WO1994018319A1 - Novel polypeptides having serotoninergic receptor activity, nucleic acids coding therefor and use thereof - Google Patents

Novel polypeptides having serotoninergic receptor activity, nucleic acids coding therefor and use thereof Download PDF

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Publication number
WO1994018319A1
WO1994018319A1 PCT/FR1994/000136 FR9400136W WO9418319A1 WO 1994018319 A1 WO1994018319 A1 WO 1994018319A1 FR 9400136 W FR9400136 W FR 9400136W WO 9418319 A1 WO9418319 A1 WO 9418319A1
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polypeptide
sequence
leu
polypeptides
ala
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PCT/FR1994/000136
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French (fr)
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Nourdine Amlaiky
Ursula Boschert
Régis GRAILHE
René HEN
Hans Matthes
Jean-Luc Plassat
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Institut National De La Sante Et De La Recherche Medicale
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Priority to JP6517723A priority Critical patent/JPH08506244A/en
Priority to EP94906247A priority patent/EP0683817A1/en
Publication of WO1994018319A1 publication Critical patent/WO1994018319A1/en

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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
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G01N33/942—Serotonin, i.e. 5-hydroxy-tryptamine

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 serotonin is affected 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 until now belong either to the family of receptors linked to ion channels (5-HT3 receptors), or to the family of receptors which interact with G proteins and which have seven transmembrane domains.
  • 5-HT receptors which have been described until now belong either to the family of receptors linked to ion channels (5-HT3 receptors), or to the family of receptors which interact with G proteins and which have seven transmembrane domains.
  • the 5-HT1 receptors comprising the mammalian subtypes 5HT1A, 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 existing, since pharmacological studies have revealed other subtypes such as 5HT4 receptors as well as certain receptors related to the 5HT1 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 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 5HT5b, 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. 2 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 No. 2.
  • 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 mention may be made, for example, of the chimeric polypeptides comprising an additional heterologous part linked to one end.
  • the term derivative also includes polypeptides homologous to polypeptide SEQ ID No. 2, 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.
  • 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 SEQ ID No. 2 peptide sequence.
  • a particular embodiment of the invention is represented by the polypeptide 5HT5b comprising the entire peptide sequence SEQ ID No. 2. 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 the sequence SEQ ID No. 2 using the 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 polypeptides 5HT5b.
  • the present invention also relates to any nucleotide sequence coding for a 5HT5b 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 5HT5b 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.
  • integrative vectors s 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 5HT5b 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 Hansen la.
  • 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 an mRNA, and therefore capable of specifically hybridizing with it, inhibiting its translation into protein.
  • the subject of the invention is therefore the antisense oligonucleotides capable of at least partially inhibiting the production of 5HT5b polypeptides as defined above.
  • the invention also relates to the antisense sequences capable of at least partially inhibiting the production of 5HT5b polypeptides.
  • Antisense sequences produce transcripts in the target cell that are complementary to cellular mRNAs.
  • oligonucleotides or sequences 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, by fragmentation or by chemical synthesis, etc. As indicated above, the invention also allows the production of nucleotide probes, synthetic or not, capable of hydriding with the nucleotide sequences defined above which code for polypeptides 5HT5b 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 5HT5b serotoninergic 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 5HT5b receptors. These probes can also be used for the detection and isolation of homologous nucleic acid sequences coding for 5HT5b polypeptides as defined above, from other cellular sources and preferably from cells of human origin.
  • the probes of the invention generally contain at least 10 bases, and they can contain up to the entire sequence SEQ ID No.
  • these probes are, prior to their use, marked.
  • 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 5HT5b 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 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 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 modulator of the activity of the polypeptides of the invention. More particularly, they can thus be used in a process for the detection and isolation of ligands or of modulator 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 5HT5b 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 under conditions allowing interaction between said polypeptide of the invention and said molecule if the latter 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 5HT5b polypeptides.
  • Another subject of the invention relates to a process for detecting and / or isolating modulators of the 5HT5b polypeptides of the invention, according to which the following steps are carried out: - a molecule or a mixture containing different is brought into contact molecules, possibly unidentified, with a recombinant cell as described above expressing on its surface a polypeptide of the invention, in the presence of 5HT, under conditions allowing the interaction between said polypeptide of the invention and 5HT , and, - the molecules capable of modulating the activity of 5HT on said polypeptide of the invention are detected and / or isolated.
  • 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 5HT5b receptors.
  • the invention also relates to any medicament comprising as active principle at least one molecule acting on a 5HT5b polypeptide of the invention.
  • the molecule is a ligand or a modulator identified and / or isolated according to the method described above.
  • Table 1 Pharmacological profile of the 5HT5b receptor. The results correspond to competitive experiments for the binding of [ 125 I] -LSD to the membranes of Cos-7 cells expressing the 5HT5b receptor.
  • the numbers in parentheses correspond to the number of independent experiments carried out, each point being carried out in triplicate.
  • Table 2 Percentages of peptide sequence homology between the 5HT5b receptor (SEQ ID No. 2) and other receptors of the receptor family coupled to G proteins. The homologies were calculated on the conserved sequences: transmembrane domain and its connection loops.
  • Fi ure 1 Saturation curve of [ 125 I] -LSD at the membranes of Cos-7 cells expressing the 5HT5b receptor. The membranes were incubated with ligand concentrations ranging from 50 p 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.
  • 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
  • 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 the nuclease Si.
  • PCR Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullis K.B. and Faloona F.A., Meth. Enzym. J ⁇ 5 (1987) 335-350] is carried out using a "DNA thermal cycler" (Perkin Elmer 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.
  • AGAACTAGTGGATCCAA (A / G) AA (A / G / C / T) GG (A / G / C / T) A (A / G) CCA (A / G) CA
  • CTTGATATCGAATTCGA (T / C) (A / G) T (A / G / C T) CT (A / G / Cy) TG (C /) TG (C / T) A C
  • GGTATCGATAAGCTTAT (C ⁇ , / A) GC (C /) CT (A / G / CT) GA (C , ) (C / A) G (A / G / C / T) TA
  • PCR reactions were carried out as follows: 5 ⁇ g of adult mouse brain RNA were subjected to a reverse transcription reaction in the presence of 500 ng of oligonucleotide (i) and 200 units of MMLV reverse transcriptase (BRL). Half of the product of this reaction was then subjected to 30 amplification cycles in the presence of 5 units of Taq polymerase (Cetus) and 1 ⁇ g of oligonucleotide (i) and of oligonucleotide (ii). 1/20 of the product of this reaction was then subjected to 30 additional amplification cycles in the presence of the oligonucleotides (i) and (iii).
  • the products thus obtained were digested with the enzymes BamHI and HindIII, inserted at the corresponding sites of the plas ide Bluescript (Stratagene), and sequences.
  • ⁇ NS and carried by the plasmid pNS, contained a 2.1 kb insert. This phage was isolated, and its insert was then introduced into the Bluescript plasmid.
  • sequence SEQ ID No. 1 The sequence of this fragment was determined on the 2 strands using the dideoxynucleotide technique using synthetic oligonucleotides.
  • the sequence thus obtained corresponds to sequence SEQ ID No. 1. It shows that the isolated cDNA carries an open reading phase of 370 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 1 N-glycosylation site , and the suspected cytoplasmic domain contains the consensus sites of phosphorylation by protein kinases C and A.
  • a genomic clone encoding the 5HT5b receptor was also isolated, by screening a genomic library using the sequence SEQ ID No. 1 as probe.
  • the library was obtained by partial digestion with Sau3A of the genomic DNA of mouse embryo stem cells, then insertion into the lambda phage EMBL3.
  • the fragments obtained were subcloned into the Bluescript plasmid and partially sequenced. Analysis of these sequences reveals the presence of an intron, located in the middle of the 3rd cytoplasmic loop.
  • the sequence of the 5HT5b receptor isolated above was compared with the sequences of the receptors coupled to the following G proteins: 5HT1B, 5HT1D, 5HT5A, 5HT1A, 5HT-dro2A, 5HT-drol, ⁇ 2, D2, ⁇ l, Dl, H2, 5HT1C and 5HT2.
  • 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 2.1 kb cDNA encoding the 5HT5b receptor was isolated from the plasmid pNS in the form of an EcoRI-.XhoI fragment, then inserted at the sites correspondents of vector p513.
  • the vector p513 is derived from the vector pSG5 [Green et al., Nucl. Acids Res. 16 (1988) 369] by adding a multisite of cloning.
  • the recombinant vector thus obtained, designated p513NS 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 different radiolabelled ligands (cf. Table 1). For this, the membrane samples (10-20 ⁇ g of proteins) 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.
  • the nucleotide sequence SEQ ID No. 1 was then used to demonstrate homologous sequences from other tissues by in situ hybridization.
  • the in situ hybridization experiments were carried out on cryostatic sections of the adult mouse brain (approximately 8 weeks) according to the technique described by Associates 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 T3 polymerase, of [ 35 S] -CTP using as matrix the plasmid pNS linearized by Xhol.
  • the human 5HT5b receptor was cloned.
  • 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 sequence SEQ ID No. 1.
  • the DNA fragments which hybridize with this probe were isolated, subcloned in a Bluescript plasmid, amplified, then sequenced in both directions according to the technique. dideoxynucleotide.

