ATG ATG GAC GTT AAC AGC AGC GGC CGC CCC GAC CTC TAC GGC CAT CTC CGT TCA CTC ATC CTG CCG GAG GTG 206

M M D V N S S G R P D L Y G H L R S L I L P E V 24

GGG CGC GGG CTG CAG GAC CTG AGC CCC GAC GGT GGC GCC CAC CCT GTG GTG AGC TCC TGG ATG CCG CAC CTG CTG AGT GGC TTC CTA 293

G R G L Q D L S P D G G A H P V V S S W M P H L L S G F L 53

GAG GTG ACG GCT AGC CCG GCG CCC ACC TGG GAC GCG CCA CCG GAC AAT GTC TCA GGC TGC GGG GAG CAG ATC AAC TAT GGC AGA GTG 380

E V T A S P A P T W D A P P D N V S G C G E Q I N Y G R V 82

GAG AAA GTT GTG ATC GGC TCC ATC CTG ACG CTC ATC ACG CTG CTG ACG ATC GCA GGC AAC TGC CTG GTG GTG ATC TCG GTG TGC TTC 467

E K V V I G S I L T L I T L L T I A G N C L V V I S V C F 111

GTC AAG AAG CTC CGC CAG CCC TCC AAC TAC CTG ATT GTG TCC CTG GCG CTG GCT GAC CTC TCG GTG GCC GTG GCG GTC ATG CCT TTC 554

V K K L R Q P S N Y L I V S L A L A D L S V A V A V M P F 140

GTT AGT GTC ACC GAC CTC ATC GGG GGC AAG TGG ATC TTC GGC CAC TTC TTC TGC AAC GTC TTC ATC GCC ATG GAC GTC ATG TGC TGC 641

V S V T D L I G G K W I F G H F F C N V F I A M D V H C C 169

ACG GCC TCG ATC ATG ACC CTG TGC GTG ATC AGC ATC GAC AGG TAC CTT GGG ATC ACG AGA CCC CTC ACG TAC CCG GTG AGG CAA AAT 728

T A S I M T L C V I S I D R Y L G I T R P L T Y P V R Q N 198

GGG AAA TGT ATG GCC AAA ATG ATT CTG TCG GTC TGG CTG CTC TCT GCC TCC ATC ACC TTA CCT CCT CTC TTC GGA TGG GCT CAG AAT 815

G K C M A K M I L S V W L L S A S I T L P P L F G W A Q N 227

GTG AAC GAT GAC AAA GTG TGC TTG ATC AGC CAG GAT TTT GGC TAC ACG ATC TAC TCC ACT GCG GTG GCG TTT TAT ATC CCC ATG TCG 902

VNDDKVCLIS ^Q DFGYTIYSTAVAFYIPMS 256

GTC ATG CTG TTC ATG TAC TAT CAG ATT TAC AAG GCC GCC AGG AAG AGT GCA GCC AAA CAC AAG TTC CCA GGC TTC CCA CGC GTG CAG 989

V M L F M Y Y Q I Y K A A R K S A A K H K F P G F P R V Q 285

CCG GAG AGT GTC ATC TCC CTG AAT GGT GTG GTG AAG CTC CAG AAG GAG GTG GAA GAG TGT GCG AAC CTT TCG AGA CTG CTC AAA CAC 1076

P E S V I S L N G V V K L Q K E V E E C A N L S R L L K H 314

GAA AGG AAA AAC ATC TCC ATC TTC AAG CGG GAA CAG AAA GCA GCC ACT ACC TTC GGG ATC ATC GTG GGA GCC TTC ACT GTG TGC TGG 1163 E R K N I S I F K R E Q K A A T T L G I 1 V G A F T V C W 343

CTG CCG TTT TTC CTC TTG TCC ACA GCC CGC CCC TTT ATC TGT GGC ACC TCC TGT AGC TGC ATT CCT CTG TGG GTG GAG AGG ACA TGT 1250

L P F F L L S T A R P F 1 C G T S C S C I P L W V E R T C 372

CTG TGG CTG GGC TAT GCA AAC TCT CTC ATT AAT CCT TTT ATA TAT GCC TTC AAC CGG GAC CTG AGG ACC ACC TAT CGT AGC CTA 1337

L W L G Y A N S L I N P F I Y A F F N R D L R T T Y R S L 401

CTC CAG TGC CAG TAC CGG AAT ATC AAC CGG AAG CTC TCT GCT GCA GGC ATG CAT GAA GCC CTG AAA CTT GCT GAG AGG CCC GAG AGA 1424

L Q C Q Y R N I N R K L S A A G M H E A L K L A E R P E R 430

TCC GAG TTT GTG CTA CAA AAC TCT GAC CAC TGT GGG AAA AAG GGT CAT GAT ACA tgatccagagtggaaccctggatgaattcatgcagaacaggtg 1521

