EP0651802A1 - Polypeptides having serotoninergic receptor activity (5ht6), nucleic acids coding for said polypeptides, and uses thereof - Google Patents

Polypeptides having serotoninergic receptor activity (5ht6), nucleic acids coding for said polypeptides, and uses thereof

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Publication number
EP0651802A1
EP0651802A1 EP93914777A EP93914777A EP0651802A1 EP 0651802 A1 EP0651802 A1 EP 0651802A1 EP 93914777 A EP93914777 A EP 93914777A EP 93914777 A EP93914777 A EP 93914777A EP 0651802 A1 EP0651802 A1 EP 0651802A1
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EP
European Patent Office
Prior art keywords
polypeptide
polypeptides
leu
sequence
ile
Prior art date
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EP93914777A
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German (de)
French (fr)
Inventor
Nourdine Amlaiky
Ursula Boschert
René HEN
Jean-Luc Plassat
Sylvie Ramboz
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Institut National de la Sante et de la Recherche Medicale INSERM
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Institut National de la Sante et de la Recherche Medicale INSERM
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to new polypeptides and the genetic material allowing their expression. More particularly, it relates to new polypeptides having a serotonergic receptor activity.
  • Serotonin is a neuromodulator capable of inducing and modulating a wide variety of behaviors such as sleep, appetite, locomotion, sexual activity or even vascular contraction. It is recognized that the activity of 0 serotonin is mediated by its interaction with receptors, designated serotonergic receptors or 5-HT receptors (for 5-hydroxytryptamine). Molecular biology studies as well as pharmacological studies have revealed that there are a large number of 5-HT receptor subtypes. The 5-HT receptors which have been described to date belong either to the family of receptors linked to 5 ion channels (5-HT3 receptors), or to the family of receptors which interact with G proteins and which possess seven transmembrane domains.
  • 5-HT receptors which have been described to date belong either to the family of receptors linked to 5 ion channels (5-HT3 receptors), or to the family of receptors which interact with G proteins and which possess seven transmembrane domains.
  • the 5-HT receptors interacting with G proteins can be subdivided into two distinct groups: the 5HT1 receptors, comprising the mammalian subtypes 5HT1A, 0 5HT1B and 5HT1D as well as three 5HT Drosophila receptors; and 5HT2 receptors including the 5HT2 and 5HT1C subtypes.
  • 5HT4 receptors are probably not the only 5HT receptors in existence, since pharmacological studies have revealed other subtypes such as the 5HT4 receptors as well as certain receptors related to the 5HT1 5 subtype ("5HT1 like" receptors ). In addition, additional molecular biology studies have also revealed heterogeneities within the 5HT1B / 1D subtypes.
  • the present invention results from the discovery of new polypeptides having a serotonergic receptor activity. Although belonging to 0 the family of receptors which interact with G proteins, these new polypeptides differ from the serotonergic receptors already described (5HT1, 5HT2, 5HT3 and 5HT4) from the structural point of view as from the pharmacological point of view. More particularly, the invention results from the isolation and characterization of these new polypeptides, designated 5HT6, as well as genetic material allowing their expression or their identification.
  • a first object of the invention therefore resides in polypeptides comprising all or part of the peptide sequence SEQ ID No. 1 or a derivative thereof.
  • the term derivative designates any molecule obtained by modification of genetic and / or chemical nature of the peptide sequence SEQ ID n ° 1.
  • modification of genetic and / or chemical nature one can hear any mutation, substitution , deletion, addition and / or modification of one or more residues.
  • Such derivatives can be generated for different purposes, such as in particular that of increasing the affinity of the peptide for its ligand (s), that of improving its production levels, that of increasing its resistance to proteases, that of increasing and / or modifying its activity, or that of giving it new pharmacokinetic and / or biological properties.
  • derivatives resulting from an addition there may be mentioned, for example, chimeric polypeptides comprising an additional heterologous part linked at one end.
  • the term derivative also includes polypeptides homologous to polypeptide SEQ ID No. 1, derived from other cellular sources and in particular from cells of human origin, or from other organisms, and having an activity of the same type. Such homologous polypeptides can be obtained by hybridization experiments as described in the examples (see SEQ ID No. 4).
  • the polypeptides of the invention are polypeptides having the capacity to bind serotonin. Even more preferably, they are polypeptides having a serotonergic receptor activity. Still according to a preferred mode, the polypeptides of the invention are capable of being recognized by antibodies recognizing the complete peptide sequence SEQ ID No. 1.
  • a particular embodiment of the invention is represented by the 5HT6 polypeptide comprising the entire peptide sequence SEQ ID No. 1. As indicated in the examples, this polypeptide can be expressed in different cell types to form a functional serotonergic receptor.
  • polypeptides of the invention can be obtained by expression in a cellular host of a nucleotide sequence as described below, by synthesis chemical, based on sequences SEQ ID No. 1 or 4 using techniques known to those skilled in the art, or by a combination of these techniques.
  • polypeptides of the invention as defined above are designated by 5HT6 polypeptides.
  • the present invention also relates to any nucleotide sequence coding for a 5HT6 polypeptide. More preferably, it is a sequence chosen from:
  • the different nucleotide sequences of the invention can be of artificial origin or not. They can be genomic sequences, cDNA, RNA, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained for example by screening DNA libraries (cDNA library, genomic DNA library) by means of probes prepared on the basis of the sequence SEQ ID No. 1. Such libraries can be prepared for starting from cells of different origins by conventional molecular biology techniques known to those skilled in the art.
  • the nucleotide sequences of the invention can also be prepared by chemical synthesis, in particular according to the phosphoramidite method, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries.
  • the nucleotide sequences of the invention can be used for the production of the 5HT6 polypeptides as defined above.
  • the part coding for said polypeptide is generally placed under the control of signals allowing its expression in a cellular host.
  • the choice of these signals can vary depending on the cell host used.
  • the nucleotide sequences of the invention can be part of a vector, which can be autonomous or integrative replication. More particularly, autonomously replicating vectors can be prepared using autonomously replicating sequences in the chosen host. As regards integrative vectors, these can be prepared for example by using sequences homologous to certain regions of the host genome, allowing, by homologous recombination, the integration of the vector.
  • the cellular hosts which can be used for the production of the 5HT6 polypeptides of the invention by the recombinant route are both eukaryotic and prokaryotic hosts.
  • suitable eukaryotic hosts there may be mentioned animal cells, yeasts, or fungi.
  • yeasts mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula.
  • yeasts mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula.
  • animal cells mention may be made of COS, CHO, C127, NIH-3T3 cells, etc.
  • the mushrooms there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp.
  • prokaryotic hosts it is preferred to use the following bacteria Kcoli, Bacillus, or Streptomyces.
  • nucleotide sequences of the present invention can also be used in the pharmaceutical field, either for the production of antisense sequences which can be used in the context of gene therapy, or else for the production of probes allowing detection, by hybridization experiments, the expression of serotonergic receptors in biological samples and the detection of genetic anomalies (polymorphism, mutations) or outliers.
  • Antisense oligonucleotides are small oliogonucleotides, complementary to the strand coding for a given gene, and therefore capable of specifically hybridizing with transcribed mRNA, inhibiting its translation into protein.
  • the subject of the invention is therefore the antisense oligonucleotides capable of at least partially inhibiting the production of 5HT6 polypeptides as defined above.
  • Such oligonucleotides can consist of all or part of the nucleotide sequences defined above. They are generally sequences or fragments of sequences complementary to sequences coding for peptides of the invention.
  • Such oligonucleotides can be obtained from the sequence SEQ ID No. 1 or 4, by fragmentation, etc., or by chemical synthesis.
  • the invention also allows the production of nucleotide probes, synthetic or not, capable of hybridizing with the nucleotide sequences defined above which code for 5HT6 polypeptides of the invention, or with the corresponding mRNAs.
  • probes can be used in vitro as a diagnostic tool, for the detection of the expression of a 5HT6 serotonergic receptor, or even for the detection of genetic anomalies (poor splicing, polymorphism, point mutations, etc.). Given the multiple activities of serotonin, the probes of the invention can thus make it possible to identify neurological, cardiovascular or psychiatric conditions as being linked to the 5HT6 receptors.
  • probes can also be used for the detection and isolation of homologous nucleic acid sequences coding for 5HT6 polypeptides as defined above, from other cellular sources and preferably from cells of human origin, as well as illustrated in the examples.
  • the probes of the invention generally contain at least 10 bases, and they can contain up to the entire sequence SEQ ID No. 1 or 4 or their complementary strand. Preferably, these probes are, prior to their use, marked. For this, different techniques known to those skilled in the art can be used (radioactive, enzymatic labeling, etc.). The hybridization conditions under which these probes can be used are indicated in the general cloning techniques below as well as in the examples.
  • Another subject of the invention relates to recombinant cells capable of expressing on their surface a 5HT6 polypeptide as defined above. These cells can be obtained by introducing a nucleotide sequence as defined above coding for a polypeptide of the invention, then culturing said cells under conditions of expression of said sequence.
  • the recombinant cells according to the invention can be both eukaryotic and prokaryotic cells.
  • eukaryotic cells which are suitable, mention may be made of animal cells, yeasts, or fungi.
  • yeasts mention may be made of yeasts of the genus Saccharomyce, Kluyveromyces, Pichia, Schwanniomyce, or Hansenula.
  • animal cells mention may be made of COS, CHO, C127, NIH-3T3 cells, etc.
  • the mushrooms there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp.
  • prokaryotic cells it is preferred to use the following bacteria E.coli, Bacillus, or Streptomyces.
  • the cells thus obtained can be used to measure the capacity of different molecules to behave as a ligand or as a modulator of the activity of the polypeptides of the invention. More particularly, they can thus be used in a process for highlighting and isolating ligands or modulators of the activity of the polypeptides of the invention, and, more preferably, of agonists and antagonists of serotonin.
  • Another object of the invention therefore relates to a process for detecting and / or isolating ligands of the 5HT6 polypeptides of the invention, according to which the following steps are carried out:
  • a molecule or a mixture containing different molecules, possibly unidentified is brought into contact with a recombinant cell as described above expressing on its surface a polypeptide of the invention under conditions allowing the interaction between said polypeptide of the invention and said molecule in the event that it has an affinity for said polypeptide, and,
  • this method of the invention is suitable for the detection and / or isolation of agonists and antagonists of serotonin for the 5HT6 polypeptides.
  • Another object of the invention relates to a method for detecting and / or isolating modulators of the 5HT6 polypeptides of the invention, according to which the following steps are carried out:
  • a molecule or mixture containing different molecules, possibly unidentified is brought into contact with a recombinant cell as described above, expressing on its surface a polypeptide of the invention, in the presence of 5HT, under conditions allowing interaction between said polypeptide of the invention and 5HT, and,
  • Another object of the invention relates to the use of a ligand or of a modulator identified and / or obtained according to the process described above as a medicament.
  • ligands or modulators can indeed make it possible to treat certain neurological, cardiovascular or psychiatric affections linked to the 5HT6 receptors.
  • the invention also relates to any medicament comprising as active principle at least one molecule acting on a 5HT6 polypeptide of the invention.
  • the molecule is a ligand or a modulator identified and / or isolated according to the method described above.
  • SEQ ID No. 1 Nucleotide and peptide sequences of the murine 5HT6 receptor.
  • the 1558 bp cDNA was sequenced on both strands from the EcoRI site to the Xhol site. The first 92 nucleotides are not shown.
  • Figure 2 Percentages of peptide sequence homology between the 5HT6 receptor presented SEQ ID No. 1 and other receptors of the family of receptors coupled to G proteins. The homologies were calculated on the conserved sequences: the transmembrane domain and its connection loops.
  • Figure 3 Saturation curve of [ 125 I] -LSD to the membranes of Cos-7 cells expressing the 5HT6 receptor. The membranes were incubated with ligand concentrations ranging from 50 pM to 1.25 nM, with or without 10 ⁇ M of 5HT. The specific link is shown. The insert represents the Scatchard analysis of the results.
  • Figure 4 Demonstration of homologous sequences by PCR on total RNA (1 ⁇ g) of different tissues.
  • Table 1 Pharmacological profile of the 5HT6 receptor. The results correspond to competitive experiments for the binding of [ 125 I] -LSD to the membranes of Cos-7 cells expressing the 5HT6 receptor transiently.
  • the numbers in parentheses correspond to the number of independent experiments carried out, each point being carried out in triplicate.
  • the filling of the prominent 5 ′ ends is carried out by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications.
  • the destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Biolabs) used according to the manufacturer's recommendations.
  • the destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease SI. Mutagenesis directed in vitro by synthetic oligodeoxynucleotides is carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 13_ (1985) 8749-8764] using the kit distributed by Amersham.
  • PCR Polymerase-catalyzed Chain Reaction, Saiki RK et al., Science 230 (1985) 1350-1354; Mullis KB and Faloona FA, Meth. ⁇ nzym. JL55_ (1987) 335-350] is carried out using a "DNA thermal cycler" (Perkin ⁇ lmer Cetus) according to the manufacturer's specifications. Verification of the nucleotide sequences is carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.
  • the normal stringency conditions are generally as follows: hybridization: 3 x SCC in the presence of 5 x Denhart's at 65 ° C; washing: 0.5 x SSC at 65 ° C.
  • This probe was used to screen a rat brain cDNA library constructed in the phage UniZap (Stratagene), under conditions of low stringency (formamide 30, 5 ⁇ SSC, 42 ° C). Among the positive phages obtained, one of them, weakly hybridizing to the probe, was isolated. This phage, called ⁇ SR and carried by the plasmid pSR, contained an insert of approximately 1.6 kb which was then introduced into the plasmid Bluescript. The sequence of this fragment was determined on the 2 strands using the dideoxynucleotide technique using synthetic oligonucleotides. The sequence thus obtained is presented in SEQ ID No. 1. It shows that the isolated cDNA carries an open reading phase of 367 amino acids.
  • hydrophobicity analysis shows that this protein carries seven hydrophobic domains, a peculiarity encountered in members of the family of receptors coupled to G proteins.
  • the N-terminal end also contains 2 N-glycosylation sites , and the suspected cytoplasmic domain contains the consensus sites of phosphorylation by protein kinases C and A.
  • the sequence of the 5HT6 receptor isolated above was compared with the sequences of the receptors coupled to the following G proteins: S31, 5HT1BB, 5HTlD ⁇ , 5HT1A, 5HT-dro2A, 5HT-drol. 5HT1C and 5HT2. These experiments revealed some homology in the potential transmembrane domain and in the connection loops, but not in the terminal regions or in the third cytoplasmic loop. Figure 2 gives the% homology at the level of the conserved regions.
