EP1112365A2 - Recepteur edg6 (gene de differentiation de cellules endotheliales) humain et murin couple a des proteines g et son utilisation - Google Patents

Recepteur edg6 (gene de differentiation de cellules endotheliales) humain et murin couple a des proteines g et son utilisation

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Publication number
EP1112365A2
EP1112365A2 EP99948712A EP99948712A EP1112365A2 EP 1112365 A2 EP1112365 A2 EP 1112365A2 EP 99948712 A EP99948712 A EP 99948712A EP 99948712 A EP99948712 A EP 99948712A EP 1112365 A2 EP1112365 A2 EP 1112365A2
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European Patent Office
Prior art keywords
edg6
receptor
human
protein
fragments
Prior art date
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Withdrawn
Application number
EP99948712A
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German (de)
English (en)
Inventor
Markus GRÄLER
Günter Bernhardt
Martin Lipp
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Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
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Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
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Priority claimed from DE19846979A external-priority patent/DE19846979A1/de
Application filed by Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft filed Critical Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Publication of EP1112365A2 publication Critical patent/EP1112365A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the G protein-coupled receptor EDG6 and its fragments, variants and mutations and its use. Fields of application of the invention are molecular biology, pharmacy and medicine.
  • G protein coupled receptors The superfamily of G protein coupled receptors (GPRs) comprises several hundred proteins. Their main task in the organism is to pass on information from the extracellular environment into the cell interior through interaction with heterotrimeric guanine nucleotide-binding proteins, which are commonly referred to as G proteins (Dohlman et al., 1987). This influences the regulatory processes within cells (Böhm et al., 1997).
  • GPRs for hormones and neurotransmitters are known, for paracrine substances and inflammatory mediators, for certain proteases, for a large number of taste and odor molecules, and for photons and calcium ions (Watson and Arkinstall, 1994).
  • the EDG (endothelial differentiation gene) receptor family belongs to the group of G-protein coupled receptors (GPRs). The first member of this family, EDG1, was cloned in 1990 as part of an investigation into the nonproliferative aspects of angiogenesis in the organization and differentiation of endothelial cells in capillaries.
  • EDG2 Another member of the EDG receptor family, occurs primarily in cortical neurogenic regions.
  • the cDNA of a third member of the EDG receptor family was isolated from human placenta, kidney and liver as well as from human heart and localized on chromosome 9q22.1-2.
  • Human EDG4 cDNA transcripts have recently been detected from the testes, prostate, pancreas and peripheral blood leukocytes and to a lesser extent in the thymus and spleen. Two other transcripts were isolated from smooth muscle cells of the rat aorta or from murine and bovine taste buds.
  • Lysophosphatidyl acid (LPA; l-acyl-2-hydroxy-s ⁇ -glycero-3-phosphate) was identified as a possible ligand for the human and murine EDG2 receptor and for the human EDG4 receptor. Furthermore, lysosphingolipids have been found as functional ligands for the human receptors EDG1 and EDG3 and for the H218 receptor of the rat.
  • the invention was based on the task of isolating a further member of the EDG receptor family, identifying them and making them usable for medical use.
  • EDG6 EDG receptor
  • the human EDG6 receptor comprises 384 amino acids of sequence 1 with seven transmembrane domains.
  • the receptor has one possible N-terminal glycosylation site, three possible palmitoylation sites located 12 to 15 amino acids C-terminal from the seventh transmembrane domain and four possible C-terminal protein kinase C phosphorylation sites.
  • a model of the receptor is shown in Figure 1.
  • the EDG6 receptor is the first member of the EDG family to be isolated from in vitro differentiated human dendritic cells. It shows a signal at around 1.7 kb in the Northern blot analysis.
  • Human edg6 mRNA is expressed in the Burkitt lymphoma cell lines JBL2, BL64 and DG75, in the promyelocytic cell line U937 and in the T cell line CEM.
  • High tissue specific expression rates of human edg ⁇ was found in human adult and fetal spleens as well as in adult peripheral leukocytes and the lungs.
  • Lower expression rates of human & dg6 were detected in adult thymus, lymph nodes, bone marrow and appendix as well as in fetal liver, thymus and lungs.
  • the scope of the invention also includes the cDNA sequence of the EDG6 receptor with sequence 2.