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Abstract

Novel 5HT5b polypeptides having serotoninergic receptor activity, genetic material for the expression thereof, any recombinant cell expressing said polypeptides, and use thereof.

Description

NOUVEAUX POLYPEPTIDES AYANT UNE ACTIVITE DE RECEPTEUR NOVEL POLYPEPTIDES HAVING RECEPTOR ACTIVITY
SEROTONINERGIQUE. ACIDES NUCLEIQUES CODANT POUR CESSEROTONINERGIC. NUCLEIC ACIDS ENCODING THESE
POLYPEPTIDES ET UTILISATIONSPOLYPEPTIDES AND USES
La présente invention concerne de nouveaux polypeptides et le matériel 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 sérotonine est édiée 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 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, 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 serotonin is affected 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 until now belong either to the family of receptors linked to ion channels (5-HT3 receptors), or to the family of receptors which interact with G proteins and which have seven transmembrane domains. Furthermore, analysis of 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, 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 (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 existing, since pharmacological studies have revealed other subtypes such as 5HT4 receptors as well as certain receptors related to the 5HT1 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 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 5HT5b, 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 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 5HT5b, 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° 2 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. 2 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° 2. 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 e.xtrêmité. Le terme dérivé comprend également les polypeptides homologues au polypeptide SEQ ID n° 2, 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.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 No. 2. 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, mention may be made, for example, of the chimeric polypeptides comprising an additional heterologous part linked to one end. The term derivative also includes polypeptides homologous to polypeptide SEQ ID No. 2, 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.
Préférentiel lement, 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 SEQ ID n° 2 complète.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 SEQ ID No. 2 peptide sequence.
Un mode de réalisation particulier de l'invention est représenté par le polypeptide 5HT5b comprenant toute la séquence peptidique SEQ ID n° 2. 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 polypeptide 5HT5b comprising the entire peptide sequence SEQ ID No. 2. 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 de la séquence SEQ ID n° 2 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 the sequence SEQ ID No. 2 using the 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 5HT5b.In what follows, the polypeptides of the invention as defined above are designated by polypeptides 5HT5b.
La présente invention a également pour objet toute séquence nucléotidique codant pour un polypeptide 5HT5b. Plus préférentiellement, il s'agit d'une séquence choisie parmi :The present invention also relates to any nucleotide sequence coding for a 5HT5b 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 5HT5b 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 intégratif s, 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 5HT5b 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 Hansen la. 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 E.coli, Bacillus, ou Streptomyces .The nucleotide sequences of the invention can be used for the production of the 5HT5b 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 the integrative vectors s, 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 5HT5b 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 Hansen la. 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 E.coli, 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, complémentaires d'un ARNm, et de ce fait capables d'hybrider spécifiquement avec celui-ci, inhibant sa traduction en protéine. L'invention a ainsi pour objet les oligonucléotides antisens capables d'inhiber au moins partiellement la production de polypeptides 5HT5b tels que définis précédemment. L'invention concerne également les séquences antisens capables d'inhiber au moins partiellement la production de polypeptides 5HT5b. Les séquences antisens produisent dans la cellule cible des transcrits qui sont complémentaires d'ARNm cellulaires. De tels oligonucléotides ou séquences 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. De tels oligonucléotides peuvent être obtenus à partir de la séquence SEQ ID n° 1, par fragmentation ou par synthèse chimique, etc. 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 5HT5b 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 5HT5b, 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 5HT5b. Ces sondes peuvent également être utilisées pour la mise en évidence et l'isolement de séquences d'acides nucléiques homologues codant pour des polypeptides 5HT5b tels que définis précédemment, à partir d'autres sources cellulaires et préférentiellement de cellules d'origines humaines. 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 de son 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.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 an mRNA, and therefore capable of specifically hybridizing with it, inhibiting its translation into protein. The subject of the invention is therefore the antisense oligonucleotides capable of at least partially inhibiting the production of 5HT5b polypeptides as defined above. The invention also relates to the antisense sequences capable of at least partially inhibiting the production of 5HT5b polypeptides. Antisense sequences produce transcripts in the target cell that are complementary to cellular mRNAs. Such oligonucleotides or sequences 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, by fragmentation or by chemical synthesis, etc. As indicated above, the invention also allows the production of nucleotide probes, synthetic or not, capable of hydriding with the nucleotide sequences defined above which code for polypeptides 5HT5b 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 5HT5b serotoninergic 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 5HT5b receptors. These probes can also be used for the detection and isolation of homologous nucleic acid sequences coding for 5HT5b polypeptides as defined above, from other cellular sources and preferably from cells of human origin. 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 its 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 5HT5b 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 5HT5b 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 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 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. Un autre objet de l'invention concerne donc un procédé de mise en évidence et/ou d'isolement de ligands des polypeptides 5HT5b de l'invention, selon lequel on réalise les étapes suivantes :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 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 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 modulator of the activity of the polypeptides of the invention. More particularly, they can thus be used in a process for the detection and isolation of ligands or of modulator 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 5HT5b polypeptides of the invention, according to which the following steps are carried out:
