JP2002525050A - Human and mouse G protein-coupled receptor EDG6 (endothelial differentiation gene) and uses thereof - Google Patents

Abstract

(57) Abstract The present invention relates to a G protein-coupled EDG6 receptor and its fragments, variants and mutations, and uses thereof. Fields of application of the invention include molecular biology, pharmaceuticals and medicine. It is an object of the present invention to isolate and identify alternative members of the EDG receptor family and make them available for medical purposes. The novel human EDG6 receptor contains 384 amino acids of sequence 1 with seven transmembrane domains. The receptor has one possible N-terminal glycolysis site, three possible palmitoylation sites of 12 to 15 amino acids at the C-terminal position of the seventh transmembrane domain, and four possible C-terminal protein kinases It has a C phosphorylation site. The invention also relates to the use of the EDG6 receptor, fragments, variants and mutations thereof, and binding partners for therapeutic methods and treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the G protein-coupled receptor EDG6 and its fragments, variants and mutations, and uses thereof. The fields of application of the invention are molecular biology, pharmaceuticals and medicine.

[0002] The G protein-coupled receptor (GPR) superfamily encompasses hundreds of proteins. Its basic task in vivo is to transfer information from the extracellular environment to the interior of the cell by interacting with a heterotrimeric guanine nucleotide binding protein, commonly referred to as the G protein (Dohlman et al., 1987). It affects the process of regulation in cells (Bohm et al., 1997).

[0003] The wide variety of GPRs is reflected in the large diversity of extracellular ligands, the so-called "primary messengers". GPRs for hormones and neurotransmitters, paracrine and inflammatory mediators, specific proteases, numerous taste and olfactory molecules and photons and calcium ions are known (Watson and Arkinstall
, 1994).

[0004] The EDG (endothelial differentiation gene) receptor family belongs to the group of G-protein coupled receptors (GPR). The first member of this family, EDG1, was cloned in 1990 in connection with the study of the nonproliferative characteristics of angiogenesis and the differentiation of capillary endothelial cells in organisms.

[0005] Another member of the EDG receptor family, EDG2, appears especially in cortical areas.

[0006] A third member of the EDG receptor family, cDNA, has been isolated from human placenta, kidney, liver and human heart and localized to chromosome 9q22.1-2. Human EDG4 cDNA transcripts have recently been detected in testis, prostate, pancreatic and peripheral blood leukocytes and, to a lesser extent, thymus and spleen. Another two transcripts were isolated from murine aortic smooth muscle cells or mouse and bovine taste buds. Possible ligands for human and mouse EDG2 receptor and human EDG4 receptor include lysophosphatidylic acid (LPA: 1-Acyl-2-hydroxy-sn-glycero-
3-phosphat) was confirmed. It was also found that lysosphingolipids were functional ligands for the human receptors EDG1 and EDG3 and the murine H218 receptor.

Underlying the present invention is the isolation of other members of the EDG receptor family,
The challenge is to identify and make available for medical applications.

[0008] It has been found that dendritic cells differentiated in vitro express another EDG receptor called EDG6.

[0009] The human EDG6 receptor comprises 384 amino acids of sequence 1 with seven transmembrane domains. This receptor has one possible N-terminal glycosylation site, three possible palmitoylation sites of 12 to 15 amino acids located at the C-terminus of the seventh transmembrane domain, and four possible C-terminal protein kinases It has a C phosphorylation site. A model of the receptor is shown in FIG.

[0010] The EDG6 receptor was isolated from human dendritic cells differentiated in vitro.
He is the first member of the G family. This receptor shows a signal at about 1.7 kb in Northern blot analysis. Human edg6 mRNA is expressed in Burkitt's lymphoma cell lines, JBL2, BL64 and DG75, promyelocytic cell line U937 and T cell line CEM. Human
High tissue-specific expression rates of edg6 were found in human adult and fetal spleen and adult peripheral leukocytes and lung. Low expression rates of human edg6 were detected in adult thymus, lymph nodes, bone marrow and colon as well as fetal liver, thymus and lung.