  • a murine clone was identified that has high homologies to the newly identified EDG6 receptor.
  • the murine cDNA clone from the EST (expressed-sequence tag) nucleic acid database comes from lymph nodes and is at amino acid level after a correction of the database entry, which leads to a change in the reading frame, over 106 amino acids to 88% homologous to the human EDG6 receptor. It follows that this cDNA clone is a fragment of the murine homologue to the human EDG6 receptor.
  • sequence 3 the entire murine cDNA sequence (sequence 3) was determined with the aid of a special polymerase chain reaction.
  • the protein derived from this cDNA sequence has sequence 4.
  • Specific anti-EDG-6 antibodies are also claimed. They are made by immunizing rats with a GST fusion protein containing a fragment of the EDG6 receptor. With the help of the spleen cells of the rat, hybridomas are produced which are examined for the production of specific antibodies by means of ELISA analysis. Additional possibilities for obtaining antibodies are immunization with whole cells which express the receptor EDG6 after the introduction of the cDNA or already naturally, and with a C-terminal 6 x histidine-coupled N-terminal EDG6 fragment.
  • EDG6-deficient mice are produced, i.e. Mice containing non-functional mutants (zero mutant ') of the EDG6. These knock out mice serve as an animal model for diseases that may be associated with the EDG6 receptor. The characterization of the phenotype of these mice can contribute decisively to the function determination.
  • a non-functional edg ⁇ gene with appropriate selection markers is integrated into the genome of murine embryonic stem cells (ES cells) using homologous recombination. The selected ES cells are then used as multipotent cells in murine embryos of early cell development (morula, blastocyst).
  • diseases can be identified that are caused directly or indirectly by the EDG6 receptor or are increased in their extent.
  • diseases include immune deficiencies, for example, against infections, and diseases based on acute and chronic inflammation.
  • This also includes autoimmune diseases, allergies and malignant diseases such as tumors, leukaemias and lymphomas.
  • the human receptor EDG6 according to the invention can be directly or indirectly involved in the occurrence of immune deficiencies, acute and chronic inflammation, autoimmune diseases, allergies and malignant diseases or increase their severity and extent.
  • the invention also relates to the use of the EDG6 nucleic acids and EDG6 polypeptides in their original, modified or synthetic form as a starting basis for the development of pharmaceutically relevant substances.
  • the EDG6 nucleic acids are used for the construction of genes and vectors, and the EDG6 polypeptides for the construction of Elisa methods, diameter and test methods.
  • the pharmaceutically relevant substances either bind themselves to the receptor polypeptide or the receptor-coding nucleic acid or they influence the binding of the physiological ligand or the binding and activity of intracellular downstream signaling molecules and thereby lead to an activation or inhibition of the receptor function.
  • These substances with an agonistic or antagonistic effect on the function of the EDG6 receptor can be organic molecules, inorganic molecules and peptides or combinations of these classes of substances.
  • the invention enables medical use for the following diagnostic or therapeutic measures.
  • diagnosis can be carried out, for example using test kits based on monoclonal antibodies or nucleic acid detection methods.
  • the functions of the receptor can be influenced in an agonistic or antagonistic manner.
  • the EDG6 receptor in its original or modified form as well as specific antibodies or binding partners can be used for therapeutic purposes, for example for gene therapy methods (for example on a cellular, liposomal or viral basis) if a malfunction of the receptor or incorrect expression of the EDG6 receptor or its ligand is present.
  • gene therapy methods for example on a cellular, liposomal or viral basis
  • PCR polymerase chain reaction
  • TM transmembrane domains
  • the possible 1560 bp full-length cDNA was gradually cloned using 5 'and 3' RACE PCR.
  • the cDNA contains an open reading frame of 1155 bp, a 22 bp 5'-untranslated region and a 383 bp 3'-untranslated region.
  • the cDNA at the 3 'end may not be complete because it does not have a typical polyadenylation signal.
  • Sequence 1 shows the resulting amino acid sequence. Sequence comparisons indicate that the newly identified receptor belongs to the EDG family of GPCRs. Therefore it was called EDG6.
  • EDG6 has 46% identity to EDG3, 44% to EDG1, 39% to EDG4 and 37% to EDG2.