  • the homolgia in the conserved regions, with the known receptors is weak, the best result being obtained with the serotonergic receptors 5HT1BB and 5HTlD ⁇ (54% homology), and with the receptor S31 which is not yet characterized.
  • Example 1 The cDNA fragment isolated in Example 1 was inserted into a eukaryotic expression vector, which was used to transfect Cos-7 cells. The membranes of the transfected cells obtained were then prepared and tested for their capacity to bind certain labeled serotonergic ligands.
  • the 1.6 kb cDNA coding for the 5HT6 receptor was isolated from the plasmid pSR in the form of an EcoRI-XhoI fragment, then inserted at the corresponding sites of the vector p513.
  • the vector p513 is derived from the vector pSG5 [Green et al., Nucl. Acids Res. l ⁇ . (1988) 369] by adding a multisite of cloning.
  • the recombinant vector thus obtained, designated p513SR was then used (20 ⁇ g per 10 cm plate) to transfect the Cos-7 cells in the presence of calcium phosphate.
  • the recombinant cells are harvested and the membranes are prepared according to the technique described by Amlaiky and Caron [J. Biol. Chem. 260 (1985) 1983]. Saturation binding and competition experiments were then carried out on these membranes in the presence of the following radiolabelled ligands: [ 125 I] -LSD; [ 12 5l] -cyanopindolol; [ 3 H] -8-OH-DPAT and [ 3 H] - spiperone. For this, the membrane samples (10-20 ⁇ g of protein) were incubated for 10 minutes at 37 ° C. in the presence of the ligand in a final volume of 250 ⁇ l of 50 mM Tris-HCl buffer (pH 7.4).
  • the reaction is then stopped by vacuum filtration on Whatman GF / C glass fiber filters, and rinsing 4 times with 4 ml of 50 mM Tris-HCl buffer (pH 7.4).
  • the non-specific binding was determined in the presence of 10 ⁇ M of 5HT. Radioactivity was measured with a ⁇ counter.
  • [ 3 H] -8-OH-DPAT and [ 3 H] -s ⁇ iperone do not bind the prepared membranes
  • the [ 125 I] -LSD did not bind the Cos-7 cells transfected with the plasmid p513.
  • the cDNA cloned in Example 1 was also expressed in NIH-3T3 cells, which do not express any serotonergic receptor endogenously.
  • the recombinant expression vector described in 3. above was used. It was introduced (20 ⁇ g per 10 cm plate) into NIH-3T3 cells by transfection in the presence of calcium phosphate, at the same time as the vector pRSVneo [Gorman et al., Science 221 (1983) 551], carrying the G418 resistance gene (1 ⁇ g per 10 cm plate).
  • the transforming clones were selected in the presence of 0.5 mg of G418.
  • the isolated clones were then amplified and the total RNAs of these clones were prepared and analyzed in Northern Blot for the expression of 5HT6 mRNA.
  • a clone was thus selected, SR4, expressing high levels of 5HT6 mRNA.
  • the cell membranes of this clone were then prepared and tested under the conditions described above for their capacity to bind certain ligands. labeled serotonergic, testifying to the presence of functional 5HT6 receptors on their surface.
  • the nucleotide sequence SEQ ID No. 1 was then used to demonstrate homologous sequences from other tissues. For this, two techniques were used:
  • tissues used for the search for homologous sequences are the following of murine origin: brain, cerebellum, kidney, liver, spinal cord, spleen, lung, intestine and heart.
  • the probe (i) corresponds to position 1174 on SEQ LD n ° 1 and the probe ( ⁇ ) at position 1394.
  • RNAs were prepared from the various tissues studied, using the technique described by Cathala et al. (DNA 2 (4) (1983)). 1 ⁇ g of these RNAs was subjected to reverse transcription in the presence of 200 units of MMLV reverse transcriptase and 300 ng of probe (i), for 1 hour at 37 ° C. Half of the product of this reaction was then amplified (20 cycles) in the presence of 5 units of the Taq polymerase (Cetus) and 500 ng of the probes (i) and (ii).
  • the in situ hybridization experiments were carried out on cryostatic sections of the adult rat brain (approximately 8 weeks) according to the technique described by Col et al. [EMBO J. 2 (1983) 617].
  • the probe used for these experiments is a single-stranded RNA obtained by transcription in the presence of T7 polymerase,
  • a human genomic DNA library was prepared from the placenta, by partial digestion with the enzyme Mbol, separation on salt gradients, and under cloning in the vector Lamda GEM 12 linearized by BamHI (host bacterium: TAP 90).
  • the library thus obtained was then screened using the 1.6 kb EcoRI-XhoI fragment described in example 3 as a probe, labeled according to the random priming technique.
  • the DNA fragments which hybridize with this probe were isolated, subcloned in a Bluescript plasmid, amplified, then sequenced in both directions according to the dideoxynucleotide technique.
  • the amplification was carried out by the PCR technique: 20 cycles in the presence of Thermus aquaticus polymerase (2.5 units; Cetus) and of oligonucleotides 1 (SEQ ID No. 5) and 2 (SEQ ID No. 6).
  • the fragment obtained was digested with the enzymes BamHI and Xhol, then subcloned into an expression vector P513.
  • sequence obtained is presented on the sequence SEQ ID No. 4. It is understood that the same experiments can be repeated using other tissues and in particular tissues of human origin, and other probes. Furthermore, the homologous sequences demonstrated during these experiments can obviously be then isolated and / or amplified by conventional techniques of molecular biology.
  • AAG ACA TTA TAC CAC AAG AGA CAA GCA AGT AGG ATT GCA AAG GAG GAG 672 Lys Thr Leu Tyr His Lys Arg Gin Ala Ser Arg Ile Ala Lys Glu Glu 210 215 220

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Abstract

Novel polypeptides designated 5HT6 having serotoninergic receptor activity, genetic material for their expression, any recombinant cell expressing said polypeptides, and uses thereof are disclosed.

Description

POLYPEPTIDES AYANT UNE ACTIVITE DE RECEPTEUR SEROTONINERGIQUE ( 5HT6) ACIDES NUCLEIQUES CODANT POUR CES POLYPEPTIDES ET UTILISATIONSPOLYPEPTIDES HAVING SEROTONINERGIC RECEPTOR ACTIVITY (5HT6) NUCLEIC ACIDS ENCODING SUCH POLYPEPTIDES AND USES
La présente invention concerne de nouveaux polypeptides et le matériel 5 génétique permettant leur expression. Plus particulièrement, elle concerne de nouveaux polypeptides ayant une activité de récepteur sérotoninergique.The present invention relates to new polypeptides and the genetic material allowing their expression. More particularly, it relates to new polypeptides having a serotonergic receptor activity.
La sérotonine est un neuromodulateur capable d'induire et de moduler une grande variété de comportements tels que le sommeil, l'appétit, la locomotion, l'activité sexuelle ou encore la contraction vasculaire. Il est admis que l'activité de la 0 sérotonine est médiée par son interaction avec des récepteurs, désignés récepteurs sérotoninergiques ou récepteurs 5-HT (pour 5-hydroxytryptamine). Des études de biologie moléculaire ainsi que des études pharmacologiques ont révélé qu'il existait un grand nombre de sous-types de récepteurs 5-HT. Les récepteurs 5-HT qui ont été décrits jusqu'à aujourd'hui appartiennent soit à la famille des récepteurs liés à des 5 canaux ioniques (récepteurs 5-HT3), soit à la famille des récepteurs qui interagissent avec des protéines G et qui possèdent sept domaines transmembranaires. Par ailleurs, l'analyse des séquences d'acides aminés a montré que les récepteurs 5-HT interagissant avec des protéines G peuvent être sous-divisés en deux groupes distincts : Les récepteurs 5HT1, comprenant les sous-types mammifères 5HT1A, 0 5HT1B et 5HT1D ainsi que trois récepteurs 5HT de drosophile ; et les récepteurs 5HT2 comprenant les sous-types 5HT2 et 5HT1C.Serotonin is a neuromodulator capable of inducing and modulating a wide variety of behaviors such as sleep, appetite, locomotion, sexual activity or even vascular contraction. It is recognized that the activity of 0 serotonin is mediated by its interaction with receptors, designated serotonergic receptors or 5-HT receptors (for 5-hydroxytryptamine). Molecular biology studies as well as pharmacological studies have revealed that there are a large number of 5-HT receptor subtypes. The 5-HT receptors which have been described to date belong either to the family of receptors linked to 5 ion channels (5-HT3 receptors), or to the family of receptors which interact with G proteins and which possess seven transmembrane domains. Furthermore, analysis of the amino acid sequences has shown that the 5-HT receptors interacting with G proteins can be subdivided into two distinct groups: the 5HT1 receptors, comprising the mammalian subtypes 5HT1A, 0 5HT1B and 5HT1D as well as three 5HT Drosophila receptors; and 5HT2 receptors including the 5HT2 and 5HT1C subtypes.
Ces récepteurs ne sont sans doute pas les seuls récepteurs 5HT existant, dans la mesure où des études pharmacologiques ont révélé d'autres sous-types tels que les récepteurs 5HT4 ainsi que certains récepteurs apparentés au sous-type 5HT1 5 (récepteurs "5HT1 like"). De plus, des études supplémentaires de biologie moléculaire ont également révélé des hétérogénéités au sein des sous-types 5HT1B/1D.These receptors are probably not the only 5HT receptors in existence, since pharmacological studies have revealed other subtypes such as the 5HT4 receptors as well as certain receptors related to the 5HT1 5 subtype ("5HT1 like" receptors ). In addition, additional molecular biology studies have also revealed heterogeneities within the 5HT1B / 1D subtypes.
La présente invention résulte de la mise en évidence de nouveaux polypeptides ayant une activité de récepteur sérotoninergique. Bien qu'appartenant à 0 la famille des récepteurs qui interagissent avec des protéines G, ces nouveaux polypeptides diffèrent des récepteurs sérotoninergiques déjà décrits (5HT1, 5HT2, 5HT3 et 5HT4) du point de vue structural comme du point de vue pharmacologique. Plus particulièrement, l'invention résulte de l'isolement et de la caractérisation de ces nouveaux polypeptides, désignés 5HT6, ainsi que du matériel génétique permettant leur expression ou leur identification.The present invention results from the discovery of new polypeptides having a serotonergic receptor activity. Although belonging to 0 the family of receptors which interact with G proteins, these new polypeptides differ from the serotonergic receptors already described (5HT1, 5HT2, 5HT3 and 5HT4) from the structural point of view as from the pharmacological point of view. More particularly, the invention results from the isolation and characterization of these new polypeptides, designated 5HT6, as well as genetic material allowing their expression or their identification.
Un premier objet de l'invention réside donc dans des polypeptides comprenant tout ou partie de la séquence peptidique SEQ ID n° 1 ou d'un dérivé de celle-ci.A first object of the invention therefore resides in polypeptides comprising all or part of the peptide sequence SEQ ID No. 1 or a derivative thereof.
Au sens de la présente invention, le terme dérivé désigne toute molécule obtenue par modification de nature génétique et/ou chimique de la séquence peptidique SEQ ID n° 1. Par modification de nature génétique et/ou chimique, on peut entendre toute mutation, substitution, délétion, addition et/ou modification d'un ou plusieurs résidus. De tels dérivés peuvent être générés dans des buts différents, tels que notamment celui d'augmenter l'affinité du peptide pour son (ses) ligand(s), celui d'améliorer ses niveaux de production, celui d'augmenter sa résistance à des protéases, celui d'augmenter et/ou de modifier son activité, ou celui de lui conférer de nouvelles propriétés pharmacocinétiques et/ou biologiques. Parmi les dérivés résultant d'une addition, on peut citer par exemple les polypeptides chimères comportant une partie hétérologue supplémentaire liée à une extrémité. Le terme dérivé comprend également les polypeptides homologues au polypeptide SEQ ID n° 1, issus d'autres sources cellulaires et notamment de cellules d'origine humaine, ou d'autres organismes, et possédant une activité de même type. De tels polypeptides homologues peuvent être obtenus par des expériences d'hybridation comme décrit dans les exemples (Cf SEQ ID n° 4).Within the meaning of the present invention, the term derivative designates any molecule obtained by modification of genetic and / or chemical nature of the peptide sequence SEQ ID n ° 1. By modification of genetic and / or chemical nature, one can hear any mutation, substitution , deletion, addition and / or modification of one or more residues. Such derivatives can be generated for different purposes, such as in particular that of increasing the affinity of the peptide for its ligand (s), that of improving its production levels, that of increasing its resistance to proteases, that of increasing and / or modifying its activity, or that of giving it new pharmacokinetic and / or biological properties. Among the derivatives resulting from an addition, there may be mentioned, for example, chimeric polypeptides comprising an additional heterologous part linked at one end. The term derivative also includes polypeptides homologous to polypeptide SEQ ID No. 1, derived from other cellular sources and in particular from cells of human origin, or from other organisms, and having an activity of the same type. Such homologous polypeptides can be obtained by hybridization experiments as described in the examples (see SEQ ID No. 4).
Préférentiellement, les polypeptides de l'invention sont des polypeptides possédant la capacité de lier la sérotonine. Encore plus préférentiellement, il s'agit de polypeptides ayant une activité de récepteur sérotoninergique. Toujours selon un mode préféré, les polypeptides de l'invention sont susceptibles d'être reconnus par des anticorps reconnaissant la séquence peptidique complète SEQ ID n° 1.Preferably, the polypeptides of the invention are polypeptides having the capacity to bind serotonin. Even more preferably, they are polypeptides having a serotonergic receptor activity. Still according to a preferred mode, the polypeptides of the invention are capable of being recognized by antibodies recognizing the complete peptide sequence SEQ ID No. 1.
Un mode de réalisation particulier de l'invention est représenté par le polypeptide 5HT6 comprenant toute la séquence peptidique SEQ ID n° 1. Comme indiqué dans les exemples, ce polypeptide peut être exprimé dans différents types cellulaires pour former un récepteur sérotoninergique fonctionnel.A particular embodiment of the invention is represented by the 5HT6 polypeptide comprising the entire peptide sequence SEQ ID No. 1. As indicated in the examples, this polypeptide can be expressed in different cell types to form a functional serotonergic receptor.