[0011] The cDNA sequence of the EDG6 receptor having sequence 2 also falls within the scope of the present invention.

When a sequence homologous to sequence 2 was determined in the nucleic acid data bank from the Husar packet of the German Cancer Research Center (DKFZ) (Heidelberg) by the program BLASTN, a mouse with high homology to the newly identified EDG6 Clones could be identified. Mouse cDNA clones from the EST (expressed-sequence tag expressed sequence tag) nucleic acid databank are derived from lymph nodes and after correction of the databank entry resulting in a change in reading frame, 88% of the 106 amino acids at the amino acid level are human Homologous to EDG6 receptor. Thus, this cDNA clone is a fragment of the mouse homolog of the human EDG6 receptor.

[0013] Starting from this partial clone, whole mouse
The cDNA sequence (sequence 3) was determined.

The protein derived from this cDNA sequence has sequence 4.

A specific anti-EDG6 antibody is also claimed. GS containing fragment of EDG6 receptor
This antibody is made by immunizing mice with the T-fusion protein. Hybridomas are made from murine spleen cells and their specific antibody production is tested by ELISA analysis.

Another method of preparing antibodies uses whole cells that express the receptor EDG6 after insertion of the cDNA or already spontaneously, and use a C-terminal 6 × histidine-linked N-terminal EDG6 fragment to immunize Sensitization.

The present invention is further extended to include EDG6-deficient mice, ie, non-functional mutants of EDG6 (“
Make mice containing the "zero mutant"). This knockout mouse is likely to be used as an animal model for EDG6-related diseases. The phenotypic characteristics of this mouse can contribute critically to functional decisions. Mouse embryonic stem cells (ES cells) by homologous recombination of a non-functional edg6 gene with an appropriate selection marker
Incorporate in Next, the selected ES cells are put into mouse embryos (morula, blast cells) of early cell development as pluripotent cells.

With this mouse strain, further statements can be made regarding the function of the EDG6 receptor. For example, a disease caused directly or indirectly by EDG6 or whose degree is enhanced can be confirmed. In particular, for example, diseases based on immunodeficiency against infection and acute and chronic inflammation belong to this category. Also included are autoimmune diseases, allergies and malignancies, such as tumors, leukemias and lymphomas. Based on the high homology of the mouse and human receptors and on the basis of highly conserved tissue-specific expression patterns, the results of the mouse model can be fully diverted to humans. The human receptor EDG6 according to the present invention is directly or indirectly involved in the onset of immunodeficiency, acute and chronic inflammation, autoimmune diseases, allergies and malignancies, or can increase the severity and magnitude thereof. .

The present invention also relates to the use of the original, modified or synthetic forms of EDG6 nucleic acids and EDG6 polypeptides as a basis for the development of pharmaceutically important substances. The EDG6 nucleic acid is then used for the construction of genes and vectors, and the EDG6 polypeptide is used for the construction of ELISAs, complete assays and assays. A pharmaceutically important substance may itself bind to a receptor polypeptide, or to a receptor-encoding nucleic acid, or affect the binding of a physiological ligand or the binding and activity of a downstream signaling molecule in a cell, thereby affecting the receptor. Activate or suppress a function. These substances having an agonist or antagonistic effect on the function of the EDG6 receptor are organic molecules, inorganic molecules and peptides or a combination of these various substances.

The present invention enables medical uses for the following diagnostic or therapeutic treatments.

1. In the case of diseases caused by abnormal expression or receptor mutations, diagnosis can be made, for example, by a test kit or a nucleic acid detection method based on monoclonal antibodies.

2. Diseases associated with receptor function can have an agonistic or antagonistic effect on receptor function.

[0023] 3. If receptor misfunction or incorrect expression of the EDG6 receptor or its ligands appears, the prototype or modified EDG6 receptor and specific antibody or binding partner may be used for therapeutic purposes, such as gene therapy (eg, cell, Liposome or virus
Base) can be used for.