- on met en contact une molécule ou un mél∑inge contenant différentes molécules, éventuellement non-identifiées, avec une cellule recombinée telle que décrite ci-dessus exprimant à sa surface un polypeptide de l'invention d.ans 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 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 interaction between said polypeptide of the invention and said molecule if the latter has an affinity for said polypeptide, and,
- on détecte et/ou isole les molécules liées au dit polypeptide de l'invention.- Molecules linked to said polypeptide of the invention are detected and / or isolated.
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 5HT5b.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 5HT5b polypeptides.
Un autre objet de l'invention concerne un procédé de mise en évidence et/ou d'isolement de modulateurs des polypeptides 5HT5b de l'invention, selon lequel on réalise les étapes suivantes : - 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, - on détecte et/ou isole les molécules capables de moduler l'activité du 5HT sur ledit polypeptide de l'invention.Another subject of the invention relates to a process for detecting and / or isolating modulators of the 5HT5b polypeptides of the invention, according to which the following steps are carried out: - a molecule or a mixture containing different is brought into contact molecules, possibly unidentified, with a recombinant cell as described above expressing on its surface a polypeptide of the invention, in the presence of 5HT, under conditions allowing the interaction between said polypeptide of the invention and 5HT , and, - the 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 5HT5b.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 5HT5b receptors.
L'invention concerne également tout médicament comprenant comme principe actif au moins une molécule agissant sur un polypeptide 5HT5b de l'invention. Préférentiellement la molécule est un ligand ou un modulateur identifié et/ou isolé selon le procédé décrit précédemment.The invention also relates to any medicament comprising as active principle at least one molecule acting on a 5HT5b polypeptide of the invention. Preferably, the molecule is a ligand or a modulator identified and / or isolated according to the method described above.
D'autres avantages de la présente invention apparaîtront à la lecture des exemples qui suivent, qui doivent être considérés comme illustratifs et non limitatifs.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
Table 1 : Profil pharmacologique du récepteur 5HT5b. Les résultats correspondent à des expériences de compétition pour la liaison du [125I]-LSD aux membranes des cellules Cos-7 exprimant le récepteur 5HT5b. 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/O + C/Kd) dans laquelle C est la concentration en [125I]-LSD (150 pM) et Kd est la constante de dissociation du [125I]-LSD. Les nombres entre parenthèses correspondent au nombre d'expériences indépendantes réalisées, chaque point étant réalisé en triple.Table 1: Pharmacological profile of the 5HT5b receptor. The results correspond to competitive experiments for the binding of [ 125 I] -LSD to the membranes of Cos-7 cells expressing the 5HT5b receptor. 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 / O + C / Kd) in which C is the concentration of [ 125 I] -LSD (150 pM) and Kd is the dissociation constant of [ 125 I] -LSD. The numbers in parentheses correspond to the number of independent experiments carried out, each point being carried out in triplicate.
Table 2 : Pourcentages d'homologie de séquence peptidique entre le récepteur 5HT5b (SEQ ID n° 2) et d'autres récepteurs de la famille des récepteuι*s couplés à des protéines G. Les homologies ont été calculées sur les séquences conservées : le domaine transmembranaire et ses boucles de connection. Fi ure 1 : Courbe de saturation du [125I]-LSD aux membranes des cellules Cos-7 exprimant le récepteur 5HT5b. Les membranes ont été incubées avec des concentrations de ligand allant de 50 p 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.Table 2: Percentages of peptide sequence homology between the 5HT5b receptor (SEQ ID No. 2) and other receptors of the receptor family coupled to G proteins. The homologies were calculated on the conserved sequences: transmembrane domain and its connection loops. Fi ure 1: Saturation curve of [ 125 I] -LSD at the membranes of Cos-7 cells expressing the 5HT5b receptor. The membranes were incubated with ligand concentrations ranging from 50 p 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.
Techniques générales de clonageGeneral 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., "Molecular Cloning, a Laboratory anual", 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, precipitation of DNA 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., "Molecular Cloning, a Laboratory anual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel FM 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.Restriction enzymes have been supplied by New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham and are used as recommended by suppliers.
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.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 p-ar un traitement ménagé par la nucléase Si.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 the nuclease Si.
La mutagénèse dirigée in vitro par oligodéoxynucléotides synthétiques est effectuée selon la méthode développée par Taylor et al. [Nucleic Acids Res. 12 (1985) 8749-8764] en utilisant le kit distribué par Amersham. L'amplification enzymatique de fragments d'ADN par la technique dite deMutagenesis directed in vitro by synthetic oligodeoxynucleotides is carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 12 (1985) 8749-8764] using the kit distributed by Amersham. The enzymatic amplification of DNA fragments by the technique known as
PCR [Polymérase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354 ; Mullis K.B. et Faloona F.A., Meth. Enzym. J^5 (1987) 335-350] est effectuée en utilisant un "DNA thermal cycler" (Perkin Elmer 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.PCR [Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullis K.B. and Faloona F.A., Meth. Enzym. J ^ 5 (1987) 335-350] is carried out using a "DNA thermal cycler" (Perkin Elmer 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 5HT5b1. Isolation of the 5HT5b receptor