Les polypeptides de l'invention peuvent être obtenus par expression dans un hôte cellulaire d'une séquence nucléotidique telle que décrite ci-dessous, par synthèse chimique, sur la base des séquences SEQ ID n° 1 ou 4 en utilisant les techniques connues de l'homme du métier, ou par une combinaison de ces techniques.The polypeptides of the invention can be obtained by expression in a cellular host of a nucleotide sequence as described below, by synthesis chemical, based on sequences SEQ ID No. 1 or 4 using techniques known to those skilled in the art, or by a combination of these techniques.
Dans ce qui suit, les polypeptides de l'invention tels que définis ci-dessus sont désignés par polypeptides 5HT6.In the following, the polypeptides of the invention as defined above are designated by 5HT6 polypeptides.
La présente invention a également pour objet toute séquence nucléotidique codant pour un polypeptide 5HT6. Plus préférentiellement, il s'agit d'une séquence choisie parmi :The present invention also relates to any nucleotide sequence coding for a 5HT6 polypeptide. More preferably, it is a sequence chosen from:
(a) tout ou partie de la séquence nucléotidique SEQ ID n° 1 ou de son brin complémentaire, (b) toute séquence hybridant avec une séquence (a) et codant pour un polypeptide tel que défini précédemment, et,(a) all or part of the nucleotide sequence SEQ ID No. 1 or its complementary strand, (b) any sequence hybridizing with a sequence (a) and coding for a polypeptide as defined above, and,
(c) les séquences dérivées des séquences (a) et (b) en raison de la dégénérescence du code génétique.(c) sequences derived from sequences (a) and (b) due to the degeneration of the genetic code.
Les différentes séquences nucléotidiques de l'invention peuvent être d'origine artificielle ou non. Il peut s'agir de séquences génomiques, d'ADNc, d'ARN, de séquences hybrides ou de séquences synthétiques ou semi-synthétiques. Ces séquences peuvent être obtenues par exemple par criblage de banques d'ADN (banque d'ADNc, banque d'ADN génomique) au moyen de sondes élaborées sur la base de la séquence SEQ ID n° 1. De telles banques peuvent être préparées à partir de cellules de différentes origines par des techniques classiques de biologie moléculaire connues de l'homme du métier. Les séquences nucléotidiques de l'invention peuvent également être préparées par synthèse chimique, notamment selon la méthode des phosphoramidites, ou encore par des méthodes mixtes incluant la modification chimique ou enzymatiqe de séquences obtenues par criblage de banques.The different nucleotide sequences of the invention can be of artificial origin or not. They can be genomic sequences, cDNA, RNA, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained for example by screening DNA libraries (cDNA library, genomic DNA library) by means of probes prepared on the basis of the sequence SEQ ID No. 1. Such libraries can be prepared for starting from cells of different origins by conventional molecular biology techniques known to those skilled in the art. The nucleotide sequences of the invention can also be prepared by chemical synthesis, in particular according to the phosphoramidite method, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries.
Les séquences nucléotidiques de l'invention peuvent être utilisées pour la production des polypeptides 5HT6 tels que définis précédemment. Dans ce cas, la partie codant pour ledit polypeptide est généralement placée sous le contrôle de signaux permettant son expression dans un hôte cellulaire. Le choix de ces signaux (promoteurs, terminateurs, etc) peut varier en fonction de l'hôte cellulaire utilisé. A cet effet, les séquences nucléotidiques de l'invention peuvent faire partie d'un vecteur, qui peut être à réplication autonome ou intégratif . Plus particulièrement, des vecteurs à réplication autonome peuvent être préparés en utilisant des séquences à réplication autonome chez l'hôte choisi. S'agissant des vecteurs intégratifs, ceux-ci peuvent être préparés par exemple en utilisant des séquences homologues à certaines régions du génome de l'hôte, permettant, par recombinaison homologue, l'intégration du vecteur. Les hôtes cellulaires utilisables pour la production des polypeptides 5HT6 de l'invention par voie recombinante sont aussi bien des hôtes eucaryotes que procaryotes. Parmi les hôtes eucaryotes qui conviennent, on peut citer les cellules animales, les levures, ou les champignons. En particulier, s'agissant de levures, on peut citer les levures du genre Saccharomyces , Kluyveromyces , Pichia, Schwanniomyces , ou Hansenula. S'agissant de cellules animales, on peut citer les cellules COS, CHO, C127, NIH-3T3, etc. Parmi les champignons, on peut citer plus particulièrement Aspergillus ssp. ou Trichoderma ssp. Comme hôtes procaryotes, on préfère utiliser les bactéries suivantes Kcoli, Bacillus, ou Streptomyces.The nucleotide sequences of the invention can be used for the production of the 5HT6 polypeptides as defined above. In this case, the part coding for said polypeptide is generally placed under the control of signals allowing its expression in a cellular host. The choice of these signals (promoters, terminators, etc.) can vary depending on the cell host used. To this end, the nucleotide sequences of the invention can be part of a vector, which can be autonomous or integrative replication. More particularly, autonomously replicating vectors can be prepared using autonomously replicating sequences in the chosen host. As regards integrative vectors, these can be prepared for example by using sequences homologous to certain regions of the host genome, allowing, by homologous recombination, the integration of the vector. The cellular hosts which can be used for the production of the 5HT6 polypeptides of the invention by the recombinant route are both eukaryotic and prokaryotic hosts. Among suitable eukaryotic hosts, there may be mentioned animal cells, yeasts, or fungi. In particular, as regards yeasts, mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula. As regards animal cells, mention may be made of COS, CHO, C127, NIH-3T3 cells, etc. Among the mushrooms, there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp. As prokaryotic hosts, it is preferred to use the following bacteria Kcoli, Bacillus, or Streptomyces.
Les séquences nucléotidiques de la présente invention sont également utilisables dans le domaine pharmaceutique, soit pour la réalisation de séquences antisens utilisables dans le cadre d'une thérapie génique, soit encore pour la réalisation de sondes permettant la détection, par des expériences d'hybridation, de l'expression de récepteurs sérotoninergiques dans des échantillons biologiques et la mise en évidence d'anomalies génétiques (polymorphisme, mutations) ou d'expressions aberrantes.The nucleotide sequences of the present invention can also be used in the pharmaceutical field, either for the production of antisense sequences which can be used in the context of gene therapy, or else for the production of probes allowing detection, by hybridization experiments, the expression of serotonergic receptors in biological samples and the detection of genetic anomalies (polymorphism, mutations) or outliers.
L'inhibition de l'expression de certains gènes par des oligonucléotides antisens s'est avérée être une stratégie prometteuse dans le contrôle de l'activité d'un gène. Les oligonucléotides antisens sont des oliogonucléotides de petite taille, complémentaire du brin codant d'un gène donné, et de ce fait capables d'hybrider spécifiquement avec l'ARNm transcrit, inhibant sa traduction en protéine. L'invention a ainsi pour objet les oligonucléotides antisens capables d'inhiber au moins partiellement la production de polypeptides 5HT6 tels que définis précédemment. De tels oligonucléotides peuvent être constitués par tout ou partie des séquences nucléotidiques définies ci-avant. Il s'agit généralement de séquences ou de fragments de séquences complémentaires de séquences codant pour des peptides de l'invention.Inhibition of the expression of certain genes by antisense oligonucleotides has proven to be a promising strategy in controlling the activity of a gene. Antisense oligonucleotides are small oliogonucleotides, complementary to the strand coding for a given gene, and therefore capable of specifically hybridizing with transcribed mRNA, inhibiting its translation into protein. The subject of the invention is therefore the antisense oligonucleotides capable of at least partially inhibiting the production of 5HT6 polypeptides as defined above. Such oligonucleotides can consist of all or part of the nucleotide sequences defined above. They are generally sequences or fragments of sequences complementary to sequences coding for peptides of the invention.
De tels oligonucléotides peuvent être obtenus à partir de la séquence SEQ ID n° 1 ou 4, par fragmentation, etc, ou par synthèse chimique.Such oligonucleotides can be obtained from the sequence SEQ ID No. 1 or 4, by fragmentation, etc., or by chemical synthesis.
Comme indiqué ci-dessus, l'invention permet également la réalisation de sondes nucléotidiques, synthétiques ou non, capables de s'hydrider avec les séquences nucléotidiques définies ci-avant qui codent pour des polypeptides 5HT6 de l'invention, ou avec les ARNm correspondant. De telles sondes peuvent être utilisées in vitro comme outil de diagnostic, pour la détection de l'expression d'un récepteur sérotoninergique 5HT6, ou encore pour la mise en évidence d'anomalies génétiques (mauvais épissage, polymorphisme, mutations ponctuelles, etc). Compte tenu des activités multiples de la sérotonine, les sondes de l'invention peuvent ainsi permettre d'identifier des affections neurologique, cardiovasculaire ou psychiatrique comme étant liées aux récepteurs 5HT6. Ces sondes peuvent également être utilisées pour la mise en évidence et l'isolement de séquences d'acides nucléiques homologues codant pour des polypeptides 5HT6 tels que définis précédemment, à partir d'autres sources cellulaires et préférentiellement de cellules d'origines humaines, ainsi qu'illustré dans les exemples. Les sondes de l'invention comportent généralement au moins 10 bases, et elles peuvent comporter jusqu'à l'intégralité de la séquence SEQ ID n° 1 ou 4 ou de leur brin complémentaire. Préférentiellement, ces sondes sont, préalablement à leur utilisation, marquées. Pour cela, différentes techniques connues de l'homme du métier peuvent être employées (marquage radioactif, enzymatique, etc). Les conditions d'hybridation dans lesquelles ces sondes peuvent être utilisées sont indiquées dans les techniques générales de clonage ci-après ainsi que dans les exemples.As indicated above, the invention also allows the production of nucleotide probes, synthetic or not, capable of hybridizing with the nucleotide sequences defined above which code for 5HT6 polypeptides of the invention, or with the corresponding mRNAs. Such probes can be used in vitro as a diagnostic tool, for the detection of the expression of a 5HT6 serotonergic receptor, or even for the detection of genetic anomalies (poor splicing, polymorphism, point mutations, etc.). Given the multiple activities of serotonin, the probes of the invention can thus make it possible to identify neurological, cardiovascular or psychiatric conditions as being linked to the 5HT6 receptors. These probes can also be used for the detection and isolation of homologous nucleic acid sequences coding for 5HT6 polypeptides as defined above, from other cellular sources and preferably from cells of human origin, as well as illustrated in the examples. The probes of the invention generally contain at least 10 bases, and they can contain up to the entire sequence SEQ ID No. 1 or 4 or their complementary strand. Preferably, these probes are, prior to their use, marked. For this, different techniques known to those skilled in the art can be used (radioactive, enzymatic labeling, etc.). The hybridization conditions under which these probes can be used are indicated in the general cloning techniques below as well as in the examples.
Un autre objet de l'invention concerne les cellules recombinées capables d'exprimer à leur surface un polypeptide 5HT6 tel que défini ci-avant. Ces cellules peuvent être obtenues par introduction d'une séquence nucléotidique telle que définie ci-dessus codant pour un polypeptide de l'invention, puis culture desdites cellules dans des conditions d'expression de ladite séquence.Another subject of the invention relates to recombinant cells capable of expressing on their surface a 5HT6 polypeptide as defined above. These cells can be obtained by introducing a nucleotide sequence as defined above coding for a polypeptide of the invention, then culturing said cells under conditions of expression of said sequence.
Les cellules recombinées selon l'invention peuvent être aussi bien des cellules eucaryotes que procaryotes. Parmi les cellules eucaryotes qui conviennent, on peut citer les cellules animales, les levures, ou les champignons. En particulier, s'agissant de levures, on peut citer les levures du genre Saccharomyce , Kluyveromyces , Pichia, Schwanniomyce , ou Hansenula. S'agissant de cellules animales, on peut citer les cellules COS, CHO, C127, NIH-3T3, etc. Parmi les champignons, on peut citer plus particulièrement Aspergillus ssp. ou Trichoderma ssp. Comme cellules procaryotes, on préfère utiliser les bactéries suivantes E.coli, Bacillus, ou Streptomyces. Les cellules ainsi obtenues peuvent être utilisées pour mesurer la capacité de différentes molécules à se comporter comme ligand ou comme modulateur de l'activité des polypeptides de l'invention. Plus particulièrement, elles peuvent ainsi être utilisées dans un procédé de mise en évidence et d'isolement de ligands ou de modulateur de l'activité des polypeptides de l'invention, et, plus préférentiellement, d'agonistes et d'antagonistes de la sérotonine.The recombinant cells according to the invention can be both eukaryotic and prokaryotic cells. Among the eukaryotic cells which are suitable, mention may be made of animal cells, yeasts, or fungi. In particular, as regards yeasts, mention may be made of yeasts of the genus Saccharomyce, Kluyveromyces, Pichia, Schwanniomyce, or Hansenula. As regards animal cells, mention may be made of COS, CHO, C127, NIH-3T3 cells, etc. Among the mushrooms, there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp. As prokaryotic cells, it is preferred to use the following bacteria E.coli, Bacillus, or Streptomyces. The cells thus obtained can be used to measure the capacity of different molecules to behave as a ligand or as a modulator of the activity of the polypeptides of the invention. More particularly, they can thus be used in a process for highlighting and isolating ligands or modulators of the activity of the polypeptides of the invention, and, more preferably, of agonists and antagonists of serotonin.
Un autre objet de l'invention concerne donc un procédé de mise en évidence et/ou d'isolement de ligands des polypeptides 5HT6 de l'invention, selon lequel on réalise les étapes suivantes :Another object of the invention therefore relates to a process for detecting and / or isolating ligands of the 5HT6 polypeptides of the invention, according to which the following steps are carried out:
- on met en contact une molécule ou un mélange contenant différentes molécules, éventuellement non-identifiées, avec une cellule recombinée telle que décrite ci-dessus exprimant à sa surface un polypeptide de l'invention dans des conditions permettant l'interaction entre ledit polypeptide de l'invention et ladite molécule dans le cas où celle-ci posséderait une affinité pour ledit polypeptide, et,a molecule or a mixture containing different molecules, possibly unidentified, is brought into contact with a recombinant cell as described above expressing on its surface a polypeptide of the invention under conditions allowing the interaction between said polypeptide of the invention and said molecule in the event that it has an affinity for said polypeptide, and,
- on détecte et/ou isole les molécules liées au dit polypeptide de l'invention. Dans un mode particulier, ce procédé de l'invention est adapté à la mise en évidence et/ou l'isolement d'agonistes et d'antagonistes de la sérotonine pour les polypeptides 5HT6.- Molecules linked to said polypeptide of the invention are detected and / or isolated. In a particular embodiment, this method of the invention is suitable for the detection and / or isolation of agonists and antagonists of serotonin for the 5HT6 polypeptides.