Next, the present invention will be described in detail with reference to examples.

Results For the determination of novel chemokine receptors and their related receptors involved in the regulation process of the immune system, the polymerase chain reaction (PCR) using degenerate primers was applied,
G protein-coupled receptors (GPCRs) were identified from human dendritic cells differentiated in vitro. PCR with degenerate primers in the regions of the second and seventh transmembrane domains (TM) of some chemokine receptors identified a 648 bp fragment that was part of a novel member of the GPCR superfamily. Possible full-length cDNA containing 1560 bp
It was cloned stepwise by 3'-RACE-PCR. cDNA has an open reading frame of 1155 bp, 22
It contains a 5'-untranslated region on a bp scale and a 3'-untranslated region on a 383 bp scale. However, the 3'-end cDNA may not be complete because there is no typical polyadenylation signal. Sequence 1 shows the obtained amino acid sequence. Sequence comparisons suggest that the newly identified receptor belongs to the EDG family of GPCRs. Therefore, this receptor was named EDG6. Comparison of the amino acid sequence of EDG6 with other EDG receptor molecules of the first to seventh transmembrane domains shows that EDG6 is 46% for EDG3, 44% for EDG1, and 39% for EDG4.
And 37% homology to EDG2. Next closely related GPCRs have 31% homology
hCB1R, a member of the cannabinoid receptor family. Computer-assisted analysis was able to determine the possible localization of the seven transmembrane domains, the possible N-glycosylation sites in the N-terminal extracellular region and some post-translational modification sites in the C-terminal cytoplasmic domain. The correct orientation of the cell membrane molecules with the N-terminus on the extracellular side was also investigated in detail. For this, the protein coding cDNA sequence was cloned into a eukaryotic expression vector while maintaining the reading frame. Eukaryotic expression vectors express the myc epitope at the C-terminus of the cloned sequence. After transfection of this vector into human embryonic kidney cells HEK293, it was possible to detect only the cells that were able to penetrate the fusion molecule with an anti-myc-specific monoclonal antibody using a flow-through hemocytometer. Also human
The last 50 bp of the edg6 (hedg6) cDNA is identical to base pairs 13 to 62 of the short sequence containing the repetitive dinucleotide polymorphism D19S120. This polymorphism was localized to chromosome 19p13.3. By PCR using gene-specific primers of hedg6 cDNA and D19S120 aplicon, a human genomic DNA fragment containing the 3'-end of hedg6 and the D19S120 polymorphism could be amplified. Thus, hedg6 was shown to be located on chromosome 19p13.3 alongside the D19S120 marker.

Further, a mouse homolog of edg6 (medg6) could be isolated by RACE-PCR.
For this purpose, a dendritic cell line 18 derived from mouse fetal skin was used. cDNA clone v
It is derived from the mouse EST sequence of a16c04.rl (Genebank Registry No. AA254425),
Gene-specific primers with high homology to the 3'-end of the coding region of the cDNA were made. Primers were selected such that the open reading frame of medg6 was amplified. Therefore, the medg6 cDNA at the 3'-end is incomplete. It contains an open reading frame of 1161 bp. The first 99 bp of the 5'-untranslated region encompassing 499 bp contains the mouse repetitive element B1. The open reading frame of medg6 is 80% homologous to the human sequence of interest. At the protein level, the two sequences have 82% homology and 91% similarity. Possible post-translational modification sites are conserved in the human and mouse edg6 sequences.

