WO2000015784A2 - Human and murine g-protein-coupled edg6 receptor (endothelial differentiation gene) and use of same - Google Patents

Abstract

The invention relates to the G protein-coupled EDG6 receptor, fragments, variants and mutations thereof, as well as to its use. Areas of application of the invention include molecular biology, pharmacy and medicine. The aim of the invention is to isolate and identify a further member of the family of EDG receptors and to permit its use for medical purposes. The new human EDG6 receptor comprises 384 amino acids of sequence 1 having seven transmembrane domains. The receptor has a possible N-terminal glycolysis site, three possible palmitoylation sites, between 12 and 15 amino acids in the C-terminal position of the seventh transmembrane domain and four possible C-terminal proteinkinase C-phosphorylation sites. The invention further relates to the use of the EDG6 receptor, its fragments, variants and mutations and possibly its binding partners for therapeutic methods and treatments.

Description

HUMAN AMERICAN G-PROTEIN COUPLED RECEPTOR EDG6 (ENDOTHELIAL DIFFERENTIATION GEN) AND ITS USE

description

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.

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).

The large number of GPR is also reflected in a large variety of extracellular ligands, the so-called ^x first messenger ^', again. 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.

Another member of the EDG receptor family, EDG2, 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-sτι-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.

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

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 edgβ 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.

When searching for homologous sequences to sequence 2 in the nucleic acid database using the BLASTN program from the Hussar package of the German Cancer Research Center (DKFZ) in Heidelberg, 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.

Starting from this partial clone, 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.

In the further development of the invention, 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. For this purpose, a non-functional edgβ 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).

With these mouse strains, further statements about the function of the EDG6 receptor can be made, e.g. For example, diseases can be identified that are caused directly or indirectly by the EDG6 receptor or are increased in their extent. In particular, these 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. Due to the high homology of the murine receptor with the human receptor and due to the highly conserved tissue-specific expression pattern, the results of the mouse model can largely be transferred to humans. 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. Furthermore, 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.

1. In the case of diseases which are caused by abnormal expression or by receptor mutations, diagnosis can be carried out, for example using test kits based on monoclonal antibodies or nucleic acid detection methods.

2. In diseases that are related to the receptor function, the functions of the receptor can be influenced in an agonistic or antagonistic manner.

3. 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. The invention will be explained in more detail below by means of exemplary embodiments.

Results

To determine new chemokine receptors and related receptors that are involved in the regulatory processes of the immune system, the polymerase chain reaction (PCR) with degenerate primers was used to identify G protein-coupled receptors (GPCRs) from in vitro differentiated human dendritic cells . Human peripheral blood mononuclear cells were differentiated into dendritic cells by treatment with the cytokines GM-CSF and IL-4. PCR with degenerate primers from regions of the second and seventh transmembrane domains (TM) of some chemokine receptors led to the identification of a 648 bp fragment, which is part of a new member of the GPCR superfamily. 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. However, 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. Comparison of the amino acid sequence of EDG6 with the other EDG receptor molecules from the first to the seventh transmembrane domain shows that 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. For this purpose, 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 jπyc epitope. After transfection of this vector into the human embryonic kidney cell line HEK 293, the fusion molecule could only be detected in permeabilized cells by means of flow cytometry using an anti-jnyc-specific monoclonal antibody. Furthermore, the last 50 bp of the human edgβ (hedgβ) 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 hedgβ and the D19S120 polymorphism. This shows that hedgβ is located on chromosome 19pl3.3 next to the D19S120 marker.

Furthermore, the murine homolog of the edgβ (medgβ) 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 hedgβ cDNA. The primers were chosen so that the open reading frame of medgβ could be amplified. Therefore, the medgβ cDNA is incomplete at the 3 'end. It contains an open reading frame of 1161 bp. The first 99 bp of the 499 bp δ'-untranslated region contain a murine repetitive element B1. The open reading frame of the medgβ 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.