S E F V L Q N S D H C G K K G H D T 448

1637 agatggcaaccτctcccttttttt 1661 SEQ ID # 2

Sequence lengther: 1124 base pairs

Type of sequenced molecule: single-strand cDNA

Partial sequence of the 5HT7 receptor

gt GCC AGT TTC TCT CGT CTG CTT AAG AAC GAC AGG AAA 38

A S F S R L L K N D R K 12

AAC ATC TCC ATC TTC AAG CGG GAG CAG AAG GCG GCC GCT 77

N I S I F K R E Q K A A A 25

ACG TTG GGG ATC GTT GTG GGG GCC TTC TCT GTC TGC TGG 116

T L G I V V G A F S V C W 38

CTG CCC TTC TTC CTG ATG TCC ACG GCC AGG CCC TTC ATC 155

L P F F L M S T A R P F I 51

TGT GGG GTA GAA TGT AGC TGC GTT CCC CTC TGG GTG GAA 194

C G V E C S C V P L W V E 64

AGG TTT CTC CTC TGG CTG GGC TAC GCC AAC TCC CTC ATC 233

R F L L W L G Y A N S L I 77

AAC CCA TTC ATC TAT GCC TTC TTC AAC AGG GAC CTG AGG 272

N P F I Y A F F N R D L R 90

ACC ACC TAC CGT AAC ATC TTG CTC TGC AGG TAC AGG AAC 311

T T Y R N I L L C R Y R N 103

ATT AAC CGC AAA CTC TCC GCC GCC TGC ATG CAC GAG GCC 350

I N R K L S A A C M H E A 116

ATC AAA CTG GCT GAG AGG CCG GAT CTG GAG ATC tagaaca 390

I E K L A R D P L E I 127 442 gtgtttcttaaacttctctaacactttgctacacaaggaccagcaaactatg gtttagatcagggtgtcaaacatttgactgcggcctgtgggtggtttgagta ataaaaaaaaactcaatgtagtgataatggacttacattttacagtctcttc 494 546 598 actctgtccatcttgttaacaatcatcaaaacgaaagccgaacagtcaggga gcatcaaaatgacaaaaataaggtgcaaaatgagtttgacacccctagttta 650 aatgaaaacttgggcttgacatatgactaaattggagcagacaaccatctgt ggtcacaggtcacagacaggggtgaaaacactactctagagatctatcagtc 702 754 806 atgaggtagagaggctactgcatgtgtatagatgctgacatatgtcatactg tatggacttacatttacagtctcttcactctgtcgtctgtacatcatcaaac gaagccgaaaaggctacatgcatgtgtatagaataactgacattattgttca 858 910 962 tacctggcacacacaagaacattagatccacatcttgtcaggttatacacag tacattttgcagtgttcatttcacttaaagagttcattttaacactacctca 1014 gataacatatagtcccactctacagagtgtaaaaagtactctgattgtagtg ttacattttgagtgttaatgtaacacttcatattataaaatattaacattcg 1066 1118 1124 atttca

Claims

1. New polypeptides having a serotonergic receptor activity (5HT6 or 5HT7) chosen from the group of polypeptides formed by sets 1 and 2 defined below:

- Set 1 of the polypeptides constituted by or containing the sequence of 448 amino acids of rat, relating to the 5HT6 receptor, defined in the sequence identifier SEQ ID nº 1 of the list of annexed sequences or peptide fragments of this sequence, said fragments being such that

either they contain the site or sites contained in said sequence of 448 amino acids and the presence of which is necessary for the fragment to be capable of binding the serotonin, its agonists or its antagonists,

either they are recognized by antibodies which also recognize said sequence of 448 amino acids but which do not recognize any of the other serotonergic receptors, including the 5HT7 receptor,

- Either they are capable of inducing antibodies recognizing said sequence of 448 amino acids but not recognizing any of the serotonergic receptors described above, including the 5HT7 receptor;

or polypeptides or similar fragments corresponding to other species, including the human species, and relating to the serotonergic receptor homologous to the 5HT6 receptor.

- Set 2 of the polypeptides consisting of or containing the sequence of 127 rat amino acids, relating to the 5HT7 receptor, defined in the sequence identifier SEQ ID No. 2 of the attached sequence list or peptide fragments of these polypeptides, said fragments being such as

- either they contain the site (s) contained in said 127 amino acid sequence or any neighboring site on a polypeptide encompassing said 127 amino acid sequence and coded by a gene expressing the 5HT7 receptor, the presence of which is necessary for the fragment either capable of binding the serotonin, its agonists or antagonists,

either they are recognized by antibodies which also recognize said sequence of 127 amino acids and / or a polypeptide encoded by a qene expressing the 5HT7 receptor but which do not recognize any of the other serotonergic receptors, including the 5HT6 receptor,

- Either they are capable of inducing antibodies recognizing said sequence of 127 amino acids and / or a polypeptide encoded by a qene expressing the 5HT7 receptor but not recognizing any of the serotonin receptors described above, including the 5HT6 receptor;

or polypeptides or similar fragments corresponding to other species, including the human species, and relating to the serotonergic receptor homologous to the 5HT7 receptor.