Next, the expression pattern of edg6 was examined. To this end, DNA fragments representing regions of low conservation in mouse and human cDNA sequences were generated. This fragment was then used as a radiolabeled probe in Northern blotting. In human cell lines, a hedg6-specific signal was found at about 1.7 kb. hedg6 mRNA is expressed in Burkitt's lymphoma cell lines JBL2, BL64 and DG75, promyelocytic cell line U937 and T cell line CEM, but not detectable in laryngeal cancer cell line Hep2, Hep2 subclone c132 and cervical cancer cell line HeLa. Did not. In general, hedg6 is weakly expressed at all positive cell sites assayed and is only detected after prolonged blotting exposure. Based on the high specificity of the hybridized samples, tissue specific expression of hedg6 could be determined by dot blotting on mRNA samples of 50 different human tissues. High expression rates of hedg6 were observed in human adult and fetal spleen and adult peripheral leukocytes and lung. Adult thymus, lymph nodes,
Low expression rates of hedg6 were detected in bone marrow and colon and fetal liver, thymus and lung.

[0028] Tissue-specific expression of medg6 mRNA within the examined organs is in very good agreement with human expression patterns. Hybridization signals were found in mouse lung, spleen, thymus and lymph nodes, but not in non-lymphoid tissues. Mouse edg6 mRNA is about 2.1 kb
And therefore 0.4 kb larger than human edg6 mRNA.

Materials and Methods Isolation of Peripheral Mononuclear Blood Cells Peripheral mononuclear blood cells (PBMC) were obtained from new primary blood cells (buffy coats buffy coat) by density centrifugation. First, add 10 ml of each new primary blood cell to four 50 ml Falcons
Each tube was mixed with 20 ml of PBS. PBS was mixed with 5 U / ml heparin. 10 ml of Bichrom's Ficoll separation solution was placed under the mixture and centrifuged at 200 xg for 20 minutes.
Subsequently, the upper 20-25 ml of the mixture were removed. The remainder of the mixture, still contaminated with thrombus, was then centrifuged again at 460 xg for another 20 minutes. The formed mesophase of all Falcon tubes was collected and washed with ice-cold PBS mixed with 1 mM EDTA three times for 1 minute each at 300 xg to avoid contamination of the thrombus.

Each 5 × 10 ⁷ peripheral mononuclear blood cells were treated with 3 ml 10 cm diameter with 15 ml RPMI culture
The mixture was placed in a petri dish and cultured at 37 ° C. for 2 hours in a CO ₂ incubator. Next, RPMI the bottom of the Petri dish
The medium was thoroughly washed with a glass pipette from all sides, and most of the non-adhered cells were detached from the bottom. Unattached cells were discarded together with the culture solution. Next, 15 ml of fresh RPMI culture preheated to 37 ° C. was added to each petri dish. RPMI culture is 800U / ml
It contained GM-CSF and 1000 U / ml IL-4. Thereafter, the culture solution was supplemented three times every other day. At that time, take out 7.5 ml of the culture solution from each Petri dish, 1600 U / ml GM-CSF and 1000 U / ml.
New RPMI broth containing ml-4 IL-4 was replenished. Cells were harvested on day 7 of cell culture.

The Burkitt's lymphoma cell lines BL64 and DG75, the lymphoblastic T cell line CEM and the promyelocytic cell line U937 were cultured in RPMI1640 medium containing 10% fetal bovine serum, and the laryngeal carcinoma cell line HEp
2 and HEp2 subclone c132 and human fetal kidney cell line HEK293 containing 10% fetal bovine serum
The cells were cultured in a DMEM culture solution.

RNA Isolation Total RNA was treated with Gibco's TRIzol reagent according to the attached protocol. mR
The treatment of NA is performed using the Pharmacia Biotech “Micro mRNA Purifica
tion Kit) ”based on the attached documents.