Les comparaisons de séquences entre les différents récepteurs sérotoni¬ nergiques connus font apparaître une certaine conservation, particulièrement dans certaines régions transmembranaires potentielles telles que les domaines III et VI. Dans le but de mettre en évidence et d'isoler un nouveau récepteur, trois oligonucléotides dégénérés correspondant à ces deux régions ont été préparés, puis utilisés dans une série de réactions de PCR sur une préparation d'ARN de cerveau de souris. La séquence des oligonucléotides dégénérés est la suivante :The comparisons of sequences between the various known serotonic receptors reveal a certain conservation, particularly in certain potential transmembrane regions such as domains III and VI. In order to identify and isolate a new receptor, three degenerate oligonucleotides corresponding to these two regions were prepared, then used in a series of PCR reactions on a preparation of mouse brain RNA. The sequence of degenerate oligonucleotides is as follows:
Oligonucléotide (i)Oligonucleotide (i)
AGAACTAGTGGATCCAA(A/G)AA(A/G/C/T)GG(A/G/C/T)A(A/G)CCA(A/G)CAAGAACTAGTGGATCCAA (A / G) AA (A / G / C / T) GG (A / G / C / T) A (A / G) CCA (A / G) CA
Oligonucléotide (ii)Oligonucleotide (ii)
CTTGATATCGAATTCGA(T/C)(A/G)T(A/G/C T)CT(A/G/Cy )TG(C/ )TG(C/T)A CCTTGATATCGAATTCGA (T / C) (A / G) T (A / G / C T) CT (A / G / Cy) TG (C /) TG (C / T) A C
Oligonucléotide (iii)Oligonucleotide (iii)
GGTATCGATAAGCTTAT(Cπ,/A)GC(C/ )CT(A/G/C T)GA(C ,)(C/A)G(A/G/C/ T)TAGGTATCGATAAGCTTAT (Cπ , / A) GC (C /) CT (A / G / CT) GA (C , ) (C / A) G (A / G / C / T) TA
Les réactions de PCR ont été réalisées de la manière suivante : 5 μg d'ARN de cerveau de souris adulte ont été soumis à une réaction de transcription inverse en présence de 500 ng d'oligonucléotide (i) et de 200 unités de transcriptase inverse MMLV (BRL). La moitié du produit de cette réaction a ensuite été soumise à 30 cycles d'amplification en présence de 5 unités de polymérase Taq (Cetus) et de 1 μg d'oligonucléotide (i) et d'oligonucléotide (ii). l/20e du produit de cette réaction a ensuite été soumis à 30 cycles d'amplification supplémentaires en présence des oligonucléotides (i) et (iii). Les produits ainsi obtenus ont été digérés avec les enzymes BamHI et HindIII, insérés aux sites correspondant du plas ide Bluescript (Stratagène), et séquences. L'un des fragments ainsi obtenus, présentant une certaine homologie avec les récepteurs sérotoninergiques, a été marqué par "random priming" (Feinberg et Vogelstein, Analytical Biochemistry 132 (1984) 6) et utilisé comme sonde pour cribler une banque de cDNA de cerveau de souris construite dans le phage UniZap (Stratagène). Parmi les phages positifs obtenus, l'un d'entre-eux, dénommé λNS et porté par le plasmide pNS, contenait un insert de 2,1 kb. Ce phage a été isolé, et son insert 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 correspond à la séquence SEQ ID n° 1. Elle montre que l'ADNc isolé porte une phase de lecture ouverte de 370 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 1 site de N-glycosylation, et le domaine cytoplasmique présumé contient les sites consensus de phosphorylation par les protéines kinases C et A.The PCR reactions were carried out as follows: 5 μg of adult mouse brain RNA were subjected to a reverse transcription reaction in the presence of 500 ng of oligonucleotide (i) and 200 units of MMLV reverse transcriptase (BRL). Half of the product of this reaction was then subjected to 30 amplification cycles in the presence of 5 units of Taq polymerase (Cetus) and 1 μg of oligonucleotide (i) and of oligonucleotide (ii). 1/20 of the product of this reaction was then subjected to 30 additional amplification cycles in the presence of the oligonucleotides (i) and (iii). The products thus obtained were digested with the enzymes BamHI and HindIII, inserted at the corresponding sites of the plas ide Bluescript (Stratagene), and sequences. One of the fragments thus obtained, having a certain homology with the serotonergic receptors, was marked by "random priming" (Feinberg and Vogelstein, Analytical Biochemistry 132 (1984) 6) and used as probe to screen a cDNA library of brain mouse constructed in the UniZap phage (Stratagene). Among the positive phages obtained, one of them, called λNS and carried by the plasmid pNS, contained a 2.1 kb insert. This phage was isolated, and its insert was then introduced into the Bluescript plasmid. The sequence of this fragment was determined on the 2 strands using the dideoxynucleotide technique using synthetic oligonucleotides. The sequence thus obtained corresponds to sequence SEQ ID No. 1. It shows that the isolated cDNA carries an open reading phase of 370 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 1 N-glycosylation site , and the suspected cytoplasmic domain contains the consensus sites of phosphorylation by protein kinases C and A.
Un clone génomique codant pour le récepteur 5HT5b a également été isolé, par criblage d'une banque génomique au moyen de la séquence SEQ ID n° 1 comme sonde. La banque avait été obtenue par digestion partielle par Sau3A de l'ADN génomique de cellules souches d'embryon de souris, puis insertion dans le phage lambda EMBL3.A genomic clone encoding the 5HT5b receptor was also isolated, by screening a genomic library using the sequence SEQ ID No. 1 as probe. The library was obtained by partial digestion with Sau3A of the genomic DNA of mouse embryo stem cells, then insertion into the lambda phage EMBL3.
Les fragments obtenus ont été sous clones dans le plasmide Bluescript et partiellement séquences. L'analyse de ces séquences révèle la présence d'un intron, localisé au milieu de la 3ème boucle cytoplasmique.The fragments obtained were subcloned into the Bluescript plasmid and partially sequenced. Analysis of these sequences reveals the presence of an intron, located in the middle of the 3rd cytoplasmic loop.
2. Etude d'homologies de séquence2. Study of sequence homologies
La séquence du récepteur 5HT5b isolé ci-dessus a été comparée avec les séquences des récepteurs couplés à des protéines G suivants : 5HT1B, 5HT1D, 5HT5A, 5HT1A, 5HT-dro2A, 5HT-drol, α2, D2, βl, Dl, H2, 5HT1C et 5HT2. Ces expériences ont révélé une certaine homologie dans le domaine transmembranaire potentiel et dans certaines boucles, mais aucune homologie dans les régions terminales ni dans la troisième boucle cytoplasmique. La table 2 donne les % d'homologie au niveau des régions conservées.The sequence of the 5HT5b receptor isolated above was compared with the sequences of the receptors coupled to the following G proteins: 5HT1B, 5HT1D, 5HT5A, 5HT1A, 5HT-dro2A, 5HT-drol, α2, D2, βl, Dl, H2, 5HT1C and 5HT2. These experiments revealed some homology in the potential transmembrane domain and in certain loops, but no homology in the terminal regions or in the third cytoplasmic loop. Table 2 gives the% homology at the level of the regions conserved.