Un autre objet de l'invention concerne un procédé de mise en évidence et/ou d'isolement de modulateurs des polypeptides 5HT6 de l'invention, selon lequel on réalise les étapes suivantes :Another object of the invention relates to a method for detecting and / or isolating modulators of the 5HT6 polypeptides of the invention, according to which the following steps are carried out:
- on met en contact une molécule ou un mélange contenant différentes molécules, éventuellement non-identifiées, avec une cellule recombinée telle que décrite ci-dessus exprimant à sa surface un polypeptide de l'invention, en présence de 5HT, dans des conditions permettant l'interaction entre ledit polypeptide de l'invention et le 5HT, et,- a molecule or mixture containing different molecules, possibly unidentified, is brought into contact with a recombinant cell as described above, expressing on its surface a polypeptide of the invention, in the presence of 5HT, under conditions allowing interaction between said polypeptide of the invention and 5HT, and,
- on détecte et/ou isole les molécules capables de moduler l'activité du 5HT sur ledit polypeptide de l'invention.- Molecules capable of modulating the activity of 5HT on said polypeptide of the invention are detected and / or isolated.
Un autre objet de l'invention concerne l'utilisation d'un ligand ou d'un modulateur identifié et/ou obtenu selon le procédé décrit ci-avant comme médicament. De tels ligands ou modulateurs peuvent en effet permettre de traiter certaines affections neurologique, cardiovasculaire ou psychiatrique liées aux récepteurs 5HT6.Another object of the invention relates to the use of a ligand or of a modulator identified and / or obtained according to the process described above as a medicament. Such ligands or modulators can indeed make it possible to treat certain neurological, cardiovascular or psychiatric affections linked to the 5HT6 receptors.
L'invention concerne également tout médicament comprenant comme principe actif au moins une molécule agissant sur un polypeptide 5HT6 de l'invention. Préférentiellement la molécule est un ligand ou un modulateur identifié et/ou isolé selon le procédé décrit précédemment. D'autres avantages de la présente invention apparaîtront à la lecture des exemples qui suivent, qui doivent être considérés comme illustratif s et non limitatifs.The invention also relates to any medicament comprising as active principle at least one molecule acting on a 5HT6 polypeptide of the invention. Preferably, the molecule is a ligand or a modulator identified and / or isolated according to the method described above. Other advantages of the present invention will appear on reading the examples which follow, which should be considered as illustrative and not limiting.
Légende des figuresLegend of figures
SEQ ID n° 1 : Séquences nucléotidique et peptidique du récepteur 5HT6 murin. L'ADNc de 1558 pb a été séquence sur les 2 brins depuis le site EcoRI jusqu'au site Xhol. Les 92 premiers nucléotides ne sont pas représentés.SEQ ID No. 1: Nucleotide and peptide sequences of the murine 5HT6 receptor. The 1558 bp cDNA was sequenced on both strands from the EcoRI site to the Xhol site. The first 92 nucleotides are not shown.
Figure 2 : Pourcentages d'homologie de séquence peptidique entre le récepteur 5HT6 présenté SEQ ID n° 1 et d'autres récepteurs de la famille des récepteurs couplés à des protéines G. Les homologies ont été calculées sur les séquences conservées : le domaine transmembranaire et ses boucles de connection.Figure 2: Percentages of peptide sequence homology between the 5HT6 receptor presented SEQ ID No. 1 and other receptors of the family of receptors coupled to G proteins. The homologies were calculated on the conserved sequences: the transmembrane domain and its connection loops.
Figure 3 : Courbe de saturation du [125I]-LSD aux membranes des cellules Cos-7 exprimant le récepteur 5HT6. Les membranes ont été incubées avec des concentrations de ligand allant de 50 pM à 1,25 nM, avec ou sans 10 μM de 5HT. La liaison spécifique est représentée. L'encart représente l'analyse en Scatchard des résultats.Figure 3: Saturation curve of [ 125 I] -LSD to the membranes of Cos-7 cells expressing the 5HT6 receptor. The membranes were incubated with ligand concentrations ranging from 50 pM to 1.25 nM, with or without 10 μM of 5HT. The specific link is shown. The insert represents the Scatchard analysis of the results.
Figure 4 : Mise en évidence de séquences homologues par PCR sur des ARN totaux (1 μg) de différents tissus.Figure 4: Demonstration of homologous sequences by PCR on total RNA (1 μg) of different tissues.
Table 1 : Profil pharmacologique du récepteur 5HT6. Les résultats correspondent à des expériences de compétition pour la liaison du [125I]-LSD aux membranes des cellules Cos-7 exprimant le récepteur 5HT6 de manière transitoire. Les valeurs d'IC50 (correspondant à la concentration en ligand nécessaire pour déplacer 50 % du [125I]-LSD lié) ont été calculées expérimentalement et converties en Ki selon l'équation suivante : Ki = IC50/Q + C Kd) dans laquelle C est la concentration en [125I]-LSD (150 pM) et Kd est la constante de dissociation du [125I]-LSD (980 pM). Les nombres entre parenthèses correspondent au nombre d'expériences indépendantes réalisées, chaque point étant réalisé en triple. Techniques générales de clonageTable 1: Pharmacological profile of the 5HT6 receptor. The results correspond to competitive experiments for the binding of [ 125 I] -LSD to the membranes of Cos-7 cells expressing the 5HT6 receptor transiently. The IC50 values (corresponding to the ligand concentration necessary to displace 50% of the bound [ 125 I] -LSD) were calculated experimentally and converted into Ki according to the following equation: Ki = IC50 / Q + C Kd) in where C is the concentration of [ 125 I] -LSD (150 pM) and Kd is the dissociation constant of [ 125 I] -LSD (980 pM). The numbers in parentheses correspond to the number of independent experiments carried out, each point being carried out in triplicate. General cloning techniques
Les méthodes classiquement utilisées en biologie moléculaire telles que les extractions préparatives d'ADN plasmidique, la centrifugation d'ADN plasmidique en gradient de chlorure de césium, l'électrophorèse sur gels d'agarose ou d'acrylamide, la purification de fragments d'ADN par électroélution, les extractions de protéines au phénol ou au phénol-chloroforme, la précipitation d'ADN en milieu salin par de l'éthanol ou de l'isopropanol, la transformation dans Escherichia coli, etc, sont bien connues de l'homme de métier et sont abondament décrites dans la littérature [Maniatis T. et al., " olecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F.M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].Classically used methods in molecular biology such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, protein extractions with phenol or phenol-chloroform, DNA precipitation in a saline medium with ethanol or isopropanol, transformation in Escherichia coli, etc., are well known to humans. trade and are abundantly described in the literature [Maniatis T. et al., "olecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel F.M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].
Les enzymes de restriction ont été fournies par New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) ou Amersham et sont utilisées selon les recommandations des fournisseurs. Pour les ligatures, les fragments d'ADN sont séparés selon leur taille par électrophorèse en gels d'agarose ou d'acrylamide, extraits au phénol ou par un mélange phénol/chloroforme, précipités à l'éthanol puis incubés en présence de l'ADN ligase du phage T4 (Biolabs) selon les recommandations du fournisseur.Restriction enzymes have been supplied by New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham and are used as recommended by suppliers. For the ligations, the DNA fragments are separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of DNA. phage T4 ligase (Biolabs) according to the supplier's recommendations.
Le remplissage des extrémités 5' proéminentes est effectué par le fragment de Klenow de l'ADN Polymérase I d'E. coli (Biolabs) selon les spécifications du fournisseur. La destruction des extrémités 3' proéminentes est effectuée en présence de l'ADN Polymérase du phage T4 (Biolabs) utilisée selon les recommandations du fabricant. La destruction des extrémités 5' proéminentes est effectuée par un traitement ménagé par la nucléase SI. La mutagénèse dirigée in vitro par oligodéoxynucléotides synthétiques est effectuée selon la méthode développée par Taylor et al. [Nucleic Acids Res. 13_ (1985) 8749-8764] en utilisant le kit distribué par Amersham.The filling of the prominent 5 ′ ends is carried out by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications. The destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Biolabs) used according to the manufacturer's recommendations. The destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease SI. Mutagenesis directed in vitro by synthetic oligodeoxynucleotides is carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 13_ (1985) 8749-8764] using the kit distributed by Amersham.
L'amplification enzymatique de fragments d'ADN par la technique dite de PCR [Polymérase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354 ; Mullis K.B. et Faloona F. A., Meth. Εnzym. JL55_ (1987) 335-350] est effectuée en utilisant un "DNA thermal cycler" (Perkin Εlmer Cetus) selon les spécifications du fabricant. La vérification des séquences nucléotidiques est effectuée par la méthode développée par Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] en utilisant le kit distribué par Amersham.The enzymatic amplification of DNA fragments by the technique known as PCR [Polymerase-catalyzed Chain Reaction, Saiki RK et al., Science 230 (1985) 1350-1354; Mullis KB and Faloona FA, Meth. Εnzym. JL55_ (1987) 335-350] is carried out using a "DNA thermal cycler" (Perkin Εlmer Cetus) according to the manufacturer's specifications. Verification of the nucleotide sequences is carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.
Pour les expériences d'hybridation, les conditions de stringence normales sont généralement les suivantes : hybridation : 3 x SCC en présence de 5 x Denhart's à 65°C ; lavage : 0,5 x SSC à 65°C.For hybridization experiments, the normal stringency conditions are generally as follows: hybridization: 3 x SCC in the presence of 5 x Denhart's at 65 ° C; washing: 0.5 x SSC at 65 ° C.
1. Isolement du récepteur 5HT61. Isolation of the 5HT6 receptor
Les comparaisons de séquences entre les différents récepteurs sérotoninergiques connus font apparaître une certaine conservation, particulièrement dans certaines régions transmembranaires potentielles telles que les domaines III et IV. Dans le but de mettre en évidence et d'isoler un nouveau récepteur, les inventeurs de la présente demande ont utilisé une sonde correspondant à un fragment génomique du récepteur 5HT1BB [Maroteaux et al., Proc. Natl. Acad. Sci. USA ≤9 (1992) 3020] pour cribler une banque d'ADN de cerveau de rat. Plus particulièrement, la sonde utilisée correspond au fragment Sacl-Bgiπ de 2,3 kb du récepteur 5HT1BB, préalablement marqué par "random priming" [Feinberg et Vogelstein, Analytical Biochemistry 132 (1984) 6]. Cette sonde a été utilisée pour cribler une banque d'ADNc de cerveau de rat construite dans le phage UniZap (Stratagène), dans des conditions de stringence faible (formamide 30 , 5 x SSC, 42°C). Parmi les phages positifs obtenus, l'un d'entre-eux, hybridant faiblement à la sonde a été isolé. Ce phage, dénommé λSR et porté par le plasmide pSR, contenait un insert de 1,6 kb environ qui a ensuite été introduit dans le plasmide Bluescript. La séquence de ce fragment a été déterminée sur les 2 brins en utilisant la technique des dideoxynucléotides au moyen d'oligonucléotides synthétiques. La séquence ainsi obtenue est présentée sur la SEQ ID n° 1. Elle montre que l'ADNc isolé porte une phase de lecture ouverte de 367 acides aminés. Par ailleurs, l'analyse d'hydrophobicité montre que cette protéine porte sept domaines hydrophobes, une particularité rencontrée chez les membres de la famille des récepteurs couplés à des protéines G. L'extrémité N-terminale contient par ailleurs 2 sites de N-glycosylation, et le domaine cytoplasmique présumé contient les sites consensus de phosphorylation par les protéines kinases C et A. 2. Etude d'homologies de séquenceSequence comparisons between the various known serotoninergic receptors show a certain conservation, particularly in certain potential transmembrane regions such as domains III and IV. In order to identify and isolate a new receptor, the inventors of the present application used a probe corresponding to a genomic fragment of the 5HT1BB receptor [Maroteaux et al., Proc. Natl. Acad. Sci. USA ≤9 (1992) 3020] to screen a rat brain DNA library. More particularly, the probe used corresponds to the SacI-Bgiπ fragment of 2.3 kb from the 5HT1BB receptor, previously marked with "random priming" [Feinberg and Vogelstein, Analytical Biochemistry 132 (1984) 6]. This probe was used to screen a rat brain cDNA library constructed in the phage UniZap (Stratagene), under conditions of low stringency (formamide 30, 5 × SSC, 42 ° C). Among the positive phages obtained, one of them, weakly hybridizing to the probe, was isolated. This phage, called λSR and carried by the plasmid pSR, contained an insert of approximately 1.6 kb which was then introduced into the plasmid Bluescript. The sequence of this fragment was determined on the 2 strands using the dideoxynucleotide technique using synthetic oligonucleotides. The sequence thus obtained is presented in SEQ ID No. 1. It shows that the isolated cDNA carries an open reading phase of 367 amino acids. Furthermore, the hydrophobicity analysis shows that this protein carries seven hydrophobic domains, a peculiarity encountered in members of the family of receptors coupled to G proteins. The N-terminal end also contains 2 N-glycosylation sites , and the suspected cytoplasmic domain contains the consensus sites of phosphorylation by protein kinases C and A. 2. Study of sequence homologies
La séquence du récepteur 5HT6 isolé ci-dessus a été comparée avec les séquences des récepteurs couplés à des protéines G suivants : S31, 5HT1BB, 5HTlDα, 5HT1A, 5HT-dro2A, 5HT-drol. 5HT1C et 5HT2. Ces expériences ont révélé une certaine homologie dans le domaine transmembranaire potentiel et dans les boucles de connection, mais pas dans les régions terminales ni dans la troisième boucle cytoplasmique. La figure 2 donne les % d'homologie au niveau des régions conservées.The sequence of the 5HT6 receptor isolated above was compared with the sequences of the receptors coupled to the following G proteins: S31, 5HT1BB, 5HTlDα, 5HT1A, 5HT-dro2A, 5HT-drol. 5HT1C and 5HT2. These experiments revealed some homology in the potential transmembrane domain and in the connection loops, but not in the terminal regions or in the third cytoplasmic loop. Figure 2 gives the% homology at the level of the conserved regions.
Comme il ressort de cette figure, l'homolgie, au niveau des régions conservées, avec les récepteurs connus est faible, le meilleur résultat étant obtenu avec les récepteurs sérotoninergiques 5HT1BB et 5HTlDα (54 % d'homologie), et avec le récepteur S31 qui n'est pas encore caractérisé.As can be seen from this figure, the homolgia, in the conserved regions, with the known receptors is weak, the best result being obtained with the serotonergic receptors 5HT1BB and 5HTlDα (54% homology), and with the receptor S31 which is not yet characterized.