Northern Blotting Transfer of RNA was performed by capillary blotting, which allows for directed transfer of RNA fragments by ion transfer. At this time, a glass plate having almost the same width as the gel to be blotted was laid on a dish filled with 20 × SSC buffer.
Two pieces of filter paper, having the length of the gel to be blotted, lying across the glass plate and having sufficient width to allow the overhanging ends to protrude deeply into the dish filled with buffer, with 20x SSC buffer It was impregnated and placed on a glass plate as shown. The RNA gel was perfectly fitted on top of it and placed on the top so that air bubbles did not enter. Nitrocellulose membranes, also of gel size, previously placed in water and subsequently 20x SSC buffer, respectively, were loaded on the gel. Since the gel also contained a significant proportion of formaldehyde, blotting was configured with bleeding. An impermeable plastic mask was placed over the nitrocellulose membrane. The plastic mask seals the edges of the blotting around the membrane in a watertight manner. Another two pieces of filter paper having the size of a nitrocellulose membrane were impregnated with a 20-fold SSC buffer, and were similarly fitted together and placed so as to prevent air bubbles from entering. The pile of dried paper napkins forms the upper seal of the composition. A weight was applied with a weight of about 0.5 kg, and blotting was performed for about 2 days. RNA was fixed at 80 ° C. for 2 hours.

Cloned human or mouse edg6 labeled with ³² P for hybridization
cDNA fragments were used. Gibco BRL “Random Primed Labeling Kit”
The labeling reaction was performed according to the instruction manual. Clontech human RNA master blotting was hybridized and washed according to the attached documentation.

Polymerase Chain Reaction 1 μm of mRNA isolated from human dendritic cells differentiated in vitro was digested with 1 pmole of a 25-30 mer oligo (dT) primer in the presence of Gibco BRL reverse transcriptase “Super
script ”. Thermoprime Plus D from Advanced Biotechnologies
PCR amplification with NA polymerase was performed 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 seconds; 48-63 ° C, 30 seconds; 72 ° C,
90 seconds; 35 cycles. For amplification of the 3'- and 5'-ends of the human edg6 cDNA, RACE-PCR was performed with the following primers: 5'hGSPRT (5'-TTG-GAG-CCA-AAG-ACG-TCG-GCC- 3 '
), 5'-hGSP1 (5'-AGG-CAG-AAG-AGG-ATG-TAG-CGC-3 '), 5'-hGSP2 (5'-GCG-CTC-CCC
-TGC-AGT-GAA-GAG-3 '), 3'-hGSP1 (5'-AGT-GAC-CTG-CTC-ACG-GGC-GCG-3'), 3'-hG
SP2 (5'-CTC-TTC-ACT-GCA-GGG-GAG-CGC-3 '). The reaction was performed according to the procedure of MAFrohman (Frohman, 1995). Similarly, by RACE-PCR, the 5'-end of mouse edg6 cDNA was amplified 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'-mGS
P2 (5'-GCC-TCG-GGC-CCA-GAT-CCT-CCA-GGG-GTG-CTG-CGG-ACG-CTG-GAA-ATG-CTG-G-
3 '). Reverse transcription was performed previously with 10 μg of total RNA from mouse cell line 18 as described above. The 5'-mGSP2 primer contains a portion of the myc-epitope sequence for extensive experimentation. Mouse EST of cDNA clone va16c04.r1 (Genbank registration number AA254425)
Primers were selected based on sequence. This EST sequence is coding human edg6 cD
It has high homology to the 3'-end of NA. The reaction was also performed according to the procedure of MAFrohman (Frohman, 1995), and "Microspin S-400" from Phrmacia Biotech.
An auxiliary purification step was performed using a 5′-polyadenylation reaction using an “HR” column according to the attached protocol. 10 μl of the undiluted DNA stock solution was used for any amplification of RACE-PCR. The mouse edg6 cDNA fragment, used as a radiolabeled probe, was isolated from the total RNA preparation of mouse cell line 18 by reverse transcriptase-polymerase chain reaction as described above with 25 pmol each of the 3'-primer (5'-CCA-CGT -CCT-CCT-GCC-CGC-CGC-3 ') and 25 pmol of 5'-mGSP2 primer (see above) Cycle: 94 ° C, 60 seconds; 50 ° C, 60 seconds; 72 ° C, 90 Seconds;
35 cycles. Amplification of the genomic 3'-sequence of human edg6 is based on 400 ng of genomic DNA with
Molar 3'-hGSP2 primer (see above) and 25 pmol CA primer (5'-CCA
-CTT-CCC-GCA-ACG-CCC-AGA-3 ') by PCR. Cycle: initial denaturation, 95
° C, 5 minutes; 95 ° C, 30 seconds; 60 ° C, 30 seconds; 72 ° C, 90 seconds; 30 cycles.