3. Expression du récepteur 5HT5b dans les cellules Cos-7 et caractérisation pharmacologique3. Expression of the 5HT5b 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 2,1 kb codant pour le récepteur 5HT5b a été isolé à partir du plasmide pNS 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. 16 (1988) 369] par addition d'un multisite de clonage. Le vecteur recombinant ainsi obtenu désigné p513NS a ensuite été utilisé (20 μg par plaque de 10 cm) pour transfecter les cellules Cos-7 en présence de phosphate de calcium.The 2.1 kb cDNA encoding the 5HT5b receptor was isolated from the plasmid pNS in the form of an EcoRI-.XhoI fragment, then inserted at the sites correspondents of vector p513. The vector p513 is derived from the vector pSG5 [Green et al., Nucl. Acids Res. 16 (1988) 369] by adding a multisite of cloning. The recombinant vector thus obtained, designated p513NS, 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 de différents ligands radiomarqués (cf. Table 1). 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 different radiolabelled ligands (cf. Table 1). For this, the membrane samples (10-20 μg of proteins) 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 le [125I]-LSD présente un site de liaison saturable avec un Kd = 470 pM et un Bmax = 170 fmol/mg de protéines membranaires (figure 1). 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.The results obtained show that the [ 125 I] -LSD has a saturable binding site with a Kd = 470 pM and a Bmax = 170 fmol / mg of membrane proteins (FIG. 1). 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 : ergotamine > 5-CT > methysergide > 5HT > RU24969 > 8-OH-DPAT > yohimbine > bufotenine (table 1). La kétansérine, le propanolol (-), le sumatriptan, la dopamine et la norépinéphrine sont inactifs.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: ergotamine>5-CT>methysergide>5HT>RU24969>8-OH-DPAT>yohimbine> bufotenine (table 1). Ketanserine, propanolol (-), sumatriptan, dopamine and norepinephrine are inactive.
4. Recherche de séquences homologues dans d'autres tissus4. 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 par hybridation in situ.The nucleotide sequence SEQ ID No. 1 was then used to demonstrate homologous sequences from other tissues by in situ hybridization.
Les expériences d'hybridation in situ ont été réalisées sur des sections cryostatées de cerveau de souris 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 T3, de [35S]-CTP en utilisant comme matrice le plasmide pNS linéarisé par Xhol .The in situ hybridization experiments were carried out on cryostatic sections of the adult mouse 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 T3 polymerase, of [ 35 S] -CTP using as matrix the plasmid pNS linearized by Xhol.
Cette étude a permis de mettre en évidence des séquences homologues selon l'invention dans l'hippocampe (région CA1), le corps d'habenula et le raphé dorsal.This study made it possible to demonstrate homologous sequences according to the invention in the hippocampus (region CA1), the body of habenula and the dorsal raphe.
5. Isolement du récepteur humain5. Isolation of the human receptor
Selon la méthodologie décrite en 4. ci-dessus, le récepteur 5HT5b humain a été clone.According to the methodology described in 4. above, the human 5HT5b 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 au moyen de la séquence SEQ ID n° 1. 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.The library thus obtained was then screened using the sequence SEQ ID No. 1. The DNA fragments which hybridize with this probe were isolated, subcloned in a Bluescript plasmid, amplified, then sequenced in both directions according to the technique. dideoxynucleotide.
La séquence obtenue est présentée sur la séquence SEQ ID n° 3.The sequence obtained is presented on the sequence SEQ ID No. 3.
Il est entendu que les même expériences peuvent être répétées en utilisant d'autres tissus, et notamment des tissus d'origine humaine, d'autres sondes et d'autres techniques (PCR, hybridation en Northern blot. 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. It is understood that the same experiments can be repeated using other tissues, and in particular tissues of human origin, other probes and other techniques (PCR, hybridization in Northern blot. In addition, the homologous sequences used Evidently during these experiments can obviously then be isolated and / or amplified by conventional molecular biology techniques.
TABLE 1TABLE 1
LIGAND pKiLIGAND pKi
5HT5b Cellules Cos-75HT5b Cos-7 cells
5-HT 6.6 (3)5-HT 6.6 (3)
5-CT 7.4 (3)5-CT 7.4 (3)
RU 24969 6.4 (2)RU 24969 6.4 (2)
TFMPP 5.4 (2)TFMPP 5.4 (2)
8-OHDPAT 6.4 (2)8-OHDPAT 6.4 (2)
Sumatriptan 5.1 (3)Sumatriptan 5.1 (3)
Bufotenine 5.8 (2)Bufotenine 5.8 (2)
Methysergide 6.9 (2)Methysergide 6.9 (2)
Ergotamine 8.5 (2)Ergotamine 8.5 (2)
2-Bromo LSD2-Bromo LSD
Methiothepin 7.8 (3)Methiothepin 7.8 (3)
Yohimbine 6.0 (2)Yohimbine 6.0 (2)
(±)Pindolol(±) Pindolol
(-)Propanolol 5.2 (2)(-) Propanolol 5.2 (2)
Ketanserin 5.8 (2)Ketanserin 5.8 (2)
SpiperoneSpiperone
Dopa ine <5 (2)Dopa ine <5 (2)
(-)Norepin <5 (2)(-) Norepin <5 (2)
TABLE 2TABLE 2
5HT5B souris 1005HT5B mouse 100
5HT5A souris 775HT5A mouse 77
5HTlBβ souris 375HTlBβ mouse 37
5HTlDα humain 375HTlDα human 37
5HTlEα souris 375HTlEα mouse 37
5HTlEβ souris 365HTlEβ mouse 36
5HT1 A humain 365HT1 A human 36
5HT-dro2A 415HT-dro2A 41
5HT-drol 38 o2 humain 345HT-drol 38 o2 human 34
D2 souris 36D2 mouse 36
H2 chien 26 βl humain 30 β2 souris 29H2 dog 26 human βl 30 β2 mouse 29
Dl humain 31Human dl 31
5HT1C souris 305HT1C mouse 30
5HT2 souris 26 LISTE DE SEQUENCES5HT2 mouse 26 LIST OF SEQUENCES
(! ) INFORMATION GENERALE:(! ) GENERAL INFORMATION:
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(ii) TITRE DE L' INVENTION: NOUVEAUX POLYPEPTIDES AYANT UNE ACTIVITE DE RECEPTEURS SEROTONINERGIQUES, ACIDES NUCLEIQUES CODANT POUR CES POLYPEPTIDES ET UTILISATIONS.(ii) TITLE OF THE INVENTION: NOVEL POLYPEPTIDES HAVING SEROTONINERGIC RECEPTOR ACTIVITY, NUCLEIC ACIDS ENCODING SUCH POLYPEPTIDES AND USES THEREOF.
(iii) NOMBRE DE SEQUENCES: 3(iii) NUMBER OF SEQUENCES: 3
(iv) FORME LISIBLE PAR ORDINATEUR: (A) TYPE DE SUPPORT: Tape(iv) FORM READABLE BY COMPUTER: (A) TYPE OF SUPPORT: Tape
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(2) INFORMATION POUR LA SEQ ID NO: 1 :(2) INFORMATION FOR SEQ ID NO: 1:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR: 2036 paires de bases (B) TYPE: acide nucléique(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2036 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 (iii) HYPOTHETIQUE: NON (iii) ANTI-SENS: NON (vi) ORIGINE:(ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (vi) ORIGIN:
(A) ORGANISME: SOURIS(A) ORGANIZATION: MOUSE
(ix) CARACTERISTIQUEADDITIONELLE: (A) NOM/CLE: CDS (B) EMPLACEMENT: 312..1424(ix) ADDITIONAL FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 312..1424
(xi) DESCRIPTION DELASEQUENCE: SEQIDNO: 1: GAΛTTCGGCA CGAGCAGGCΛ CCAGTCCCCA ΛTCCTCTGCA GTCGGTTACC CTGAAGACCA 60(xi) DESCRIPTION OF THE BASIS: SEQIDNO: 1: GAΛTTCGGCA CGAGCAGGCΛ CCAGTCCCCA ΛTCCTCTGCA GTCGGTTACC CTGAAGACCA 60