3. Expression transitoire du récepteur 5HT6 dans les cellules Cos-7 et caractérisation pharmacologique3. Transient expression of the 5HT6 receptor in Cos-7 cells and pharmacological characterization
Le fragment d'ADNc isolé dans l'exemple 1 a été inséré dans un vecteur d'expression eucaryote, qui a été utilisé pour transfecter des cellules Cos-7. Les membranes des cellules transfectées obtenues ont ensuite été préparées et testées pour leur capacité à lier certains ligands sérotoninergiques marqués.The cDNA fragment isolated in Example 1 was inserted into a eukaryotic expression vector, which was used to transfect Cos-7 cells. The membranes of the transfected cells obtained were then prepared and tested for their capacity to bind certain labeled serotonergic ligands.
L'ADNc de 1,6 kb codant pour le récepteur 5HT6 a été isolé à partir du plasmide pSR sous forme d'un fragment EcoRI-XhoI, puis inséré aux sites correspondants du vecteur p513. Le vecteur p513 dérive du vecteur pSG5 [Green et al., Nucl. Acids Res. lβ. (1988) 369] par addition d'un multisite de clonage. Le vecteur recombinant ainsi obtenu désigné p513SR a ensuite été utilisé (20 μg par plaque de 10 cm) pour transfecter les cellules Cos-7 en présence de phosphate de calcium.The 1.6 kb cDNA coding for the 5HT6 receptor was isolated from the plasmid pSR in the form of an EcoRI-XhoI fragment, then inserted at the corresponding sites of the vector p513. The vector p513 is derived from the vector pSG5 [Green et al., Nucl. Acids Res. lβ. (1988) 369] by adding a multisite of cloning. The recombinant vector thus obtained, designated p513SR, was then used (20 μg per 10 cm plate) to transfect the Cos-7 cells in the presence of calcium phosphate.
48 heures après la transfection, les cellules recombinantes sont récoltées et les membranes sont préparées selon la technique décrite par Amlaiky et Caron [J. Biol. Chem. 260 (1985) 1983]. Des expériences de liaison à saturation et de compétition ont ensuite été réalisées sur ces membranes en présence des ligands radiomarqués suivants : [125I]-LSD; [125l]-cyanopindolol; [3H]-8-OH-DPAT et [3H]- spiperone. Pour cela, les échantillons de membrane (10-20 μg de protéines) ont été incubés 10 minutes à 37°C en présence du ligand dans un volume final de 250 μl de tampon Tris-HCl 50 mM (pH 7,4). La réaction est ensuite stopée par filtration sous vide sur filtres en fibre de verre Whatman GF/C, et rinçage 4 fois avec 4 ml de tampon Tris-HCl 50 mM (pH 7,4). La liaison non- spécifique a été déterminée en présence de 10 μM de 5HT. La radioactivité a été mesurée avec un compteur γ.48 hours after transfection, the recombinant cells are harvested and the membranes are prepared according to the technique described by Amlaiky and Caron [J. Biol. Chem. 260 (1985) 1983]. Saturation binding and competition experiments were then carried out on these membranes in the presence of the following radiolabelled ligands: [ 125 I] -LSD; [ 12 5l] -cyanopindolol; [ 3 H] -8-OH-DPAT and [ 3 H] - spiperone. For this, the membrane samples (10-20 μg of protein) were incubated for 10 minutes at 37 ° C. in the presence of the ligand in a final volume of 250 μl of 50 mM Tris-HCl buffer (pH 7.4). The reaction is then stopped by vacuum filtration on Whatman GF / C glass fiber filters, and rinsing 4 times with 4 ml of 50 mM Tris-HCl buffer (pH 7.4). The non-specific binding was determined in the presence of 10 μM of 5HT. Radioactivity was measured with a γ counter.
Les résultats obtenus montrent que, bien que le [125I]-cyanopindolol; leThe results obtained show that, although [ 125 I] -cyanopindolol; the
[3H]-8-OH-DPAT et le [3H]-sρiperone ne lient pas les membranes préparées, le [125I]-LSD présente un site de liaison saturable avec un Kd = 980 pM et un Bmax = 2,2 pmol/mg de protéines membranaires (figure 3). Dans une exprérience contrôle, il a par ailleurs été montré que le [125I]-LSD ne liait pas les cellules Cos-7 transfectées par le plasmide p513.[ 3 H] -8-OH-DPAT and [ 3 H] -sρiperone do not bind the prepared membranes, [ 125 I] -LSD has a saturable binding site with a Kd = 980 pM and a Bmax = 2, 2 pmol / mg of membrane proteins (Figure 3). In a control experiment, it was also shown that the [ 125 I] -LSD did not bind the Cos-7 cells transfected with the plasmid p513.
Pour déterminer le profil pharmacologique de ce récepteur, le [125I]-LSD lié aux membranes a été déplacé en présence de différentes drogues sérotoninergiques (table 1). Ces différentes drogues montrent l'ordre d'efficacité de déplacement suivant : méthylsergide > bufotenine > sumatriptan > 5HT (table 1). La kétansérine, le (+) cyanopindolol et le 5-CT possèdent une faible affinité, tant dis que la norépinéphrine est inactive.To determine the pharmacological profile of this receptor, the [ 125 I] -LSD bound to the membranes was displaced in the presence of various serotonergic drugs (table 1). These different drugs show the following order of displacement efficiency: methylsergide>bufotenine>sumatriptan> 5HT (table 1). Ketanserine, (+) cyanopindolol and 5-CT have a low affinity, as long as norepinephrine is inactive.
4. Expression du récepteur 5HT6 dans les cellules NIH-3T3 et étude pharmacologique4. Expression of the 5HT6 receptor in NIH-3T3 cells and pharmacological study
L'ADNc clone dans l'exemple 1 a également été exprimé dans les cellules NIH-3T3, qui n'expriment aucun récepteur sérotoninergique de manière endogène. Pour cela, le vecteur d'expression recombinant décrit en 3. ci-dessus a été utilisé. Il a été introduit (20 μg par plaque de 10 cm) dans les cellules NIH-3T3 par transfection en présence de phosphate de calcium, en même temps que le vecteur pRSVnéo [Gorman et al., Science 221 (1983) 551], portant le gène de résistance au G418 (1 μg par plaque de 10 cm). Les clones transformants ont été sélectionnés en présence de 0,5 mg de G418. Les clones isolés ont ensuite été amplifiés et les RNA totaux de ces clones ont été préparés et analysés en Northern Blot pour l'expression d'ARNm du 5HT6. Un clones a ainsi été sélectionné, SR4, exprimant des niveaux élevés d'ARNm du 5HT6.The cDNA cloned in Example 1 was also expressed in NIH-3T3 cells, which do not express any serotonergic receptor endogenously. For this, the recombinant expression vector described in 3. above was used. It was introduced (20 μg per 10 cm plate) into NIH-3T3 cells by transfection in the presence of calcium phosphate, at the same time as the vector pRSVneo [Gorman et al., Science 221 (1983) 551], carrying the G418 resistance gene (1 μg per 10 cm plate). The transforming clones were selected in the presence of 0.5 mg of G418. The isolated clones were then amplified and the total RNAs of these clones were prepared and analyzed in Northern Blot for the expression of 5HT6 mRNA. A clone was thus selected, SR4, expressing high levels of 5HT6 mRNA.
Les membranes des cellules de ce clone ont ensuite été préparées et testées dans les conditions décrites ci-dessus pour leur capacité à lier certains ligands sérotoninergiques marqués, témoignant de la présence de récepteurs 5HT6 fonctionnels à leur surface.The cell membranes of this clone were then prepared and tested under the conditions described above for their capacity to bind certain ligands. labeled serotonergic, testifying to the presence of functional 5HT6 receptors on their surface.
5. Recherche de séquences homologues dans d'autres tissus5. Search for homologous sequences in other tissues
La séquence nucléotidique SEQ ID n° 1 a ensuite été utilisée pour la mise en évidence de séquences homologues à partir d'autres tissus. Pour cela, deux techniques ont été utilisées :The nucleotide sequence SEQ ID No. 1 was then used to demonstrate homologous sequences from other tissues. For this, two techniques were used:
- la PCR- PCR
- l'hybridation in situ.- in situ hybridization.
Les tissus utilisés pour la recherche de séquences homologues sont les suivants d'origine murine : cerveau, cervelet, rein, foie, moelle épinière, rate, poumon, intestin et coeur.The tissues used for the search for homologous sequences are the following of murine origin: brain, cerebellum, kidney, liver, spinal cord, spleen, lung, intestine and heart.
5.1. Recherche par PCR5.1. Research by PCR
Pour la recherche par PCR, les sondes suivantes ont été utilisées :For PCR research, the following probes were used:
Sonde (i) : SEQ ID n° 2 Sonde (ii) : SEQ ID n° 3Probe (i): SEQ ID n ° 2 Probe (ii): SEQ ID n ° 3
La sonde (i) correspond à la position 1174 sur la SEQ LD n° 1 et la sonde (ϋ) à la position 1394.The probe (i) corresponds to position 1174 on SEQ LD n ° 1 and the probe (ϋ) at position 1394.
Les ARN totaux ont été préparés à partir des différents tissus étudiés, en utilisant la technique décrite par Cathala et al. (DNA 2(4) (1983)). 1 μg de ces ARN a été soumis à une transcription inverse en présence de 200 unités de transcriptase inverse MMLV et de 300 ng de la sonde (i), pendant 1 heure à 37°C. La moitié du produit de cette réaction a ensuite été amplifiée (20 cycles) en présence de 5 unités de la polymérase Taq (Cetus) et de 500 ng des sondes (i) et (ii). Les produits ainsi obtenus ont ensuite été transférés sur filtres de nitro-cellulose et hybrides dans les conditions de stringence élevée suivantes : 42°C, dans un tampon phosphate de sodium 20 mM (pH 6,5) contenant 50 % de formamide, 5 x SSC, 1 x Denhardt's, 0,1 % de SDS et 100 μg/ml d'ARNt. Les lavages ont été effectués à 60°C dans un tampon 0,1 x SSC, 0,1 % SDS. Cette étude a permis de mettre en évidence des fragments d'ADN spécifiques homologues dans la moelle épinière et le cerveau (figure 4).Total RNAs were prepared from the various tissues studied, using the technique described by Cathala et al. (DNA 2 (4) (1983)). 1 μg of these RNAs was subjected to reverse transcription in the presence of 200 units of MMLV reverse transcriptase and 300 ng of probe (i), for 1 hour at 37 ° C. Half of the product of this reaction was then amplified (20 cycles) in the presence of 5 units of the Taq polymerase (Cetus) and 500 ng of the probes (i) and (ii). The products thus obtained were then transferred to nitro-cellulose and hybrid filters under the following high stringency conditions: 42 ° C., in a 20 mM sodium phosphate buffer (pH 6.5) containing 50% formamide, 5 x SSC, 1 x Denhardt's, 0.1% SDS and 100 μg / ml tRNA. The washes were carried out at 60 ° C. in a buffer 0.1 x SSC, 0.1% SDS. This study revealed specific homologous DNA fragments in the spinal cord and the brain (Figure 4).
5.2. Recherche par hybridation in situ5.2. In situ hybridization research
Les expériences d'hybridation in situ ont été réalisées sur des sections cryostatées de cerveau de rat adulte (8 semaines environ) selon la technique décrite par Hafen et al. [EMBO J. 2 (1983) 617]. La sonde utilisée pour ces expériences est un ARN simple brin obtenu par transcription en présence de polymérase T7, deThe in situ hybridization experiments were carried out on cryostatic sections of the adult rat brain (approximately 8 weeks) according to the technique described by Hafen et al. [EMBO J. 2 (1983) 617]. The probe used for these experiments is a single-stranded RNA obtained by transcription in the presence of T7 polymerase,
[35S]-CTP en utilisant le plasmide PSR comme matrice.[ 35 S] -CTP using the plasmid PSR as a template.
Cette étude a permis de mettre en évidence des séquences homologues selon l'invention dans les couches CAl, CA2 et CA3 de l'hippocampe. Une expérience contrôle réalisée dans les mêmes conditions avec différentes sondes d'ARN de même longueur mais non spécifique des récepteurs de l'invention n'a révélé aucun signal positif.This study made it possible to demonstrate homologous sequences according to the invention in the layers CA1, CA2 and CA3 of the hippocampus. A control experiment carried out under the same conditions with different RNA probes of the same length but not specific for the receptors of the invention did not reveal any positive signal.
6. Isolement du récepteur humain6. Isolation of the human receptor
Selon la méthodologie décrite en 5. ci-dessus, le récepteur 5HT6 humain a été clone.According to the methodology described in 5. above, the human 5HT6 receptor was cloned.
Pour cela, une banque d'ADN génomique humain a été préparée à partir de placenta, par digestion partielle par l'enzyme Mbol, séparation sur gradients de sels, et sous clonage dans le vecteur Lamda GEM 12 linéarisé par BamHI (bactérie hôte:TAP 90).For this, a human genomic DNA library was prepared from the placenta, by partial digestion with the enzyme Mbol, separation on salt gradients, and under cloning in the vector Lamda GEM 12 linearized by BamHI (host bacterium: TAP 90).
La banque ainsi obtenue a ensuite été criblée en utilisant comme sonde le fragment EcoRI-XhoI de 1,6 kb décrit dans l'exemple 3., marqué selon la technique de random priming. Les fragments de DNA qui hybrident avec cette sonde ont été isolés, sous clones dans un plasmide Bluescript, amplifiés, puis séquences dans les deux sens selon la technique dideoxynucleotide. L'amplification a été réalisée par la technique PCR : 20 cycles en présence de Thermus aquaticus polymerase(2,5 unités; Cetus) et d'oligonucléotides 1 (SEQ ID n° 5) et 2 (SEQ ID n° 6). Le fragment obtenu a été digéré par les enzymes BamHI et Xhol, puis sous clones dans un vecteur d'expression P513. La séquence obtenue est présentée sur la séquence SEQ ID n° 4. Il est entendu que les même expériences peuvent être répétées en utilisant d'autres tissus et notamment des tissus d'origine humaine, et d'autres sondes. Par ailleurs, les séquences homologues mises en évidence lors de ces expériences peuvent évidemment être ensuite isolées et/ou amplifiées par les techniques classiques de biologie moléculaire. The library thus obtained was then screened using the 1.6 kb EcoRI-XhoI fragment described in example 3 as a probe, labeled according to the random priming technique. The DNA fragments which hybridize with this probe were isolated, subcloned in a Bluescript plasmid, amplified, then sequenced in both directions according to the dideoxynucleotide technique. The amplification was carried out by the PCR technique: 20 cycles in the presence of Thermus aquaticus polymerase (2.5 units; Cetus) and of oligonucleotides 1 (SEQ ID No. 5) and 2 (SEQ ID No. 6). The fragment obtained was digested with the enzymes BamHI and Xhol, then subcloned into an expression vector P513. The sequence obtained is presented on the sequence SEQ ID No. 4. It is understood that the same experiments can be repeated using other tissues and in particular tissues of human origin, and other probes. Furthermore, the homologous sequences demonstrated during these experiments can obviously be then isolated and / or amplified by conventional techniques of molecular biology.