Next, the expression pattern of edgβ was examined. For this purpose, 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 hedgβ-specific signal was found at around 1.7 kb in human cell lines, hedgβ 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. In general, hedgβ 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 hedgβ with mRNA samples from 50 different human tissues using a dot blot. High hedgβ expression rates were found in human adult and fetal spleens, as well as in adult peripheral leukocytes and the lungs. Lower hedgβ expression rates were detected in adult thymus, lymph nodes, bone marrow and appendix as well as in fetal liver, thymus and lungs.

The tissue-specific expression of medgβ 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 edgβ mRNA is approximately 2.1 kb in size and thus 0.4 kb larger than the human edgβ mRNA. material and methods

Isolation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were obtained from fresh primary blood cells (buffy coats) by means of density centrifugation. First, 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.

In each case 5 × 10 ⁷ 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. From now on, 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 isolation

Total 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.

Northernblot

The transfer of the 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. In the same way, 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.

Polymerase chain reaction

A μg 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. ; 48-63 ° C, 30 sec; 72 ° C, 90 sec .; 35 cycles. For the amplification of the 3 'and 5' ends of the human edg6 cDNA, 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 reactions were carried out according to the protocol of MA Froh (Frohman, 1995). 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 μg 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. Furthermore, 10 μl undiluted template DNA was used for both amplifications of the RACE-PCR. 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.

Cloning and sequencing

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.

Construction, expression and FACS analysis of the myc epitope-labeled human EDG6 receptor

The construction of the C-terminal myc epitope-labeled human EDG6 receptor and its expression in HEK293 cells and its analysis by means of flow cytometry was carried out as described (Emrich et al., 1993).

Computer analysis

Sequence comparisons, database searches and statistical calculations were carried out using the HUSAR package V4.0 at the German Cancer Research Institute in Heidelberg and the PC program ClustalX VI.62b.

Legend for the pictures:

Figure 1: Schematic representation of a GPR in the cell membrane (after Emrich, 1995). The arrangement of the (α - 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.

Claims

claims 1. Human G-protein coupled receptor EDG6 with sequence 1 and its fragments, variants and mutations. 2. Muriner G protein-coupled receptor EDG6 with sequence 4 and its fragments, variants and mutations. 3. DNA sequence encoding the human G protein-coupled receptor EDG6 and its fragments, variants and mutations. 4. DNA according to claim 3, characterized by sequence 2. 5. DNA sequence encoding the murine G protein-coupled receptor EDG-6 and its fragments, variants and mutations. 6. DNA according to claim 5, characterized by sequence 3. 7. Vectors which contain a DNA sequence optionally coupled to a suitable promoter according to claims 3-6. 8. host cells containing vectors according to claim 7. 9. Antibodies against human or murine EDG6 G protein-coupled receptors. 10. Monoclonal antibodies according to claim 9. 11. Test kit for the detection of the EDG6 receptor based on monoclonal antibodies according to claim 10. 12. Test kit for the detection of the EDG6 receptor based on nucleic acid diagnostics. 13. Use of the EDG6 receptor as well as its fragments, variants and mutations and possibly its binding partner for therapeutic methods and treatments. 14. Use according to claim 13, characterized in that the therapeutic measures influence the function of blood and body cells, for example lead to the inhibition of acute and chronic inflammation. 15. Use of the EDG6 receptor and its fragments, variants and mutations and possibly its binding partner according to claim 13-14, characterized by the use for gene therapy methods and treatments. 16. Use of the monoclonal antibodies according to claim 10, optionally coupled to other molecules and substances, for example therapeutic agents, toxins or antibodies, for therapeutic methods and treatments. 17. Use according to claim 16, characterized in that the therapeutic measures influence the function of the EDG6 receptor, for example in immune and inflammatory reactions. 18. EDG6-deficient mouse strains that contain non-functional mutants (zero mutant ') of EDG6. 19. mouse strains according to claim 18, in which further gene deficiencies are crossed, for example for immunomodulatory and iramunregulatory gene functions, such as. B. receptors or signaling molecules. 20. Use of mice according to claim 18-19 as an animal model for diseases associated with the EDG6 receptor.