CAAAGGGACT GΛGAGΛTTGΛ TGCGCTGGGC ΛΛAGCTGGΛC TAΛGGAGTCT CATCTGGAAA 120CAAAGGGACT GΛGAGΛTTGΛ TGCGCTGGGC ΛΛAGCTGGΛC TAΛGGAGTCT CATCTGGAAA 120
AGAGGGTCCΛ TGAAAAAΛGC CAAAAGAAGC GCCAAAΛGGA GGCTGCAGTT TGGAAAAGGG 180 ACAAGGGTGG CGCGGTTTGG ACATCTTTTT GCGTAGCTGG GCGCGCGGAG TGCCTCTCTT 240AGAGGGTCCΛ TGAAAAAΛGC CAAAAGAAGC GCCAAAΛGGA GGCTGCAGTT TGGAAAAGGG 180 ACAAGGGTGG CGCGGTTTGG ACATCTTTTT GCGTAGCTGG GCGCGCGGAG TGCCTCTCTT 240
GCTTCGCCAC CTΛTCCACΛG TTCATGCAAC CACGGGCACA TCTCCTGCCC CAGAGCCCAA 300GCTTCGCCAC CTΛTCCACΛG TTCATGCAAC CACGGGCACA TCTCCTGCCC CAGAGCCCAA 300
GTCCCTGAAT Λ ATG GAA GTT TCT AAC CTC TCA GGC GCC ACT CCC GGC CTT 350 Met Glu Val Ser Asn Leu Ser Gly Ala Thr Pro Gly Leu 1 5 10 GCC TTT CCT CCG GGA CCT GAG AGC TGC AGT GAC AGC CCA AGT TCC GGC 398 Ala Phe Pro Pro Gly Pro Glu Ser Cys Ser Asp Ser Pro Ser Ser Gly 15 20 25GTCCCTGAAT Λ ATG GAA GTT TCT AAC CTC TCA GGC GCC ACT CCC GGC CTT 350 Met Glu Val Ser Asn Leu Ser Gly Ala Thr Pro Gly Leu 1 5 10 GCC TTT CCT CCG GGA CCT GAG AGC TGC AGT GAC AGC CCA AGT TCC GGC 398 Ala Phe Pro Pro Gly Pro Glu Ser Cys Ser Asp Ser Pro Ser Ser Gly 15 20 25
AGG AGC ATG GGA TCC ACC CCA GGT GGG CTC ATC TTG CCC GGC CGC GAG 446 Arg Ser Met Gly Ser Thr Pro Gly Gly Leu Ile Leu Pro Gly Arg Glu 30 35 40 45AGG AGC ATG GGA TCC ACC CCA GGT GGG CTC ATC TTG CCC GGC CGC GAG 446 Arg Ser Met Gly Ser Thr Pro Gly Gly Leu Ile Leu Pro Gly Arg Glu 30 35 40 45
CCG CCC TTC TCT GCT TTC ACC GTG CTT GTG GTG ACT CTA CTG GTG TTG 494 Pro Pro Phe Ser Ala Phe Thr Val Leu Val Val Thr Leu Leu Val Leu 50 55 60CCG CCC TTC TCT GCT TTC ACC GTG CTT GTG GTG ACT CTA CTG GTG TTG 494 Pro Pro Phe Ser Ala Phe Thr Val Leu Val Val Thr Leu Leu Val Leu 50 55 60
CTG ATC GCT GCC ACT TTC TTΛ TGG ΛΛT CTG CTA GTT CTG GTG ACT ATC 542 Leu Ile Ala Ala Thr Phe Leu Trp Asn Leu Leu Val Leu Val Thr Ile 65 70 75CTG ATC GCT GCC ACT TTC TTΛ TGG ΛΛT CTG CTA GTT CTG GTG ACT ATC 542 Leu Ile Ala Ala Thr Phe Leu Trp Asn Leu Leu Val Leu Val Thr Ile 65 70 75
CTG CGC GTC CGC GCC TTC CAC CGC GTG CCA CΛT AAC TTG GTG GCC TCG 590 Leu Arg Val Arg Ala Phe H s Arg Val Pro His Asn Leu Val Ala Ser 80 85 90 ACA GCC GTC TCG GΛT GTC CTG GTG GCG GTT CTG GTG ATG CCT CTG AGC 638 Thr Ala Val Ser Asp Val Leu Val Ala Val Leu Val Met Pro Leu Ser 95 100 105CTG CGC GTC CGC GCC TTC CAC CGC GTG CCA CΛT AAC TTG GTG GCC TCG 590 Leu Arg Val Arg Ala Phe H s Arg Val Pro His Asn Leu Val Ala Ser 80 85 90 ACA GCC GTC TCG GΛT GTC CTG GTG GCG GTT CTG GTG ATG CCT CTG AGC 638 Thr Ala Val Ser Asp Val Leu Val Ala Val Leu Val Met Pro Leu Ser 95 100 105
CTG GTG AGC GAG TTG TCC GCT GGG CGA CGT TGG CAG CTA GGC AGG AGT 686 Leu Val Ser Glu Leu Ser Ala Gly Arg Arg Trp Gin Leu Gly Arg Ser 110 115 120 125CTG GTG AGC GAG TTG TCC GCT GGG CGA CGT TGG CAG CTA GGC AGG AGT 686 Leu Val Ser Glu Leu Ser Ala Gly Arg Arg Trp Gin Leu Gly Arg Ser 110 115 120 125
CTG TGC CAC GTG TGG ATC TCC TTC GAC GTG TTG TGC TGC ACC GCC AGC 734 Leu Cys His Val Trp Ile Ser Phe Asp Val Leu Cys Cys Thr Ala Ser 130 135 140CTG TGC CAC GTG TGG ATC TCC TTC GAC GTG TTG TGC TGC ACC GCC AGC 734 Leu Cys His Val Trp Ile Ser Phe Asp Val Leu Cys Cys Thr Ala Ser 130 135 140
ATC TGG AAC GTG GCG GCC ΛTC GCC CTG GΛT CGC TAC TGG ACT ATC ACG 782 Ile Trp Asn Val Ala Ala Ile Λla Leu Asp Λrg Tyr Trp Thr Ile Thr 145 150 155ATC TGG AAC GTG GCG GCC ΛTC GCC CTG GΛT CGC TAC TGG ACT ATC ACG 782 Ile Trp Asn Val Ala Ala Ile Λla Leu Asp Λrg Tyr Trp Thr Ile Thr 145 150 155
CGC CAC CTG CAG TAC ACG CTG CGC ΛCC CGG AGC CGT GCT TCT GCG CTC 830 Λrg His Leu Gin Tyr Thr Leu Λrg Thr Λrg Ser Arg Ala Ser Ala Leu 160 165 170 ATG ATC GCG ΛTC ΛCC TGG GCΛ CTG TCC GCG CTC ATT GCT CTC GCC CCG 878 Met Ile Ala Ile Thr Trp Λla Leu Ser Ala Leu Ile Ala Leu Ala Pro 175 180 185CGC CAC CTG CAG TAC ACG CTG CGC ΛCC CGG AGC CGT GCT TCT GCG CTC 830 Λrg His Leu Gin Tyr Thr Leu Λrg Thr Λrg Ser Arg Ala Ser Ala Leu 160 165 170 ATG ATC GCG ΛTC ΛCC TGG GCΛ CTG TCC GCG CTC ATT GCT CTC GCC CCG 878 Met Ile Ala Ile Thr Trp Λla Leu Ser Ala Leu Ile Ala Leu Ala Pro 175 180 185
CTG CTT TTT GGC TGG GGC GAA GCC TΛT GΛT GCT CGG CTG CAG CGT TGC 926 Leu Leu Phe Gly Trp Gly Glu Λla Tyr Λsp Ala Arg Leu Gin Arg Cys 190 195 200 205CTG CTT TTT GGC TGG GGC GAA GCC TΛT GΛT GCT CGG CTG CAG CGT TGC 926 Leu Leu Phe Gly Trp Gly Glu Λla Tyr Λsp Ala Arg Leu Gin Arg Cys 190 195 200 205
CAG GTG AGC CΛG GΛG CCC TCC TΛT CCT GTC TTC TCC ACC TGC GGA GCC 974 Gin Val Ser Gin Glu Pro Ser Tyr Λla Val Phe Ser Thr Cys Gly Ala 210 215 220CAG GTG AGC CΛG GΛG CCC TCC TΛT CCT GTC TTC TCC ACC TGC GGA GCC 974 Gin Val Ser Gin Glu Pro Ser Tyr Λla Val Phe Ser Thr Cys Gly Ala 210 215 220
TTC TΛC CTG CCT CTA GCG GTG GTG CTC TTC GTC TAC TGG AAA ATA TAC 1022 Phe Tyr Leu Pro Leu Ala Val Val Leu Phe Val Tyr Trp Lys Ile Tyr 225 230 235TTC TΛC CTG CCT CTA GCG GTG GTG CTC TTC GTC TAC TGG AAA ATA TAC 1022 Phe Tyr Leu Pro Leu Ala Val Val Leu Phe Val Tyr Trp Lys Ile Tyr 225 230 235
AAA GCC GCC AAG TTT CGA TTC GGT CGC AGA CGG CGG GCG GTG GTA CCG 107 Lys Ala Ala Lys Phe Arg Phe Gly Arg Arg Arg Arg Ala Val Val Pro 240 245 250AAA GCC GCC AAG TTT CGA TTC GGT CGC AGA CGG CGG GCG GTG GTA CCG 107 Lys Ala Ala Lys Phe Arg Phe Gly Arg Arg Arg Arg Ala Val Val Pro 240 245 250
CTT CCT GCC ACC ACG CAG GCA AAG GAA GCA CCT CCG GAG TCT GAG ATG 111 Leu Pro Ala Thr Thr Gin Ala Lys Glu Ala Pro Pro Glu Ser Glu Met 255 260 265CTT CCT GCC ACC ACG CAG GCA AAG GAA GCA CCT CCG GAG TCT GAG ATG 111 Leu Pro Ala Thr Thr Gin Ala Lys Glu Ala Pro Pro Glu Ser Glu Met 255 260 265
GTG TTC ACA GCC CGT CGC CGA GCA ACA GTG ACC TTC CAG ACA AGC GGA 116 Val Phe Thr Ala Arg Arg Arg Ala Thr Val Thr Phe Gin Thr Ser Gly 270 275 280 285 GAC TCC TGG CGG GAG CAG AAG GAG AAG CGG GCA GCC ATG ATG GTC GGG 1214 Asp Ser Trp Arg Glu Gin Lys Glu Lys Arg Ala Ala Met Met Val Gly 290 295 300GTG TTC ACA GCC CGT CGC CGA GCA ACA GTG ACC TTC CAG ACA AGC GGA 116 Val Phe Thr Ala Arg Arg Arg Ala Thr Val Thr Phe Gin Thr Ser Gly 270 275 280 285 GAC TCC TGG CGG GAG CAG AAG GAG AAG CGG GCA GCC ATG ATG GTC GGG 1214 Asp Ser Trp Arg Glu Gin Lys Glu Lys Arg Ala Ala Met Met Val Gly 290 295 300
ATC TTG ATT GGC GTG TTT GTG CTT TGT TGG ATC CCC TTC TTC CTG ACG 1262 Ile Leu Ile Gly Val Phe Val Leu Cys Trp Ile Pro Phe Phe Leu Thr 305 310 315ATC TTG ATT GGC GTG TTT GTG CTT TGT TGG ATC CCC TTC TTC CTG ACG 1262 Ile Leu Ile Gly Val Phe Val Leu Cys Trp Ile Pro Phe Phe Leu Thr 305 310 315
GAG CTC ATC AGC CCG CTC TGT GCC TGC AGC CTG CCA CCC ATC TGG AAA 1310 Glu Leu Ile Ser Pro Leu Cys Ala Cys Ser Leu Pro Pro Ile Trp Lys 320 325 330GAG CTC ATC AGC CCG CTC TGT GCC TGC AGC CTG CCA CCC ATC TGG AAA 1310 Glu Leu Ile Ser Pro Leu Cys Ala Cys Ser Leu Pro Pro Ile Trp Lys 320 325 330
AGC ATA TTC CTG TGG CTT GGA TAT TCC AAT TCG TTC TTC AAC CCC TTG 1358 Ser Ile Phe Leu Trp Leu Gly Tyr Ser Asn Ser Phe Phe Asn Pro Leu 335 340 345AGC ATA TTC CTG TGG CTT GGA TAT TCC AAT TCG TTC TTC AAC CCC TTG 1358 Ser Ile Phe Leu Trp Leu Gly Tyr Ser Asn Ser Phe Phe Asn Pro Leu 335 340 345
ATT TAC ACT GCC TTT AAT AAG AAT TAC AAC AAT GCC TTC AAG AGC CTC 1406ATT TAC ACT GCC TTT AAT AAG AAT TAC AAC AAT GCC TTC AAG AGC CTC 1406