LISTE DE SEQUENCESLIST OF SEQUENCES
(1 ) INFORMATION GENERALE:(1) GENERAL INFORMATION:
(i) DEPOSANT:(i) DEPOSITOR:
(A) NOM: INSERM(A) NAME: INSERM
(B) RUE: 101 , rue de Tolbiac(B) STREET: 101, rue de Tolbiac
(C) VILLE: PARIS (E) PAYS: France(C) CITY: PARIS (E) COUNTRY: France
(F) CODE POSTAL: 75654(F) POSTAL CODE: 75654
(ii) TITRE DE L1 INVENTION: Nouveaux polypeptides ayant une activité de récepteur sérotoninergique, acides nucléiques codant pour ces polypeptides et utilisations.(ii) TITLE OF INVENTION 1: New polypeptides having serotonin receptor activity, nucleic acids encoding the polypeptides and uses.
(iii) NOMBRE DE SEQUENCES: 6(iii) NUMBER OF SEQUENCES: 6
(iv) FORME LISIBLE PAR ORDINATEUR: (A) TYPE DE SUPPORT: Tape(iv) FORM READABLE BY COMPUTER: (A) TYPE OF SUPPORT: Tape
(B) ORDINATEUR: IBM PC compatible(B) COMPUTER: IBM PC compatible
(C) SYSTEME D' EXPLOITATION: PC-DOS/MS-DOS(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) LOGICIEL: Patentln Release #1.0, Version #1.25 (OEB)(D) SOFTWARE: Patentln Release # 1.0, Version # 1.25 (EPO)
(2) INFORMATION POUR LA SEQ ID NO: 1(2) INFORMATION FOR SEQ ID NO: 1
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 1557 paires de bases (B) TYPE: acide nucléique(A) LENGTH: 1557 base pairs (B) TYPE: nucleic acid
(C) NOMBRE DE BRINS: double(C) NUMBER OF STRANDS: double
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADNc(ii) TYPE OF MOLECULE: cDNA
(iii) HYPOTHETIQUE: NON(iii) HYPOTHETIC: NO
(iii) ANTI-SENS: NON (vi) ORIGINE:(iii) ANTI-SENSE: NO (vi) ORIGIN:
(A) ORGANISME: Souris(A) ORGANIZATION: Mouse
(ix) CARACTERISTIQUE ADDITIONELLE: (A) NOM/CLE: CDS (B) EMPLACEMENT: 310..1410(ix) ADDITIONAL FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 310..1410
(D) AUTRES RENSEIGNEMENTS: /product= "Gène récepteur 5HT6 souris"(D) OTHER INFORMATION: / product = "5HT6 mouse receptor gene"
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 1 :(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
GTCGGCCTCG AGTGGACTGG CGTCTGGAAC CCGCCCTAGA GCTGCGCCCC AAGCTGCAGC 60GTCGGCCTCG AGTGGACTGG CGTCTGGAAC CCGCCCTAGA GCTGCGCCCC AAGCTGCAGC 60
GCGCATTCAG CTCGCCACCC AAGAGGCAGC CGGGACGCGC TGTTGTCGCC AGAGAACGAC 120GCGCATTCAG CTCGCCACCC AAGAGGCAGC CGGGACGCGC TGTTGTCGCC AGAGAACGAC 120
CGCGGCGGGC TAGGGACCAG AGCCCCTTAG CTTCGCTCTG GGGAAGCTGA GTTGAGATGG 180CGCGGCGGGC TAGGGACCAG AGCCCCTTAG CTTCGCTCTG GGGAAGCTGA GTTGAGATGG 180
CATTGAACTG TGAATGGCTG ACTAATTTCT CACCAGATCA GGAGGTGAAG TGAGAATGAA 240 GACCAACAGT TGAGCCTGCC ACACCACGGT ATTCATTTCT TCAACTATAT TAACATTTTA 300CATTGAACTG TGAATGGCTG ACTAATTTCT CACCAGATCA GGAGGTGAAG TGAGAATGAA 240 GACCAACAGT TGAGCCTGCC ACACCACGGT ATTCATTTCT TCAACTATAT TAACATTTTA 300
ACAAAAAAA ATG GAT TTT TTA AAC GCA TCA GAC CAA AAC TTG ACC TCT 348ACAAAAAAA ATG GAT TTT TTA AAC GCA TCA GAC CAA AAC TTG ACC TCT 348
Met Asp Phe Leu Asn Ala Ser Asp Gin Asn Leu Thr Ser 1 5 10Met Asp Phe Leu Asn Ala Ser Asp Gin Asn Leu Thr Ser 1 5 10
GAG GAA CTG TTA AAC CGA ATG CCA TCC AAA ATT CTG GTA TCC CTC ACT 39 Glu Glu Leu Leu Asn Arg Met Pro Ser Lys Ile Leu Val Ser Leu Thr 15 20 25GAG GAA CTG TTA AAC CGA ATG CCA TCC AAA ATT CTG GTA TCC CTC ACT 39 Glu Glu Leu Leu Asn Arg Met Pro Ser Lys Ile Leu Val Ser Leu Thr 15 20 25
CTG TCT GGG CTG GCA TTG ATG ACA ACC ACC ATC AAC TCC CTC GTG ATC 44 Leu Ser Gly Leu Ala Leu Met Thr Thr Thr Ile Asn Ser Leu Val Ile 30 35 40 45CTG TCT GGG CTG GCA TTG ATG ACA ACC ACC ATC AAC TCC CTC GTG ATC 44 Leu Ser Gly Leu Ala Leu Met Thr Thr Thr Ile Asn Ser Leu Val Ile 30 35 40 45
GCT GCG ATC ATT GTG ACT CGG AAG CTG CAC CAC CCA GCC AAC TAT TTA 49 Ala Ala Ile Ile Val Thr Arg Lys Leu His His Pro Ala Asn Tyr Leu 50 55 60 ATT TGT TCC TTG GCA GTT ACA GAT TTT CTT GTA GCT GTC CTG GTG ATG 54 Ile Cys Ser Leu Ala Val Thr Asp Phe Leu Val Ala Val Leu Val Met 65 70 75GCT GCG ATC ATT GTG ACT CGG AAG CTG CAC CAC CCA GCC AAC TAT TTA 49 Ala Ala Ile Ile Val Thr Arg Lys Leu His His Pro Ala Asn Tyr Leu 50 55 60 ATT TGT TCC TTG GCA GTT ACA GAT TTT CTT GTA GCT GTC CTG GTG ATG 54 Ile Cys Ser Leu Ala Val Thr Asp Phe Leu Val Ala Val Leu Val Met 65 70 75
CCC TTC AGT ATT GTG TAC ATT GTG AGA GAG AGC TGG ATT ATG GGA CAA 58 Pro Phe Ser Ile Val Tyr Ile Val Arg Glu Ser Trp Ile Met Gly Gin 80 85 90CCC TTC AGT ATT GTG TAC ATT GTG AGA GAG AGC TGG ATT ATG GGA CAA 58 Pro Phe Ser Ile Val Tyr Ile Val Arg Glu Ser Trp Ile Met Gly Gin 80 85 90
GTA CTC TGT GAC ATT TGG CTG AGT GTC GAC ATC ATC TGT TGT ACG TGT 63 Val Leu Cys Asp Ile Trp Leu Ser Val Asp Ile Ile Cys Cys Thr Cys 95 100 105GTA CTC TGT GAC ATT TGG CTG AGT GTC GAC ATC ATC TGT TGT ACG TGT 63 Val Leu Cys Asp Ile Trp Leu Ser Val Asp Ile Ile Cys Cys Thr Cys 95 100 105
TCC ATC TTG CAT CTG TCG GCT ATA GCC TTG GAT AGG TAC CGC GCC ATC 68 Ser Ile Leu His Leu Ser Ala Ile Ala Leu Asp Arg Tyr Arg Ala Ile 110 115 120 125TCC ATC TTG CAT CTG TCG GCT ATA GCC TTG GAT AGG TAC CGC GCC ATC 68 Ser Ile Leu His Leu Ser Ala Ile Ala Leu Asp Arg Tyr Arg Ala Ile 110 115 120 125
ACA GAT GCA GTT GAA TAC GCC AGG AAG AGG ACT CCC AGG CAT GCC GGC 73 Thr Asp Ala Val Glu Tyr Ala Arg Lys Arg Thr Pro Arg His Ala Gly 130 135 140 ATC ATG ATC ACG ATC GTG TGG GTT ATA TCT GTG TTC ATC TCT ATG CCT 78 Ile Met Ile Thr Ile Val Trp Val Ile Ser Val Phe Ile Ser Met Pro 145 150 155ACA GAT GCA GTT GAA TAC GCC AGG AAG AGG ACT CCC AGG CAT GCC GGC 73 Thr Asp Ala Val Glu Tyr Ala Arg Lys Arg Thr Pro Arg His Ala Gly 130 135 140 ATC ATG ATC ACG ATC GTG TGG GTT ATA TCT GTG TTC ATC TCT ATG CCT 78 Ile Met Ile Thr Ile Val Trp Val Ile Ser Val Phe Ile Ser Met Pro 145 150 155
CCT CTC TTC TGG AGG CAC CAA GGA ACT AGC CGT GAT GAT GAG TGT GTC 82 Pro Leu Phe Trp Arg His Gin Gly Thr Ser Arg Asp Asp Glu Cys Val 160 165 170CCT CTC TTC TGG AGG CAC CAA GGA ACT AGC CGT GAT GAT GAG TGT GTC 82 Pro Leu Phe Trp Arg His Gin Gly Thr Ser Arg Asp Asp Glu Cys Val 160 165 170
ATC AAA CAT GAC CAC ATT GTT TCC ACA ATT TAC TCC ACG TTT GGA GCT 87 Ile Lys His Asp His Ile Val Ser Thr Ile Tyr Ser Thr Phe Gly Ala 175 180 185ATC AAA CAT GAC CAC ATT GTT TCC ACA ATT TAC TCC ACG TTT GGA GCT 87 Ile Lys His Asp His Ile Val Ser Thr Ile Tyr Ser Thr Phe Gly Ala 175 180 185
TTC TAC ATC CCG CTT GTA TTG ATA TTG ATC CTC TAC TAC AAA ATA TAC 92 Phe Tyr Ile Pro Leu Val Leu Ile Leu Ile Leu Tyr Tyr Lys Ile Tyr 190 195 200 205TTC TAC ATC CCG CTT GTA TTG ATA TTG ATC CTC TAC TAC AAA ATA TAC 92 Phe Tyr Ile Pro Leu Val Leu Ile Leu Ile Leu Tyr Tyr Lys Ile Tyr 190 195 200 205
AGA GCA GCA AGG ACA CTG TAC CAC AAG AGA CAA GCG AGT CGG ATG ATA 97 Arg Ala Ala Arg Thr Leu Tyr His Lys Arg Gin Ala Ser Arg Met Ile 210 215 220 AAG GAG GAG CTG AAC GGT CAA GTC TTC TTG GAG AGC GGT GAG AAG AGC 102AGA GCA GCA AGG ACA CTG TAC CAC AAG AGA CAA GCG AGT CGG ATG ATA 97 Arg Ala Ala Arg Thr Leu Tyr His Lys Arg Gin Ala Ser Arg Met Ile 210 215 220 AAG GAG GAG CTG AAC GGT CAA GTC TTC TTG GAG AGC GGT GAG AAG AGC 102
Lys Glu Glu Leu Asn Gly Gin Val Phe Leu Glu Ser Gly Glu Lys Ser 225 230 235Lys Glu Glu Leu Asn Gly Gin Val Phe Leu Glu Ser Gly Glu Lys Ser 225 230 235
ATT AAA CTG GTC TCC ACA TCC TAC ATG TTA GAA AAA TCC TTG TCC GAT 106 Ile Lys Leu Val Ser Thr Ser Tyr Met Leu Glu Lys Ser Leu Ser Asp 240 245 250ATT AAA CTG GTC TCC ACA TCC TAC ATG TTA GAA AAA TCC TTG TCC GAT 106 Ile Lys Leu Val Ser Thr Ser Tyr Met Leu Glu Lys Ser Leu Ser Asp 240 245 250
CCA TCA ACG GAC TTT GAT AGA ATT CAC AGC ACC GTG AAA AGT CCC AGA 111 Pro Ser Thr Asp Phe Asp Arg Ile His Ser Thr Val Lys Ser Pro Arg 255 260 265CCA TCA ACG GAC TTT GAT AGA ATT CAC AGC ACC GTG AAA AGT CCC AGA 111 Pro Ser Thr Asp Phe Asp Arg Ile His Ser Thr Val Lys Ser Pro Arg 255 260 265
TCG GAA CTG AAG CAT GAG AAA TCT TGG AGA AGA CAG AAA ATC TCA GGC 1164 Ser Glu Leu Lys His Glu Lys Ser Trp Arg Arg Gin Lys Ile Ser Gly 270 275 280 285TCG GAA CTG AAG CAT GAG AAA TCT TGG AGA AGA CAG AAA ATC TCA GGC 1164 Ser Glu Leu Lys His Glu Lys Ser Trp Arg Arg Gin Lys Ile Ser Gly 270 275 280 285
ACC AGA GAA CGC AAA GCA GCC ACT ACC CTG GGA TTG ATC TTG GGT GCA 1212 Thr Arg Glu Arg Lys Ala Ala Thr Thr Leu Gly Leu Ile Leu Gly Ala 290 295 300ACC AGA GAA CGC AAA GCA GCC ACT ACC CTG GGA TTG ATC TTG GGT GCA 1212 Thr Arg Glu Arg Lys Ala Ala Thr Thr Leu Gly Leu Ile Leu Gly Ala 290 295 300
TTT GTA ATA TGT TGG CTG CCC TTT TTT GTA AAA GAA TTG GTT GTT AAT 1260 Phe Val Ile Cys Trp Leu Pro Phe Phe Val Lys Glu Leu Val Val Asn 305 310 315TTT GTA ATA TGT TGG CTG CCC TTT TTT GTA AAA GAA TTG GTT GTT AAT 1260 Phe Val Ile Cys Trp Leu Pro Phe Phe Val Lys Glu Leu Val Val Asn 305 310 315
GTC TGT GAA AAA TGT AAA ATT TCT GAA GAA ATG TCA AAC TTT TTG GCA 1308 Val Cys Glu Lys Cys Lys Ile Ser Glu Glu Met Ser Asn Phe Leu Ala 320 325 330 TGG CTT GGT TAC CTG AAT TCC CTT ATA AAT CCA CTG ATT TAT ACC ATC 1356 Trp Leu Gly Tyr Leu Asn Ser Leu Ile Asn Pro Leu Ile Tyr Thr Ile 335 340 345GTC TGT GAA AAA TGT AAA ATT TCT GAA GAA ATG TCA AAC TTT TTG GCA 1308 Val Cys Glu Lys Cys Lys Ile Ser Glu Glu Met Ser Asn Phe Leu Ala 320 325 330 TGG CTT GGT TAC CTG AAT TCC CTT ATA AAT CCA CTG ATT TAT ACC ATC 1356 Trp Leu Gly Tyr Leu Asn Ser Leu Ile Asn Pro Leu Ile Tyr Thr Ile 335 340 345
TTT AAT GAA GAC TTC AAG AAA GCT TTC CAA AAA CTT GTA CGA TGC CGA 1404 Phe Asn Glu Asp Phe Lys Lys Ala Phe Gin Lys Leu Val Arg Cys Arg 350 355 360 365TTT AAT GAA GAC TTC AAG AAA GCT TTC CAA AAA CTT GTA CGA TGC CGA 1404 Phe Asn Glu Asp Phe Lys Lys Ala Phe Gin Lys Leu Val Arg Cys Arg 350 355 360 365
TAT TAGGATAAAA GAAACCTAAT TTTAAAGTGC GGAGGCTTTA TTTGTTGGGG 1457TAT TAGGATAAAA GAAACCTAAT TTTAAAGTGC GGAGGCTTTA TTTGTTGGGG 1457
TyrTyr
GGAGGGGCAG GGATAATTAA ATGAATGTAA AGTAAGAAAA CATTTAAATT TTTAGAGAAA 1517GGAGGGGCAG GGATAATTAA ATGAATGTAA AGTAAGAAAA CATTTAAATT TTTAGAGAAA 1517
ATATATTAAA ACTGCTAAAA TTAAAAAAAA AAAAAAAAAA 1557ATATATTAAA ACTGCTAAAA TTAAAAAAAA AAAAAAAAAA 1557
2) INFORMATION POUR LA SFO ID NO: ?.:2) INFORMATION FOR SFO ID NO:?.:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR: 20 paires de bases (B) TYPE: acide nucléique(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADNc(ii) TYPE OF MOLECULE: cDNA