  • the next related GPCR is hCBlR, a member of the cannabinoid receptor family, with 31% identity.
  • Computer-aided analyzes showed the possible localization of the seven transmembrane domains, a possible N-glycosylation site in the N-terminal extracellular region and several post-translational modification sites in the C-terminal cytoplasmic domain can be determined. Furthermore, the correct orientation of the molecule in the cell membrane with the N-terminus on the extracellular side was investigated.
  • the protein-encoding cDNA sequence was cloned into a eukaryotic expression vector while maintaining the reading frame, the C-terminal of the cloned-in sequence expressing a j ⁇ yc epitope.
  • the fusion molecule could only be detected in permeabilized cells by means of flow cytometry using an anti-jnyc-specific monoclonal antibody.
  • the last 50 bp of the human edg ⁇ (hedg ⁇ ) cDNA are identical to the base pairs 13 to 62 of a short sequence that contains the repetitive dinucleotide polymorphism D19S120.
  • This polymorphism was located on chromosome 19pl3.3.
  • a PCR with gene-specific primers of hedg6 cDNA and the D19S120 amplicon was able to amplify a human genomic DNA fragment which contains the 3 'end of hedg ⁇ and the D19S120 polymorphism. This shows that hedg ⁇ is located on chromosome 19pl3.3 next to the D19S120 marker.
  • the murine homolog of the edg ⁇ (medg ⁇ ) cDNA could be isolated with the help of RACE-PCR.
  • Total RNA of the dendritic cell line 18 originating from murine fetal skin was used for this.
  • Gene-specific primers were produced which originate from the murine EST sequence of the cDNA clone val6c04.rl (GenBank entry no. AA254425) and have a high identity to the 3 'end of the coding region of the hedg ⁇ cDNA.
  • the primers were chosen so that the open reading frame of medg ⁇ could be amplified. Therefore, the medg ⁇ cDNA is incomplete at the 3 'end. It contains an open reading frame of 1161 bp.
  • the first 99 bp of the 499 bp ⁇ '-untranslated region contain a murine repetitive element B1.
  • the open reading frame of the medg ⁇ cDNA is 80% homologous to the corresponding human sequence. At the protein level, both sequences have an identity of 82% and a similarity of 91%. The possible post-translational modification sites are conserved both in the human and in the murine e g6 sequence.
  • DNA fragments were produced which represent regions with low conservation in the murine and in the human cDNA sequence. These fragments were then used as radiolabelled probes in Northern blots.
  • a hedg ⁇ -specific signal was found at around 1.7 kb in human cell lines, hedg ⁇ itiRNA is expressed in the Burkitt lymphoma cell lines JBL2, BL64 and DG75, in the promyelocytic cell line U937 and in the T cell line CEM, while it is expressed in the Throat cancer cell line HEp2 and the HEp2 subclone cl32 and in the cervical carcinoma cell line HeLa could not be detected.
  • hedg ⁇ is weakly expressed in all positive cell lines tested and can only be demonstrated by prolonged exposure times of the blots. Due to the high specificity of the hybridization samples, it was possible to determine the tissue-specific expression of hedg ⁇ with mRNA samples from 50 different human tissues using a dot blot. High hedg ⁇ expression rates were found in human adult and fetal spleens, as well as in adult peripheral leukocytes and the lungs. Lower hedg ⁇ expression rates were detected in adult thymus, lymph nodes, bone marrow and appendix as well as in fetal liver, thymus and lungs.
  • tissue-specific expression of medg ⁇ mRNA agrees very well with the human expression pattern within the examined organs. Hybridization signals were found in murine lungs, spleen, thymus and lymph nodes while they did not appear in non-lymphatic tissue.
  • the murine edg ⁇ mRNA is approximately 2.1 kb in size and thus 0.4 kb larger than the human edg ⁇ mRNA. material and methods
  • Peripheral blood mononuclear cells were obtained from fresh primary blood cells (buffy coats) by means of density centrifugation.
  • 10 ml of the fresh primary blood cells were mixed in four 50 ml Falcon tubes with 20 ml PBS each.
  • the PBS was mixed with 5 U / ml heparin.
  • This mixture was underlaid with 10 ml of Ficoll separation solution from Biochrom and centrifuged at 200 x g for 20 min.