Ile Tyr Thr Ala Phe Asn Lys Asn Tyr Asn Asn Ala Phe Lys Ser Leu 350 355 360 365 TTT ACT AAG CAG AGA TAAGAAGGGC TGGGGAGAGA AAAGGGAAGA CCTGGGAAGA 1461 Phe Thr Lys Gin Arg 370Ile Tyr Thr Ala Phe Asn Lys Asn Tyr Asn Asn Ala Phe Lys Ser Leu 350 355 360 365 TTT ACT AAG CAG AGA TAAGAAGGGC TGGGGAGAGA AAAGGGAAGA CCTGGGAAGA 1461 Phe Thr Lys Gin Arg 370
GAAAGGGGAC CTGTGATCCT CATTTCACCC GAGACCTTCT CCCCACCACC CACGGCCCGA 1521GAAAGGGGAC CTGTGATCCT CATTTCACCC GAGACCTTCT CCCCACCACC CACGGCCCGA 1521
TGATGACACT CCAAAAGTCA TACCATTGGC CCTGGACGTT GAAGAGTTCT CATGGGGGTT 1581TGATGACACT CCAAAAGTCA TACCATTGGC CCTGGACGTT GAAGAGTTCT CATGGGGGTT 1581
GGCAACTCTT TGCCAAACAC ACCCATGCCT TCTACAGAAG GACGTGACAG ACATTATTAG 1641 TGAATTGTGC TCCATTTCTG CACTAGGCAG AAACCCTGCC AAACACTTTC AGGATAGATT 1701GGCAACTCTT TGCCAAACAC ACCCATGCCT TCTACAGAAG GACGTGACAG ACATTATTAG 1641 TGAATTGTGC TCCATTTCTG CACTAGGCAG AAACCCTGCC AAACACTTTC AGGATAGATT 1701
TTAGGTATTG GTGAGCATTT GTTAGACCTC ACATGTGACA AAGTTGATTT GCTTTTCCAT 1761TTAGGTATTG GTGAGCATTT GTTAGACCTC ACATGTGACA AAGTTGATTT GCTTTTCCAT 1761
TATTTAGTAC TGTGTCCTCT CTCAGGAGTT TCCTGAGTCT GTCTCCTTGA CACAGCCTCT 1821TATTTAGTAC TGTGTCCTCT CTCAGGAGTT TCCTGAGTCT GTCTCCTTGA CACAGCCTCT 1821
CCTCTATCCC TTATCCATCA GAGGAGTTTC CCTTTCTTAG CCTCCTATAC AACCTCCACA 1881CCTCTATCCC TTATCCATCA GAGGAGTTTC CCTTTCTTAG CCTCCTATAC AACCTCCACA 1881
GGACΛATGAT TCTCAGCTTA GAΛGCGGGCC TCACCTGAAG CTTTGATAAA ACGTGTTCCA 1941 CGCAGGTGTC TTCAAGATGG CTCAGTAGGT GAAGGCACCT GCCCCTGAGC CTGGTGGCCT 2001GGACΛATGAT TCTCAGCTTA GAΛGCGGGCC TCACCTGAAG CTTTGATAAA ACGTGTTCCA 1941 CGCAGGTGTC TTCAAGATGG CTCAGTAGGT GAAGGCACCT GCCCCTGAGC CTGGTGGCCT 2001
GCTTTTGΛTG GAGΛCACACG TAATGGAAGG AAAGG 2036GCTTTTGΛTG GAGΛCACACG TAATGGAAGG AAAGG 2036
(2) INFORMATION POURLA SEQID NO: 2:(2) INFORMATION FOR SEQID NO: 2:
(i) CARACTERISTIQUES DELA SEQUENCE: (A) LONGUEUR: 370 acides amin.s(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 370 amino acids
(B) TYPE: acide amin,(B) TYPE: amino acid,
(D) CONFIGURATION: lin, aire (ii) TYPE DE MOLECULE: prot.ine(D) CONFIGURATION: linen, area (ii) TYPE OF MOLECULE: prot.ine
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 2:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
Met Glu Val Ser Asn Leu Ser Gly Ala Thr Pro Gly Leu Ala Phe Pro 1 5 1 0 1 5Met Glu Val Ser Asn Leu Ser Gly Ala Thr Pro Gly Leu Ala Phe Pro 1 5 1 0 1 5
Pro Gly Pro Glu Ser Cys Ser Asp Ser Pro Ser Ser Gly Arg Ser Met 20 25 30 Gly Ser Thr Pro Gly Gly Leu Ile Leu Pro Gly Arg Glu Pro Pro Phe 35 40 45Pro Gly Pro Glu Ser Cys Ser Asp Ser Pro Ser Ser Gly Arg Ser Met 20 25 30 Gly Ser Thr Pro Gly Gly Leu Ile Leu Pro Gly Arg Glu Pro Pro Phe 35 40 45
Ser Ala Phe Thr Val Leu Val Val Thr Leu Leu Val Leu Leu Ile Ala 50 55 60Ser Ala Phe Thr Val Leu Val Val Thr Leu Leu Val Leu Leu Ile Ala 50 55 60
Ala Thr Phe Leu Trp Asn Leu Leu Val Leu Val Thr Ile Leu Arg ValAla Thr Phe Leu Trp Asn Leu Leu Val Leu Val Thr Ile Leu Arg Val
65 70 75 8065 70 75 80
Arg Ala Phe His Arg Val Pro His Asn Leu Val Ala Ser Thr Ala Val 85 90 95Arg Ala Phe His Arg Val Pro His Asn Leu Val Ala Ser Thr Ala Val 85 90 95
Ser Asp Val Leu Val Ala Val Leu Val Met Pro Leu Ser Leu Val Ser 100 105 110 Glu Leu Ser Ala Gly Arg Arg Trp Gin Leu Gly Arg Ser Leu Cys His 115 120 125Ser Asp Val Leu Val Ala Val Leu Val Met Pro Leu Ser Leu Val Ser 100 105 110 Glu Leu Ser Ala Gly Arg Arg Trp Gin Leu Gly Arg Ser Leu Cys His 115 120 125
Val Trp Ile Ser Phe Asp Val Leu Cys Cys Thr Ala Ser Ile Trp AsnVal Trp Ile Ser Phe Asp Val Leu Cys Cys Thr Ala Ser Ile Trp Asn
130 135 140130 135 140
Val Ala Ala Ile Ala Leu Asp Arg Tyr Trp Thr Ile Thr Arg His LeuVal Ala Ala Ile Ala Leu Asp Arg Tyr Trp Thr Ile Thr Arg His Leu
145 150 155 160145 150 155 160
Gin Tyr Thr Leu Arg Thr Arg Ser Arg Ala Ser Ala Leu Met Ile Ala 165 170 175Gin Tyr Thr Leu Arg Thr Arg Ser Arg Ala Ser Ala Leu Met Ile Ala 165 170 175
Ile Thr Trp Ala Leu Ser Ala Leu Ile Ala Leu Ala Pro Leu Leu Phe 180 185 190 Gly Trp Gly Glu Λla Tyr Asp Ala Λrg Leu Gin Arg Cys Gin Val Ser 195 200 205Ile Thr Trp Ala Leu Ser Ala Leu Ile Ala Leu Ala Pro Leu Leu Phe 180 185 190 Gly Trp Gly Glu Λla Tyr Asp Ala Λrg Leu Gin Arg Cys Gin Val Ser 195 200 205
Gin Glu Pro Ser Tyr Ala Val Phe Ser Thr Cys Gly Ala Phe Tyr Leu 210 215 220Gin Glu Pro Ser Tyr Ala Val Phe Ser Thr Cys Gly Ala Phe Tyr Leu 210 215 220
Pro Leu Ala Val Val Leu Phe Val Tyr Trp Lys Ile Tyr Lys Ala Ala 225 230 235 240Pro Leu Ala Val Val Leu Phe Val Tyr Trp Lys Ile Tyr Lys Ala Ala 225 230 235 240
Lys Phe Arg Phe Gly Arg Arg Λrg Arg Ala Val Val Pro Leu Pro Ala 245 250 255Lys Phe Arg Phe Gly Arg Arg Λrg Arg Ala Val Val Pro Leu Pro Ala 245 250 255
Thr Thr Gin Ala Lys Glu Λla Pro Pro Glu Ser Glu Met Val Phe Thr 260 265 270 Ala Arg Arg Arg Λla Thr Val Thr Phe Gin Thr Ser Gly Asp Ser Trp 275 280 285 Arg Glu Gin Lys Glu Lys Arg Ala Ala Met Met Val Gly Ile Leu Ile 290 295 300Thr Thr Gin Ala Lys Glu Λla Pro Pro Glu Ser Glu Met Val Phe Thr 260 265 270 Ala Arg Arg Arg Λla Thr Val Thr Phe Gin Thr Ser Gly Asp Ser Trp 275 280 285 Arg Glu Gin Lys Glu Lys Arg Ala Ala Met Met Val Gly Ile Leu Ile 290 295 300
Gly Val Phe Val Leu Cys Trp Ile Pro Phe Phe Leu Thr Glu Leu Ile 305 310 315 320Gly Val Phe Val Leu Cys Trp Ile Pro Phe Phe Leu Thr Glu Leu Ile 305 310 315 320
Ser Pro Leu Cys Ala Cys Ser Leu Pro Pro Ile Trp Lys Ser Ile Phe 325 330 335 Leu Trp Leu Gly Tyr Ser Asn Ser Phe Phe Asn Pro Leu Ile Tyr Thr 340 345 350Ser Pro Leu Cys Ala Cys Ser Leu Pro Pro Ile Trp Lys Ser Ile Phe 325 330 335 Leu Trp Leu Gly Tyr Ser Asn Ser Phe Phe Asn Pro Leu Ile Tyr Thr 340 345 350
Ala Phe Asn Lys Asn Tyr Asn Asn Ala Phe Lys Ser Leu Phe Thr Lys 355 360 365Ala Phe Asn Lys Asn Tyr Asn Asn Ala Phe Lys Ser Leu Phe Thr Lys 355 360 365
Gin Arg 370Gin Arg 370
(2) INFORMATION POUR LA SEQ ID NO: 3:(2) INFORMATION FOR SEQ ID NO: 3:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(B) TYPE: acide nucléique(B) TYPE: nucleic acid
(C) NOMBRE DE BRINS: double (D) CONFIGURATION: linéaire(C) NUMBER OF STRANDS: double (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: ADNg (iii) HYPOTHETIQUE: NON (iii) ANTI-SENS: NON (vi) ORIGINE:(ii) TYPE OF MOLECULE: gDNA (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (vi) ORIGIN:
(A) ORGANISME: Homo Sapiens(A) ORGANIZATION: Homo Sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 3: \ΕEAPDEAEV\O AHCKATNSFQVSGDSWREQKERRAAMMVGILIGVFVLCWIP FFLTELISPLCACSLPPIWKSIFLWLGYSΝSFFΝPLIYTAFΝKΝYΝΝAFKSLFTKQ R*(xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: \ ΕEAPDEAEV \ O AHCKATNSFQVSGDSWREQKERRAAMMVGILIGVFVLCWIP FFLTELISPLCACSLPPIWKSIFLWLGYSΝSFFΝPLIYTAFΝKΝYΝΝAFKSLFTKQ R *
GTAAAGGAAGCACCTGATGAGGCTGAAGTGGTGTTCACGGCACATTGCAAAGCAACGGTG TCCTTCCAGGTGAGCGGGGACTCCTGGCGGGAGCAGAAGGAGAGGCGAGCAGCCATGATG GTGGGGATTCTGATTGGCGTGTTTGTGCTGTGCTGGATCCCCTTCTTCCTGACGGAACTC ATCAGCCCACTCTGTGCCTGCAGCCTGCCCCCCATCTGGAAAAGCATATTTCTGTGGCTT GGCTACTCCAATTCTTTCTTCAACCCCCTGATTTACACAGCTTTTAACAAGAACTACAAC AATGCCTTCAAGAGCCTCTTTACTAAGCAGAGATGA GTAAAGGAAGCACCTGATGAGGCTGAAGTGGTGTTCACGGCACATTGCAAAGCAACGGTG TCCTTCCAGGTGAGCGGGGACTCCTGGCGGGAGCAGAAGGAGAGGCGAGCAGCCATGATG GTGGGGATTCTGATTGGCGTGTTTGTGCTGTGCTGGATCCCCTTCTTCCTGACGGAACTC ATCAGCCCACTCTGTGCCTGCAGCCTGCCCCCCATCTGGAAAAGCATATTTCTGTGGCTT GGCTACTCCAATTCTTTCTTCAACCCCCTGATTTACACAGCTTTTAACAAGAACTACAAC AATGCCTTCAAGAGCCTCTTTACTAAGCAGAGATGA