(vi) ORIGINE: (A) ORGANISME: Sonde (i)(vi) ORIGIN: (A) ORGANIZATION: Probe (i)
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 2: AAGAATTGGT TGTTAATGTC 20(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: AAGAATTGGT TGTTAATGTC 20
(2) INFORMATION POUR LA SEQ ID NO: 3: (i) CARACTERISTIQUES DE LA SEQUENCE:(2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 19 paires de bases(A) LENGTH: 19 base pairs
(B) TYPE: acide nucléique (C) NOMBRE DE BRINS: simple(B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADNc(ii) TYPE OF MOLECULE: cDNA
(vi) ORIGINE:(vi) ORIGIN:
(A) ORGANISME: Sonde (ii)(A) ORGANIZATION: Probe (ii)
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 3: TACATTCATT TAATTATCC 19(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: TACATTCATT TAATTATCC 19
(2) INFORMATION POUR LA SEQ ID NO: 4:(2) INFORMATION FOR SEQ ID NO: 4:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 1101 paires de bases (B) TYPE: acide nucléique(A) LENGTH: 1101 base pairs (B) TYPE: nucleic acid
(C) NOMBRE DE BRINS: double(C) NUMBER OF STRANDS: double
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADNc(ii) TYPE OF MOLECULE: cDNA
(iii) HYPOTHETIQUE: NON(iii) HYPOTHETIC: NO
(iii) ANTI-SENS: NON (vi) ORIGINE:(iii) ANTI-SENSE: NO (vi) ORIGIN:
(A) ORGANISME: Homo sapiens(A) ORGANIZATION: Homo sapiens
(ix) CARACTERISTIQUE ADDITIONELLE: (A) NOM/CLE: CDS (B) EMPLACEMENT: 1..1101(ix) ADDITIONAL FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 1..1101
(D) AUTRES RENSEIGNEMENTS: /product= "Gène récepteur 5HT6 humain" É (D) OTHER INFORMATION: / product = "Human 5HT6 receptor gene" É
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 4:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
ATG GAT TTC TTA AAT TCA TCT GAT CAA AAC TTG ACC TCA GAG GAA CTG 48ATG GAT TTC TTA AAT TCA TCT GAT CAA AAC TTG ACC TCA GAG GAA CTG 48
Met Asp Phe Leu Asn Ser Ser Asp Gin Asn Leu Thr Ser Glu Glu LeuMet Asp Phe Leu Asn Ser Ser Asp Gin Asn Leu Thr Ser Glu Glu Leu
1 5 10 151 5 10 15
TTA AAC AGA ATG CCA TCC AAA ATT CTG GTG TCC CTC ACT CTG TCT GGG 96TTA AAC AGA ATG CCA TCC AAA ATT CTG GTG TCC CTC ACT CTG TCT GGG 96
Leu Asn Arg Met Pro Ser Lys Ile Leu Val Ser Leu Thr Leu Ser GlyLeu Asn Arg Met Pro Ser Lys Ile Leu Val Ser Leu Thr Leu Ser Gly
20 25 30 CTG GCA CTG ATG ACA ACA ACT ATC AAC TCC CTT GTG ATC GCT GCA ATT 144 Leu Ala Leu Met Thr Thr Thr Ile Asn Ser Leu Val Ile Ala Ala Ile 35 40 4520 25 30 CTG GCA CTG ATG ACA ACA ACT ATC AAC TCC CTT GTG ATC GCT GCA ATT 144 Leu Ala Leu Met Thr Thr Thr Ile Asn Ser Leu Val Ile Ala Ala Ile 35 40 45
ATT GTG ACC CGG AAG CTG CAC CAT CCA GCC AAT TAT TTA ATT TGT TCC 192 Ile Val Thr Arg Lys Leu His His Pro Ala Asn Tyr Leu Ile Cys Ser 50 55 60ATT GTG ACC CGG AAG CTG CAC CAT CCA GCC AAT TAT TTA ATT TGT TCC 192 Ile Val Thr Arg Lys Leu His His Pro Ala Asn Tyr Leu Ile Cys Ser 50 55 60
CTT GCA GTC ACA GAT TTT CTT GTG GCT GTC CTG GTG ATG CCC TTC AGC 240 Leu Ala Val Thr Asp Phe Leu Val Ala Val Leu Val Met Pro Phe Ser 65 70 75 80CTT GCA GTC ACA GAT TTT CTT GTG GCT GTC CTG GTG ATG CCC TTC AGC 240 Leu Ala Val Thr Asp Phe Leu Val Ala Val Leu Val Met Pro Phe Ser 65 70 75 80
ATT GTG TAT ATT GTG AGA GAG AGC TGG ATT ATG GGG CAA GTG GTC TGT 288ATT GTG TAT ATT GTG AGA GAG AGC TGG ATT ATG GGG CAA GTG GTC TGT 288
Ile Val Tyr Ile Val Arg Glu Ser Trp Ile Met Gly Gin Val Val Cys 85 90 95 GAC ATT TGG CTG AGT GTT GAC ATT ACC TGC TGC ACG TGC TCC ATC TTG 336Ile Val Tyr Ile Val Arg Glu Ser Trp Ile Met Gly Gin Val Val Cys 85 90 95 GAC ATT TGG CTG AGT GTT GAC ATT ACC TGC TGC ACG TGC TCC ATC TTG 336
Asp Ile Trp Leu Ser Val Asp Ile Thr Cys Cys Thr Cys Ser Ile Leu 100 105 110 CAT CTC TCA GCT ATA GCT TTG GAT CGG TAT CGA GCA ATC ACA GAT GCT 384Asp Ile Trp Leu Ser Val Asp Ile Thr Cys Cys Thr Cys Ser Ile Leu 100 105 110 CAT CTC TCA GCT ATA GCT TTG GAT CGG TAT CGA GCA ATC ACA GAT GCT 384
His Leu Ser Ala Ile Ala Leu Asp Arg Tyr Arg Ala Ile Thr Asp Ala 115 120 125His Leu Ser Ala Ile Ala Leu Asp Arg Tyr Arg Ala Ile Thr Asp Ala 115 120 125
GTT GAG TAT GCC AGG AAA AGG ACT CCA AAG CAT GCT GGC ATT ATG ATT 432 Val Glu Tyr Ala Arg Lys Arg Thr Pro Lys His Ala Gly Ile Met Ile 130 135 140GTT GAG TAT GCC AGG AAA AGG ACT CCA AAG CAT GCT GGC ATT ATG ATT 432 Val Glu Tyr Ala Arg Lys Arg Thr Pro Lys His Ala Gly Ile Met Ile 130 135 140
ACA ATA GTT TGG ATT ATA TCT GTT TTT ATC TCT ATG CCT CCT CTA TTC 480ACA ATA GTT TGG ATT ATA TCT GTT TTT ATC TCT ATG CCT CCT CTA TTC 480
Thr Ile Val Trp Ile Ile Ser Val Phe Ile Ser Met Pro Pro Leu Phe 145 150 155 160Thr Ile Val Trp Ile Ile Ser Val Phe Ile Ser Met Pro Pro Leu Phe 145 150 155 160
TGG AGG CAC CAA GGA ACT AGC AGA GAT GAT GAA TGC ATC ATC AAG CAC 528TGG AGG CAC CAA GGA ACT AGC AGA GAT GAT GAA TGC ATC ATC AAG CAC 528
Trp Arg His Gin Gly Thr Ser Arg Asp Asp Glu Cys Ile Ile Lys HisTrp Arg His Gin Gly Thr Ser Arg Asp Asp Glu Cys Ile Ile Lys His
165 170 175165 170 175
GAC CAC ATT GTT TCC ACC ATT TAC TCA ACA TTT GGA GCT TTC TAC ATC 576GAC CAC ATT GTT TCC ACC ATT TAC TCA ACA TTT GGA GCT TTC TAC ATC 576
Asp His Ile Val Ser Thr Ile Tyr Ser Thr Phe Gly Ala Phe Tyr Ile 180 185 190 CCA CTG GCA TTG ATT TTG ATC CTT TAC TAC AAA ATA TAT AGA GCA GCA 624Asp His Ile Val Ser Thr Ile Tyr Ser Thr Phe Gly Ala Phe Tyr Ile 180 185 190 CCA CTG GCA TTG ATT TTG ATC CTT TAC TAC AAA ATA TAT AGA GCA GCA 624
Pro Leu Ala Leu Ile Leu Ile Leu Tyr Tyr Lys Ile Tyr Arg Ala Ala 195 200 205Pro Leu Ala Leu Ile Leu Ile Leu Tyr Tyr Lys Ile Tyr Arg Ala Ala 195 200 205
AAG ACA TTA TAC CAC AAG AGA CAA GCA AGT AGG ATT GCA AAG GAG GAG 672 Lys Thr Leu Tyr His Lys Arg Gin Ala Ser Arg Ile Ala Lys Glu Glu 210 215 220AAG ACA TTA TAC CAC AAG AGA CAA GCA AGT AGG ATT GCA AAG GAG GAG 672 Lys Thr Leu Tyr His Lys Arg Gin Ala Ser Arg Ile Ala Lys Glu Glu 210 215 220
GTG AAT GGC CAA GTC CTT TTG GAG AGT GGT GAG AAA AGC ACT AAA TCA 720GTG AAT GGC CAA GTC CTT TTG GAG AGT GGT GAG AAA AGC ACT AAA TCA 720
Val Asn Gly Gin Val Leu Leu Glu Ser Gly Glu Lys Ser Thr Lys Ser 225 230 235 240Val Asn Gly Gin Val Leu Leu Glu Ser Gly Glu Lys Ser Thr Lys Ser 225 230 235 240
GTT TCC ACA TCC TAT GTA CTA GAA AAG TCT TTA TCT GAC CCA TCA ACA 768GTT TCC ACA TCC TAT GTA CTA GAA AAG TCT TTA TCT GAC CCA TCA ACA 768
Val Ser Thr Ser Tyr Val Leu Glu Lys Ser Leu Ser Asp Pro Ser ThrVal Ser Thr Ser Tyr Val Leu Glu Lys Ser Leu Ser Asp Pro Ser Thr
245 250 255245 250 255
GAC TTT GAT AAA ATT CAT AGC ACA GTG AGA AGT CTC AGG TCT GAA TTC 816GAC TTT GAT AAA ATT CAT AGC ACA GTG AGA AGT CTC AGG TCT GAA TTC 816
Asp Phe Asp Lys Ile His Ser Thr Val Arg Ser Leu Arg Ser Glu Phe 260 265 270 AAG CAT GAG AAA TCT TGG AGA AGG CAA AAG ATC TCA GGT ACA AGA GAA 864Asp Phe Asp Lys Ile His Ser Thr Val Arg Ser Leu Arg Ser Glu Phe 260 265 270 AAG CAT GAG AAA TCT TGG AGA AGG CAA AAG ATC TCA GGT ACA AGA GAA 864
Lys His Glu Lys Ser Trp Arg Arg Gin Lys Ile Ser Gly Thr Arg Glu 275 280 285Lys His Glu Lys Ser Trp Arg Arg Gin Lys Ile Ser Gly Thr Arg Glu 275 280 285
CGG AAA GCA GCC ACT ACC CTG GGA TTA ATC TTG GGT GCA TTT GTA ATA 912 Arg Lys Ala Ala Thr Thr Leu Gly Leu Ile Leu Gly Ala Phe Val Ile 290 295 300CGG AAA GCA GCC ACT ACC CTG GGA TTA ATC TTG GGT GCA TTT GTA ATA 912 Arg Lys Ala Ala Thr Thr Leu Gly Leu Ile Leu Gly Ala Phe Val Ile 290 295 300
TGT TGG CTT CCT TTT TTT GTA AAA GAA TTA GTT GTT AAT GTC TGT GAC 960TGT TGG CTT CCT TTT TTT GTA AAA GAA TTA GTT GTT AAT GTC TGT GAC 960
Cys Trp Leu Pro Phe Phe Val Lys Glu Leu Val Val Asn Val Cys Asp 305 310 315 320Cys Trp Leu Pro Phe Phe Val Lys Glu Leu Val Val Asn Val Cys Asp 305 310 315 320
AAA TGT AAA ATT TCT GAA GAA ATG TCC AAT TTT TTG GCA TGG CTT GGG 1008AAA TGT AAA ATT TCT GAA GAA ATG TCC AAT TTT TTG GCA TGG CTT GGG 1008
Lys Cys Lys Ile Ser Glu Glu Met Ser Asn Phe Leu Ala Trp Leu GlyLys Cys Lys Ile Ser Glu Glu Met Ser Asn Phe Leu Ala Trp Leu Gly
325 330 335325 330 335
TAT CTC AAT TCC CTT ATA AAT CCA CTG ATT TAC ACA ATC TTT AAT GAA 1056 Tyr Leu Asn Ser Leu Ile Asn Pro Leu Ile Tyr Thr Ile Phe Asn Glu 340 345 350 GAC TTC AAG AAA GCA TTC CAA AAG CTT GTG CGA TGT CGA TGT TA 1101TAT CTC AAT TCC CTT ATA AAT CCA CTG ATT TAC ACA ATC TTT AAT GAA 1056 Tyr Leu Asn Ser Leu Ile Asn Pro Leu Ile Tyr Thr Ile Phe Asn Glu 340 345 350 GAC TTC AAG AAA GCA TTC CAA AAG CTT GTG CGA TGT CGA TGT TA 1101
Asp Phe Lys Lys Ala Phe Gin Lys Leu Val Arg Cys Arg Cys 355 360 365Asp Phe Lys Lys Ala Phe Gin Lys Leu Val Arg Cys Arg Cys 355 360 365
2 ) INFORMATION POUR LA SEQ ID NO: 5:2) INFORMATION FOR SEQ ID NO: 5:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 37 paires de bases(A) LENGTH: 37 base pairs
(B) TYPE: acide nucléique (C) NOMBRE DE BRINS: simple(B) TYPE: nucleic acid (C) NUMBER OF STRANDS: simple
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADNc(ii) TYPE OF MOLECULE: cDNA
(vi) ORIGINE:(vi) ORIGIN:
(A) ORGANISME: Oligonucléotide 1 (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 5:(A) ORGANISM: Oligonucleotide 1 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
AAGGATCCCA GCCACCATGG ATTTCTTAAA TTCATCT 37AAGGATCCCA GCCACCATGG ATTTCTTAAA TTCATCT 37
(2 INFORMATION POUR LA SEQ ID NO: 6:(2 INFORMATION FOR SEQ ID NO: 6:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR: 37 paires de bases(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs
(B) TYPE: acide nucléique(B) TYPE: nucleic acid
(C) NOMBRE DE BRINS: simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADNc (vi) ORIGINE:(ii) TYPE OF MOLECULE: cDNA (vi) ORIGIN:
(A) ORGANISME: Oligonucléotide 2(A) ORGANISM: Oligonucleotide 2
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 6: TTCGAACACG CTACAGCTAC AATCCTCGAG GTACCAA 37 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: TTCGAACACG CTACAGCTAC AATCCTCGAG GTACCAA 37
TABLE 1TABLE 1

Claims

REVENDICATIONS
1. Polypeptide comprenant tout ou partie de la séquence peptidique SEQ ID n° 1 ou d'un dérivé de celle-ci.1. Polypeptide comprising all or part of the peptide sequence SEQ ID No. 1 or a derivative thereof.