  • the top 20 to 25 ml of the mixture was then removed.
  • the rest of the mixture which was still contaminated with thrombocytes, was then centrifuged once more for 20 min at 460 x g.
  • the interphase formed from all Falcon tubes was collected and washed three times for 15 min at 300 ⁇ g with ice-cold PBS mixed with 1 mM EDTA in order to largely avoid contamination with platelets.
  • peripheral mononuclear blood cells were placed together with 15 ml of RPMI medium in 3 sterile Petri dishes with a diameter of 10 cm and cultured for 2 hours in a CO 2 incubator at 37 ° C. The bottom of the petri dish was then carefully washed several times with the RPMI medium using a glass pipette from all sides, with a large part of the non-adherent cells detaching from the bottom. The non-adherent cells were discarded together with the medium. 15 ml of fresh RPMI medium, prewarmed to 37 ° C., which now contained 800 U / ml GM-CSF and 1000 U / ml IL-4, were then added to each Petri dish.
  • the medium was refreshed three more times every other day. 7.5 ml of the medium were removed from each Petri dish and replaced with new RPMI medium, which now contained 1600 U / ml GM-CSF and 1000 U / ml IL-4. The cells were harvested on the 7th day of cell culture.
  • the Burkitt lymphoma cell lines BL64 and DG75 as well as the lymphoblastoid T cell line CEM and the promyelocytic Cell line U937 was cultured in RPMI1640 medium with 10% fetal calf serum, the larynx cancer cell line HEp2 and the HEp2 subclone cl32 and the human embryonic kidney cell line HEK293 were cultured in DMEM medium with 10% fetal calf serum.
  • RNA was prepared with the TRIzol reagent from Gibco BRL according to the protocol supplied. The preparation of mRNA was carried out using the "Micro mRNA Purification Kit” from Pharmacia Biotech on the basis of the enclosed documents.
  • RNA was carried out according to the capillary blot method, which enables a directed transfer of RNA fragments by ion migration.
  • a glass plate which was about as wide as the gel to be blotted, was placed across a dish filled with 20x SSC buffer.
  • Two filter papers which had the length of the gel to be blotted and were wide enough to protrude across the glass pane with both projecting ends deep into the bowl filled with buffer, were impregnated with 20x SSC buffer and stacked on top of one another as described put the glass pane.
  • the RNA gel was placed on top of it with a precise fit and free of air bubbles.
  • the nitrocellulose membrane which was the size of the gel and was previously placed in water for 10 minutes and then in 20 ⁇ SSC buffer, was placed on the gel. Since the gel still contained a considerable amount of formaldehyde, the blot was set up under the hood. A water-impermeable plastic mask was placed on the nitrocellulose membrane, which sealed off the edges of the blot around the membrane. Two other filter papers the size of the nitrocellulose membrane were soaked in 20x SSC buffer and also placed in a precise fit and free of air bubbles. A stack of dry paper towels formed the top of the structure. Weighed down with a weight of about 0.5 kg blotted for about 2 days. The RNA was fixed at 80 ° C for 2 hours.
  • a 32P-labeled cloned human or murine edg6 cDNA fragment was used for the hybridization.
  • the labeling reaction was carried out using the "Random Primed Labeling Kit” from Gibco BRL according to their instructions.
  • the human RNA master blot from Clontech was hybridized and washed in accordance with the documents supplied.
  • a ⁇ g of the mRNA isolated from in vitro differentiated human dendritic cells was reverse transcribed with the reverse transcriptase "Superscript" from Gibco BRL in the presence of a pmol of a 25 to 30 mer oligo (dT) primer.
  • the PCR amplification using Thermoprime Plus DNA polymerase from Advanced Biotechnologies was carried out with 100 pmol of the following primers: R1 (5'-C-CGG-ATC-CGC-VTD-VTS-GGM-AAY-KBV-YTS-GT-3 '), R3 (5'-CG-GGA-TCC-GAA-RGY-RTA-SAD-SAD-RGG-RTT-3'). Cycle: 94'C, 60 sec.