Cloning and Sequencing The cDNA fragment of the PCR reaction with degenerate primers was digested with Bam HI, followed by Invitro
It was cloned with the pZEro-2 vector from gen. HIND II for human edg6 RACE-PCR product
After I / PstI digestion, it was cloned in the same vector. This was ligated to the Pst I nick for a full-length clone. The 5'-RACE-PCR product of mouse edg6 was cloned into pZEro-2 vector after HIND III / Eco RV restriction. For this purpose, RACE-PCR products
After T4 polymerase reaction, it was digested with HIND III. A human cDNA fragment for radiolabeling was isolated after Pst I / Aat II restriction of a full-length clone (bp 438-842).
The amplified mouse cDNA fragment (bp 328-637) was cloned into Apa I cut pZEro-2 vector. After radiolabeling, this fragment was used as a probe for Northern blotting. All fragments from Amersham I
nternational “Thermo Sequenase fluorescent labeled primer cycle sequ
sequencing kit with 7-deaza-dGTP ”, and MWG Biotech's Li-Cor Seque
Analysis was performed by ncer according to the attached protocol.

Structure, Expression and FACS Analysis of Myc Epitope-Labeled Human EDG6 Receptor The structure of human EDG6 receptor labeled with a myc epitope at the C-terminus, its expression in HEK293 cells, and its analysis were determined by a flow-through hemocytometer as described above. (Emrich et al., 1993).

Computer Analysis Sequence comparisons, databank searches and statistical calculations were performed with the HUSAR packet V4.0 and the PC program ClustalX V1.62b from the German Cancer Institute (Heidelberg).

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a schematic of the GPR of the cell membrane (according to Emrich, 1995). (The sequence of the α-helical transmembrane domain (I-VII) is shown as a cylinder. Possible glycosylation sites (●●) and phosphorylation sites (P) are identified as well as possible palmitoylation sites (◇). Described.