2. Polypeptide selon la revendication 1 caractérisé en ce qu'il possède la capacité de lier la sérotonine.2. Polypeptide according to claim 1 characterized in that it has the capacity to bind serotonin.
3. Polypeptide selon la revendication 2 caractérisé en ce qu'il possède une activité de récepteur sérotoninergique.3. Polypeptide according to claim 2 characterized in that it has a serotonergic receptor activity.
4. Polypeptide selon l'une des revendications 1 à 3 caractérisé en ce qu'il peut être reconnu par des anticorps reconnaissant la séquence peptidique complète SEQ ID n° l.4. Polypeptide according to one of claims 1 to 3 characterized in that it can be recognized by antibodies recognizing the complete peptide sequence SEQ ID No. 1.
5. Polypeptide selon l'une des revendications 1 à 4 caractérisé en ce qu'il comprend toute la séquence peptidique SEQ ID n° 1.5. Polypeptide according to one of claims 1 to 4 characterized in that it comprises the entire peptide sequence SEQ ID No. 1.
6. Séquence nucléotidique codant pour un polypeptide selon l'une des revendications 1 à 5.6. Nucleotide sequence coding for a polypeptide according to one of claims 1 to 5.
7. Séquence selon la revendication 6 caractérisée en ce qu'elle est choisie parmi :7. Sequence according to claim 6 characterized in that it is chosen from:
(a) tout ou partie de la séquence nucléotidique SEQ ID n° 1 ou de son brin complémentaire,(a) all or part of the nucleotide sequence SEQ ID No. 1 or its complementary strand,
(b) toute séquence hybridant avec une séquence (a) et codant pour un polypeptide selon l'une des revendications 1 à 5, et,(b) any sequence hybridizing with a sequence (a) and coding for a polypeptide according to one of claims 1 to 5, and,
(c) les séquences dérivées des séquences (a) et (b) en raison de la dégénérescence du code génétique.(c) sequences derived from sequences (a) and (b) due to the degeneration of the genetic code.
8. Séquence selon la revendication 7 caractérisée en ce qu'elle est choisie parmi les séquences génomiques, d'ADNc, d'ARN, les séquences hybrides ou les séquences synthétiques ou semi-synthétiques.8. Sequence according to claim 7 characterized in that it is chosen from genomic, cDNA, RNA, hybrid sequences or synthetic or semi-synthetic sequences.
9. Séquence selon l'une des revendications 6 à 8 caractérisée en ce que la partie codant pour ledit polypeptide est placée sous le contrôle de signaux permettant son expression dans un hôte cellulaire. 9. Sequence according to one of claims 6 to 8 characterized in that the part coding for said polypeptide is placed under the control of signals allowing its expression in a cellular host.
10. Oligonucléotide antisens capable d'inhiber au moins partiellement la production de polypeptide selon l'une des revendications 1 à 5.10. Antisense oligonucleotide capable of at least partially inhibiting the production of polypeptide according to one of claims 1 to 5.
11. Oligonucléotide selon la revendication 10 caractérisé en ce qu'il est constitué par tout ou partie d'une séquence nucléotidique selon la revendication 7.11. Oligonucleotide according to claim 10 characterized in that it consists of all or part of a nucleotide sequence according to claim 7.
12. Sonde nucléotidique capable de s'hydrider avec une séquence selon la revendication 6 ou avec l'ARNm correspondant.12. Nucleotide probe capable of hydriding with a sequence according to claim 6 or with the corresponding mRNA.
13 Sonde selon la revendication 12 caractérisée en ce qu'elle comporte au moins 10 bases.13 A probe according to claim 12 characterized in that it comprises at least 10 bases.
14. Sonde selon la revendication 13 caractérisée en ce qu'elle comporte l'intégralité de la séquence SEQ ID n° 1 ou de son brin complémentaire.14. Probe according to claim 13 characterized in that it comprises the entire sequence SEQ ID No. 1 or its complementary strand.
15. Sonde selon la revendication 13 caractérisée en ce qu'elle est choisie parmi les séquences SEQ ID n° 2 et 3.15. A probe according to claim 13 characterized in that it is chosen from the sequences SEQ ID No. 2 and 3.
16. Utilisation d'une sonde selon l'une des revendications 12 à 15 pour la détection de l'expression d'un récepteur sérotoninergique 5HT6; ou pour la mise en évidence d'anomalies génétiques (mauvais épissage, polymoφhisme, mutations ponctuelles, etc) ; ou pour identifier des affections neurologique, cardiovasculaire ou psychiatrique comme étant liées aux récepteurs 5HT6; ou encore pour la mise en évidence et l'isolement de séquences d'acides nucléiques homologues codant pour des polypeptides 5HT6.16. Use of a probe according to one of claims 12 to 15 for the detection of the expression of a 5HT6 serotoninergic receptor; or for the highlighting of genetic anomalies (bad splicing, polymoφhism, point mutations, etc.); or to identify neurological, cardiovascular or psychiatric conditions as being linked to the 5HT6 receptors; or for the detection and isolation of homologous nucleic acid sequences encoding 5HT6 polypeptides.
17. Cellule recombinée capable d'exprimer à sa surface un polypeptide selon l'une des revendications 1 à 5.17. Recombinant cell capable of expressing on its surface a polypeptide according to one of claims 1 to 5.
18. Cellule selon la revendication 17 caractérisée en ce qu'elle est choisie parmi les cellules eucaryotes ou procaryotes.18. Cell according to claim 17 characterized in that it is chosen from eukaryotic or prokaryotic cells.
19. Procédé de mise en évidence et/ou d'isolement de ligands des polypeptides tels que définis dans les revendications 1 à 5, caractérisé en ce que l'on réalise les étapes suivantes :19. Method for detecting and / or isolating ligands of the polypeptides as defined in claims 1 to 5, characterized in that the following steps are carried out:
- on met en contact une molécule ou un mélange contenant différentes molécules, éventuellement non-identifiées, avec une cellule recombinée selon la revendication 17 exprimant à sa surface un polypeptide tel que défini dans les revendications 1 à 5 dans des conditions permettant l'interaction entre ledit polypeptide et ladite molécule dans le cas où celle-ci posséderait une affinité pour ledit polypeptide, et,- a molecule or a mixture containing different molecules, possibly unidentified, is brought into contact with a recombinant cell according to claim 17 expressing on its surface a polypeptide as defined in Claims 1 to 5 under conditions allowing interaction between said polypeptide and said molecule in the event that the latter has an affinity for said polypeptide, and,
- on détecte et/ou isole les molécules liées au dit polypeptide.- the molecules linked to said polypeptide are detected and / or isolated.
20. Procédé selon la revendication 19 pour la mise en évidence et/ou l'isolement d'agonistes ou d'antagonistes de la sérotonine.20. The method of claim 19 for the detection and / or isolation of agonists or antagonists of serotonin.
21. Procédé de mise en évidence et/ou d'isolement de modulateurs des polypeptides tels que définis dans les revendications 1 à 5, caractérisé en ce que l'on réalise les étapes suivantes : - on met en contact une molécule ou un mélange contenant différentes molécules, éventuellement non-identifiées,' avec une cellule recombinée selon la revendication 17 exprimant à sa surface un polypeptide tel que défini dans les revendications 1 à 5, en présence de 5HT, dans des conditions permettant l'interaction entre ledit polypeptide et le 5HT, et, - on détecte et/ou isole les molécules capables de moduler l'activité du 5HT sur ledit polypeptide.21. Method for detecting and / or isolating modulators of the polypeptides as defined in claims 1 to 5, characterized in that the following steps are carried out: - a molecule or a mixture containing is brought into contact different molecules, possibly unidentified, 'with a recombinant cell according to claim 17 expressing on its surface a polypeptide as defined in claims 1 to 5, in the presence of 5HT, under conditions allowing the interaction between said polypeptide and the 5HT, and, - molecules and molecules capable of modulating the activity of 5HT on said polypeptide are detected and / or isolated.
22 Ligand ou modulateur d'un polypeptide tel que défini dans les revendications 1 à 5, susceptible d'être obtenu selon les procédés des revendications 19 à 21.22 Ligand or modulator of a polypeptide as defined in claims 1 to 5, capable of being obtained according to the methods of claims 19 to 21.
23. Utilisation d'un ligand ou modulateur identifié et/ou obtenu selon les procédé des revendications 19 à 21 pour la préparation d'un médicament destiné au traitement des affections neurologique, cardiovasculaire ou psychiatrique liées aux récepteurs 5HT6.23. Use of a ligand or modulator identified and / or obtained according to the methods of claims 19 to 21 for the preparation of a medicament intended for the treatment of neurological, cardiovascular or psychiatric affections linked to 5HT6 receptors.
24. Médicament comprenant comme principe actif au moins une molécule agissant sur un polypeptide selon l'une des revendications 1 à 5.24. A medicament comprising as active ingredient at least one molecule acting on a polypeptide according to one of claims 1 to 5.
25. Médicament selon la revendication 24 caractérisé en ce que la molécule est un ligand ou un modulateur identifié et/ou isolé selon le procédé des revendications 19 à 21. 25. Medicament according to claim 24 characterized in that the molecule is a ligand or a modulator identified and / or isolated according to the method of claims 19 to 21.
EP93914777A 1992-07-01 1993-06-29 Polypeptides having serotoninergic receptor activity (5ht6), nucleic acids coding for said polypeptides, and uses thereof Withdrawn EP0651802A1 (en)

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FR9208082A FR2693201B1 (en) 1992-07-01 1992-07-01 New polypeptides having serotonergic receptor activity, nucleic acids encoding these polyptides and uses.
FR9208082 1992-07-01
PCT/FR1993/000651 WO1994001556A1 (en) 1992-07-01 1993-06-29 Polypeptides having serotoninergic receptor activity (5ht6), nucleic acids coding for said polypeptides, and uses thereof

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WO1994009828A1 (en) 1992-11-03 1994-05-11 Synaptic Pharmaceutical Corporation Dna encoding a human serotonin receptor (5-ht4b) and uses thereof
US6300087B1 (en) 1992-11-03 2001-10-09 Synaptic Pharmaceutical Corporation DNA encoding a human serotonin receptor (5-HT4B) and uses thereof
EP0946551A2 (en) * 1996-12-19 1999-10-06 Smithkline Beecham Plc N-piperazin-1-ylphenyl-benzamide derivatives

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US5155218A (en) * 1990-05-08 1992-10-13 Neurogenetic Corporation Dna encoding human 5-ht1d receptors
DE4041464A1 (en) * 1990-12-22 1992-06-25 Basf Ag 5-HT (DOWN ARROW) 1 (ARROW DOWN) RECEPTOR

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FR2693201B1 (en) 1994-08-19

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