  • a RACE-PCR was carried out with the following primers: 5'hGSPRT (5'-TTG-GAG-CCA-AAG-ACG-TCG-GCC-3 ' ), 5 '-hGSPl (5' -AGG-CAG-AAG-AGG-ATG- TAG-CGC-3 '), 5'-hGSP2 (5'-GCG-CTC-CCC-TGC-AGT-GAA-GAG- 3 '), 3' -hGSPl (5 '-AGT-GAC-CTG-CTC-ACG-GGC-GCG-3'), 3 '-hGSP2 (5'- CTC-TTC-ACT-GCA-GGG-GAG- CGC-3 ').
  • the 5 'end of the murine edg6 cDNA was also amplified using RACE-PCR with the following primers: 5'-mGSPRT (5' -CTC-ACC-TCG-TCT-GGG-AGG-GCC-TGC-3 '), 5' -mGSP1 (5 '-TGG-GCA-ACT-GGC-TGG-TCC-AAG-CTC-3'), 5 '-W.GSP2 (5' -GCC-TCG-GGC-CCA-GAT -CCT-CCA-GGG-GTG-CTG-CGG-ACG-CTG-GAA-ATG-CTG-G-3 ').
  • a reverse transcription with 10 ⁇ g total RNA of the murine cell line 18 was previously carried out as described above.
  • the 5 'mGSP2 primer contains part of the myc epitope sequence for further experiments.
  • the primers were based on the murine EST sequence of the cDNA clone val6c04.rl (GenBank entry no. AA254425), which has a high homology with the 3 'end of the coding human edg6 cDNA.
  • the reactions were also carried out according to the protocol of MA Frohman (Frohman, 1995) with an additional cleaning step using "MicroSpin S-400 HR" columns from Pharmacia Biotech using the protocol supplied after the 5'-polyadenylation reaction.
  • the murine edg6 cDNA fragment which was used as a radioactively labeled sample in the Northern blot, was amplified by the reverse transcriptase polymerase chain reaction from a total RNA preparation of the murine cell line 18 as described above with 25 pmol each of the 3 'primer (5th '- CCA-CGT-CCT-CCT-GCC-CGC-CGC-3') and 25 pmol of the 5'-mGSP2 primer (see above). Cycle: 94 ° C, 60 sec .; 50 ° C, 60 sec .; 72 ° C, 90 sec .; 35 cycles.
  • the amplification of the genomic 3 'sequence of the human edg6 was carried out by means of PCR from 400 ng HEp2 genomic DNA with 25 pmol of the 3'-hGSP2 primer (see above) and 25 pmol of the CA primer (5'-CCA-CTT-CCC- GCA-ACG-CCC-AGA- 3 '). Cycle: initial denaturation, 95 ° C, 5 min .; 95 ° C, 30 sec .; 60 ° C, 30 sec .; 72 ° C, 90 sec .; 30 cycles.
  • the cDNA fragments of the PCR reactions with the degenerate primers were cloned into the pZErO-2 vector from Invitrogen after Barn HI digestion.
  • the human edg6 RACE-PCR products were cloned into the same vector after HIND III / Pst I digestion. They were ligated to a full length clone at the Pst I interface.
  • the murine edg6 5'-RACE-PCR product was cloned into the pZErO-2 vector after HIND III / Eco RV restriction.
  • the RACE-PCR product was HIND III-digested after a T4 polymerase reaction.
  • the human cDNA fragment for the radioactive labeling was isolated after Pst I / Aat II restriction of the full length clone (bp 438-842).
  • the amplified murine cDNA fragment (bp 328-637) was cloned into the Apa I cut pZErO-2 vector. After radioactive labeling, this fragment was probed in Northern blots used. All fragments were sequenced with the "Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP" from Amersham International and analyzed using the Li-Cor sequencer from MWG Biotech in accordance with the protocols provided.
  • Figure 1 Schematic representation of a GPR in the cell membrane (after Emrich, 1995). The arrangement of the ( ⁇ - helical transmembrane domains (I-VII) is shown by cylinders. Possible glycosylation (••) and phosphorylation sites (P) are shown as well as a possible palmitoylation site (0).
  • Figure 2A Northern blot with total RNA of the human Burkitt lymphoma cell lines BL64 and DG75, the promyelocytic cell line U937 and the lymphoblastoid T cell line CEM as well as with mRNA of the larynx cancer cell lines HEp2 and cl32, hybridized with a radioactively labeled probe of the human edg6 cDNA. Ethidium bromide stained rRNA is shown as a control.