2. Polypeptides of set 1 of claim 1, characterized in that they contain the sites forming part of said sequence of 448 amino acids, the presence of which is necessary, when the polypeptides are presented on a cell membrane in the presence of tritiated serotonin and in the presence of an aqonist or a serotonin antagonist, so that the apparent affinity of said antagonists or agonists corresponds to that which is shown in Table 1.

3. Nucleic acids containing or constituted by the sequences of nuc le ti coding for any of the polypeptides according to claim 1.

4. Nucleic acids consisting of or comprising the complete sequence coding for the 5HT6 or 5HT7 receptors in humans or in animals.

5. Nucleic acids comprising or consisting of the sequences SEQ ID No. 1 or No. 2 or fragments of these sequences placed in the reading frame, as well as the homologous sequences, in particular in humans.

6. Nucleic acid according to claim 5 constituted by the sequence extending from the nucieotide in position 135 to that in position 1478 in the identifier SEQ ID nº 1 or to its counterparts in humans or in animals.

7. Recombinant vectors, usable for cloning or for the expression of nucleic acids, and containing a nucleic acid according to one of claims 3 to 6.

8. Cell hosts transformed by a recombinant vector according to claim 7 and capable of expressing the coding sequence for the synthesis of a polypeptide according to one of claims 1 and 2.

9. Monoclonal or polyclonal antibodies directed against a polypeptide according to one of claims 1 and 2.

10. Nucleotide probes hybridizing with one of the nucleic acids according to one of claims 3 to 6 or with their complementary sequences or corresponding RNA, being specific for the 5HT6 receptor or for the 5HT7 receptor, and which do not hybridize with the corresponding nucleic sequences or RNAs of the other serotonergic receptors.

11. Probes according to claim 10 for the diagnosis of neurological, psychiatric, gastroenterological or neuroendocrine disorders linked to qualitative or quantitative anomalies of the 5HT6 or 5HT7 receptors.

12. Method for screening drugs intended for the treatment of neurological conditions, in particular Parkinson's disease, Alzheimer's disease, sleep disorders, migraine, and psychiatric disorders such as mood disorders, depression, psychotic, or neuroendocrine states, and in particular dysfunctions of the hypothal amo-pituitary axis, or gastroenterological, in particular functional disorders of the intestine, motor or secretory disorders, in which one puts in contact, in a suitable environment , a candidate molecule with the membranes of a cell host transformed by a vector comprising an insert coding for a polypeptide according to one of claims 1 and 2 and capable of expressing said polypeptide so as to present specific sites on the membrane surface of this polypeptide, the possible ligand-polypeptide complex which is formed then being detected.

13. Method for screening drugs intended for the treatment of neurological conditions, in particular Parkinson's disease, Alzheimer's disease, sleep disorders, migraine, and psychiatric disorders such as mood disorders, depression, psychotic, or neuroendocrine states, and in particular dysfunctions of the hypothalamic-pituitary axis, or gastroenterological, in particular functional disorders of the intestine, motor or secretory disorders in which measures a response induced by the binding of a candidate molecule with a cell, transformed by a vector comprising an insert coding for a polypeptide according to one of claims 1 and 2 and capable of expressing said polypeptide fagon to be presented on the surface menbranar one or more specific sites of this polypeptide in such a way that the measurement of a signal such as the production of cyclic AMP makes it possible to differentiate the serotonergic molecules which behave as antagonists and those which behave as agonists.

14. A method of screening for labeled ligands such as radioactive or fluorescent ligands in which the specific binding of candidate ligands is measured with the membrane of a cell transformed by a vector comprising an insert coding for a polypeptide according to one of claims 1 and 2 and capable of expressing said polypeptide so as to express on the membrane surface one or more specific sites of this polypeptide in such a way that the specific binding of the candidate ligand can be evaluated or indirectly estimated.

15. A method of identifying tissues or cells spontaneously expressing at their membrane surface a polypeptide according to one of claims 1 and 2 in such a way that said tissues or cells can be used for the development of drugs or ligands labeled according to claims 12 to 14.

16. Medicines containing, in a dose intended to be administered, a therapeutically effective amount of an active substance on a polypeptide having a 5HT6 or 5HT7 receptor activity in a pharmaceutically acceptable vehicle.

17. Medicines containing, in a pharmaceutically acceptable vehicle, an effective amount of a substance active on the other serotonergic receptors but not active on at least one of the 5HT6 receptors and

5HT7.

18. Use of a molecule according to any one of claims 12 to 14, for the production of a drug which is not active on at least one of the 5HT6 and 5HT7 receptors, but active on the other serotonergic receptors.