FIG. 2. (A) Human Burkitt's lymphoma cell lines BL64 and DG75 hybridized with a radiolabeled probe of human edg6 cDNA, promyelocytic cell line U937, lymphoblasts.
Northern blotting of total RNA of T cell line CEM and mRNA of laryngeal cancer cell lines HEp2 and C132. RRNA stained with ethidium bromide is shown as a control. (B) Human RNA master blotting (Clontech) hybridized with a radiolabeled probe of human edg6 cDNA. A1: testis; A2: ovary; A3: pancreas; A4: pituitary; A5: adrenal gland; A6: thyroid; A7: salivary gland; A8: thymus; B1: kidney; B2: liver; : Thymus; B6: peripheral leukocytes; B7: lymph node; B8: bone marrow; C1: colon; C2: lung; C3: trachea; C4: placenta; D1: fetal brain; D2: fetal heart; D3: fetal kidney D4: fetal liver; D5: fetal spleen; D6: fetal thymus; D7: fetal lung. No edg6-specific hybridization signal was obtained from mRNA of the following human tissues (not shown). Whole brain, cerebellum, cerebral cortex, frontal lobe, hippocampus, pituitary, occipital lobe, putamen,
Black nucleus, temporal lobe, thalamus, spinal cord, heart, aorta, skeletal muscle, large intestine, bladder, uterus, prostate, stomach. (C) Diagram of the relative intensity of the dot blotting signal of the selected organ. (D) Northern blotting of mouse organs hybridized with a radiolabeled probe of mouse edg6 cDNA with total RNA. Ly: lymph node; sp: spleen; th: thymus; l
u: lung; si: small intestine; li: large intestine; st: stomach. No edg6-specific hybridization signal was obtained from the following total RNA preparations of mouse tissues (not shown).
Heart, liver, kidney, skeletal muscle, pancreas, cerebellum.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 9/00 A61P 37/00 4C085 29/00 43/00 105 4H045 37/00 C07K 14/705 43/00 105 16/28 C07K 14/705 C12N 1/15 16/28 1/19 C12N 1/15 1/21 1/19 C12Q 1/68 A 1/21 G01N 33/53 S 5/10 33/566 C12Q 1 / 68 33/577 B G01N 33/53 C12P 21/08 33/566 C12N 15/00 ZNAA 33/577 5/00 B // C12P 21/08 A61K 37/02 (81) Designated country EP (AT, BE, CH CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), JP, US (72) Inventor Rip, Martin D-16548 Gri Enichケ, Turpenstrasse 31 F term (reference) 4B024 AA01 AA11 BA41 BA63 CA04 DA03 FA02 GA11 HA15 4B063 QA19 QQ42 QQ79 QQ91 QR32 QR56 QS34 4B064 AG20 AG27 CA10 CA19 CC24 DA01 DA13 4B065 AA91X AA14A02A14A14A14A14 AA07 AA13 AA17 BA01 BA08 BA22 BA23 BA33 BA34 BA44 CA18 CA23 CA53 DC50 NA14 ZA362 ZB212 4C085 AA14 AA21 EE01 4H045 AA10 AA11 AA30 BA10 CA40 DA50 DA75 DA76 EA22 EA50 FA72 FA74 HA05

Claims (20)

[Claims] 1. A human G protein-coupled receptor EDG6 having sequence 1 and fragments, variants and mutations thereof. 2. A mouse G protein-coupled receptor EDG6 having sequence 4 and its fragments, variants and mutations. 3. A DNA sequence encoding the human G protein-coupled receptor EDG6 and fragments, variants and mutations thereof. 4. The DNA according to claim 3, wherein the DNA is sequence 2. 5. A DNA sequence encoding the mouse G protein-coupled receptor EDG6 and its fragments, variants and mutations. 6. The DNA according to claim 5, wherein the DNA has the sequence 3. 7. The method as claimed in claim 3, wherein the promoter is optionally linked to an appropriate promoter.
A vector comprising the DNA sequence described in 1. (8) A host cell comprising the vector according to (7). 9. An antibody against human or mouse G protein-coupled receptor EDG6. 10. The monoclonal antibody according to claim 9. A test kit for detecting the EDG6 receptor based on the monoclonal antibody according to claim 10. 12. A test kit for detecting an EDG6 receptor based on nucleic acid diagnosis. 13. Use of the EDG6 receptor and its fragments, variants and mutations, and optionally its binding partner, for therapeutic methods and treatment. 14. The use according to claim 13, wherein the therapeutic treatment interferes with the function of blood cells or body cells, for example for the suppression of acute and chronic inflammation. 15. Use of the EDG6 receptor and its fragments, variants and mutations, optionally a binding partner, according to claims 13 and 14, characterized in that it is used for gene therapy methods and therapy. 16. A method and therapeutic use of a monoclonal antibody according to claim 10, optionally conjugated to other molecules and substances, such as therapeutics, toxins or antibodies. 17. Use according to claim 16, characterized in that the therapeutic treatment interferes with the function of the EDG6 receptor, for example in immune and inflammatory responses. 18. EDG6 comprising a non-functional mutant of EDG6 ("zero mutant")
Deletion mouse strain. 19. The mouse strain of claim 18, wherein another gene deletion is crossed, for example, for a receptor or signal molecule, eg, for immunoregulatory and immunoregulatory gene function. 20. Use of the mouse according to claims 18 and 19 as an animal model for a disease associated with the receptor EDG6.