  • Figure 2B Human RNA master blot (Clontech), hybridized with a radioactively labeled probe of the human edg6 cDNA.
  • AI testicles; A2: ovaries; A3: pancreas; A4: pituitary; A5: adrenal gland; A6: thyroid; A7: salivary gland; A8: mammary gland; Bl: kidney; B2: liver; B3: small intestine; B4: spleen; B5: thymus; B6: peripheral leukocytes; B7: lymph nodes; B8: bone marrow; Cl: appendix; C2: lungs; C3: trachea; C4: placenta; Dl: fetal brain; D2: fetal heart; D3: fetal kidney; D4: fetal liver; D5: fetal spleen; D6: fetal thymus; D7: Fetal lungs.
  • No edg6-specific hybridization signals were obtained from the mRNA of the following human tissues (not shown): whole brain, cerebellum, cortex, frontal lobe, hippocampus, pituitary gland, occipital lobe, putamen, substantia nigra, temporal lobe, thala us, spinal cord, heart, aorta, Skeletal muscle, colon, urinary bladder, uterus, prostate, stomach.
  • Figure 2C Diagram of the relative intensity of the dot blot signals from selected organs.
  • Figure 2D Northern blot with total RNA of murine organs, hybridized with a radiolabelled probe of the murine edg6 cDNA. Ly: lymph nodes; sp: spleen; th: thymus; lu: lungs; si: small intestine; left: colon; st: stomach. No edg6-specific hybridization signal was obtained from the following total RNA preparations of murine tissue (not shown): heart, liver, kidney, skeletal muscle, pancreas, cerebellum, cerebrum.

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Abstract

L'invention concerne le récepteur EDG6 couplé à des protéines G, et ses fragments, variantes et mutations. ainsi que son utilisation. Les domaines d'application de l'invention sont la biologie moléculaire, la pharmacie et la médecine. L'invention vise à isoler et à identifier un membre supplémentaire de la famille des récepteurs EDG, et à permettre l'utilisation de ce nouveau membre à des fins médicales. Ce nouveau récepteur EDG6 humain comprend 384 acides aminés de la séquence 1 comportant sept domaines transmembranaires. Ce récepteur présente un site de glycosylation potentielle N-terminal, trois sites de palmitoylation potentielle, 12 à 15 acides aminés placés à l'extrémité C-terminale du septième domaine transmembranaire, et quatre sites C-terminaux de phosphorylation potentielle catalysée par la protéine kinase C. L'invention concerne également l'utilisation du récepteur EDG6, de ses fragments, variantes et mutations et éventuellement de ses partenaires de liaison pour des méthodes et traitements thérapeutiques.
EP99948712A 1998-09-11 1999-09-10 Recepteur edg6 (gene de differentiation de cellules endotheliales) humain et murin couple a des proteines g et son utilisation Withdrawn EP1112365A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19843240 1998-09-11
DE19843240 1998-09-11
DE19846979A DE19846979A1 (de) 1998-09-11 1998-10-13 G-Protein gekoppelter Rezeptor EDG6 und seine Verwendung
DE19846979 1998-10-13
PCT/DE1999/002871 WO2000015784A2 (fr) 1998-09-11 1999-09-10 Recepteur edg6 (gene de differentiation de cellules endotheliales) humain et murin couple a des proteines g et son utilisation

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US6812335B1 (en) 1999-03-23 2004-11-02 The Regents Of The University Of California Human polypeptide receptors for lysophospholipids and sphingolipids and nucleic acids encoding the same
CA2509218C (fr) 2002-12-20 2010-09-07 Merck & Co., Inc. 1-(amino)indanes et (1,2-dihydro-3-amino)-benzofuranes, benzothiophenes et indoles utilises en tant qu'agonistes du recepteur edg
US20050079549A1 (en) * 2003-07-23 2005-04-14 John Castracane Methods for modeling GPCRs and for producing ligand blocking and receptor activating antibodies for same

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US5891720A (en) * 1997-04-17 1999-04-06 Millennium Pharmaceuticals, Inc. Isolated DNA encoding a novel human G-protein coupled receptor
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