Claims

REVENDICATIONS
1. Polypeptide comprenant tout ou partie de la séquence peptidique SEQ ID n° 2 ou d'un dérivé de celle-ci.1. Polypeptide comprising all or part of the peptide sequence SEQ ID No. 2 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 SEQ ID n° 2 complète.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. 2.
5. Polypeptide selon l'une des revendications 1 à 4 caractérisé en ce qu'il comprend toute la séquence peptidique SEQ ID n° 2 ou SEQ ID n° 3.5. Polypeptide according to one of claims 1 to 4 characterized in that it comprises the entire peptide sequence SEQ ID No. 2 or SEQ ID No. 3.
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. Séquence antisens capable d'inhiber au moins partiellement la production de polypeptides selon l'une des revendications 1 à 5.10. Antisense sequence capable of at least partially inhibiting the production of polypeptides according to one of claims 1 to 5.
11. Séquence selon la revendication 10 caractérisée en ce qu'elle est constituée par tout ou partie d'une séquence nucléotidique selon la revendication 7.11. Sequence 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. Utilisation d'une sonde selon l'une des revendications 12 à 13 pour la détection de l'expression d'un récepteur sérotoninergique 5HT5b; ou pour la mise en évidence d'anomalies génétiques (mauvais épissage, polymorphisme, mutations ponctuelles, etc); ou pour identifier des affections neurologique, cardiovasculaire ou psychiatrique comme étant liées aux récepteurs 5HT5b; ou encore pour la mise en évidence et l'isolement de séquences d'acides nucléiques homologues codant pour des polypeptides 5HT5b.15. Use of a probe according to one of claims 12 to 13 for the detection of the expression of a 5HT5b serotoninergic receptor; or for highlighting genetic anomalies (poor splicing, polymorphism, point mutations, etc.); or to identify neurological, cardiovascular or psychiatric conditions as being linked to the 5HT5b receptors; or also for the detection and isolation of homologous nucleic acid sequences coding for 5HT5b polypeptides.
16. Cellule recombinée capable d'exprimer à sa surface un polypeptide selon l'une des revendications 1 à 5.16. Recombinant cell capable of expressing on its surface a polypeptide according to one of claims 1 to 5.
17. Cellule selon la revendication 16 caractérisée en ce qu'elle est choisie parmi les cellules eucaryotes ou procaryotes.17. Cell according to claim 16 characterized in that it is chosen from eukaryotic or prokaryotic cells.
18. 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 : - on met en contact une molécule ou un mélange contenant différentes molécules, éventuellement non-identifiées, avec une cellule recombinée selon la revendication 16 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, - on détecte et/ou isole les molécules liées au dit polypeptide.18. 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: - a molecule or a mixture containing is brought into contact different molecules, possibly unidentified, with a recombinant cell according to claim 16 expressing on its surface a polypeptide as defined in claims 1 to 5 under conditions allowing the interaction between said polypeptide and said molecule in the case where the latter ci would have an affinity for said polypeptide, and, - the molecules linked to said polypeptide are detected and / or isolated.
19. Procédé selon la revendication 18 pour la mise en évidence et/ou l'isolement d'agonistes ou d'antagonistes de la sérotonine.19. The method of claim 18 for the detection and / or isolation of agonists or antagonists of serotonin.
20. 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 :20. 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:
- on met en contact une molécule ou un mélange contenant différentes molécules, éventuellement non-identifiées, avec une cellule recombinée selon la revendication 16 exprimant à sa surface un polypeptide tel que défini dans les revendications 1 à 5, en présence de 5HT, dans des conditions permett.ant l'interaction entre ledit polypeptide et le 5HT, et,- a molecule or mixture containing different molecules, possibly unidentified, is brought into contact with a recombinant cell according to claim 16 expressing on its surface a polypeptide as defined in claims 1 to 5, in the presence of 5HT, in conditions allowing the interaction between said polypeptide and 5HT, and,
- on détecte et/ou isole les molécules capables de moduler l'activité du 5HT sur ledit polypeptide.- Molecules capable of modulating the activity of 5HT on said polypeptide are detected and / or isolated.
21. Ligand ou modulateur d'un polypeptide tel que défini dans les revendications 1 à 5, susceptible d'être obtenu selon les procédés des revendications21. Ligand or modulator of a polypeptide as defined in claims 1 to 5, capable of being obtained according to the methods of the claims
18 à 20.18 to 20.
22. Utilisation d'un ligand ou modulateur identifié et/ou obtenu selon les procédés des revendications 18 à 20 pour la préparation d'un médicament destiné au traitement des affections neurologique, cardiovasculaire ou psychiatrique liées aux récepteurs 5HT5b.22. Use of a ligand or modulator identified and / or obtained according to the methods of claims 18 to 20 for the preparation of a medicament intended for the treatment of neurological, cardiovascular or psychiatric conditions linked to the 5HT5b receptors.
23. Médicament comprenant comme principe actif au moins une molécule agissant sur un polypeptide selon l'une des revendications 1 à 5.23. A medicament comprising as active ingredient at least one molecule acting on a polypeptide according to one of claims 1 to 5.
24. Médicament selon la revendication 23 caractérisé en ce que la molécule est un ligand ou un modulateur identifié et/ou isolé selon le procédé des revendications 18 à 20. 24. Medicament according to claim 23 characterized in that the molecule is a ligand or a modulator identified and / or isolated according to the method of claims 18 to 20.
PCT/FR1994/000136 1993-02-09 1994-02-07 Novel polypeptides having serotoninergic receptor activity, nucleic acids coding therefor and use thereof WO1994018319A1 (en)

Priority Applications (2)

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JP6517723A JPH08506244A (en) 1993-02-09 1994-02-07 Novel polypeptides having serotonin-like receptor activity, nucleic acids encoding these polypeptides, and uses
EP94906247A EP0683817A1 (en) 1993-02-09 1994-02-07 Novel polypeptides having serotoninergic receptor activity, nucleic acids coding therefor and use thereof

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FR9301392A FR2701265B1 (en) 1993-02-09 1993-02-09 New polypeptides having serotoninergic receptor activity, nucleic acids encoding these polypeptides and uses.
FR93/01392 1993-02-09

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EP0791660A1 (en) * 1996-02-22 1997-08-27 Smithkline Beecham Corporation A novel diagnostic marker for splicing variants of genes associated with neurological function
WO2000041472A2 (en) * 1999-01-11 2000-07-20 Knoll Aktiengesellschaft Binding partners for 5-ht5-receptors for the treatment of migraine
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J-L PLASSAT ET AL: "The mouse 5HT5 receptor reveals a remarkable heterogeneity within the 5HT1D receptor family", EMBO JOURNAL., vol. 11, no. 13, 1992, EYNSHAM, OXFORD GB, pages 4779 - 4786 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0791660A1 (en) * 1996-02-22 1997-08-27 Smithkline Beecham Corporation A novel diagnostic marker for splicing variants of genes associated with neurological function
WO2000041472A2 (en) * 1999-01-11 2000-07-20 Knoll Aktiengesellschaft Binding partners for 5-ht5-receptors for the treatment of migraine
WO2000041472A3 (en) * 1999-01-11 2001-10-11 Knoll Ag Binding partners for 5-ht5-receptors for the treatment of migraine
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US10167636B2 (en) 2014-06-26 2019-01-01 Onduline Method of designing a corrugated sheet and corregated sheet obtained

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FR2701265B1 (en) 1995-04-07
JPH08506244A (en) 1996-07-09
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CA2153162A1 (en) 1994-08-18
FR2701265A1 (en) 1994-08-12

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