Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the invention relates to a new angiotensin receptor, the production and use thereof.
• the Renin Angiotensin System is significantly involved in the regulation of the cardiovascular system, the electrolyte and the fluid balance.
• the physiologically effective effector hormones e.g. Angiotensin II (Angll) and Angiotensin 1-7 (Ang1-7) are produced by proteolytic cleavage of the precursor angiotensinogen synthesized in the liver or the angiotensin I formed therefrom.
• a large number of proteases are involved in these cleavages (e.g. renin, angiotensin converting enzyme (ACE)).
• AT1 and AT2 Two human angiotensin receptors have been described, designated AT1 and AT2. Both receptors belong to the angiotensin II (Ang ll) receptors, i.e. these receptors preferably bind Ang II.
• Ang ll angiotensin II
• the cDNA sequences of these two receptors (Takayanagi, R. et al. (1992). Biochemical and Biophysical Research Communications 183, 910-915; Tsuzuki, S. et al. (1994). Biochemical and Biophysical Research Communications 200, 1449-1454) each have seven hydrophobic areas, which represent potential transmembrane domains.
• angiotensin receptors AT1 and AT2 belong to the family of 7-transmembrane receptors, which mediate the action of their ligand angiotensin II via signal transduction cascades coupled via intracellular G protein.
• the present invention relates to a new angiotensin receptor which has at least one subunit which has the amino acid sequence SEQ ID NO. 10 contains, this amino acid sequence optionally having further amino acids at the C-terminal end.
• the angiotensin receptor is a transmembrane receptor. In contrast to the angiotensin receptors described so far, it preferably has fewer than 7 transmembrane domains.
• the transmembrane domain preferably has the sequence “MSIVIPL” (indicated in the one-letter code for amino acids).
• the angiotensin receptor can have one or more subunits.
• the receptor can be used either as a) monomer (a subunit that contains sequence SEQ ID NO. 10) or as b) dimer with an identical or another subunit or as c) tetramer with three further identical subunits or with an identical and two other subunits.
• angiotensin receptor can also be present as a monomer (one subunit) or as a dimer (two different subunits) until the ligand is bound and only change to a dimeric or tetrameric form when the ligand is bound.
• Receptors that have a similar structure to the new angiotensin receptor include the insulin receptor, growth factors such as nerve growth factor (NGF), endothelial growth factor (EGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF) , T-cell antigen receptors, haematopoietic cytokine receptors (eg IL1, IL5), and adhesion molecules (integrins, selectins).
• the new angiotensin receptor can optionally be post-translationally modified, for example the receptor can be glycosylated and / or phosphorylated.
• the angiotensin receptor can bind its ligand and mediate the action of this ligand or ligands intracellularly.
• the angiotensin receptor can bind angiotensin 1-7 and / or a derivative of angiotensin 1-7.
• Derivatives of angiotensin 1-7 are preferably truncated peptides derived from Ang1-7, e.g. Degradation products from Ang1 -7.
• Examples of derivatives of Ang1-7 are angiotensin III and angiotensin IV.
• the receptor preferably binds Ang 1-7.
• the invention relates to a receptor which the amino acid sequence in Table 2, SEQ ID NO. 10 has or contains.
• the angiotensin receptor or the subunit that has the sequence SEQ ID NO. 10 contains, has a length of at least 175 amino acids.
• the invention furthermore relates to a nucleic acid which has a sequence which codes for the new angiotensin receptor (“receptor”).
• the receptor can be coded in whole or in part by a nucleic acid which has the sequence SEQ ID NO. 9
• a nucleic acid which codes for the receptor or a subunit of the receptor can have the sequence SEQ ID NO.9 and may have further nucleotides at the 3 ' end which belong to the coding sequence.
• the nucleic acid can be, for example, a DNA, a cDNA or an RNA.
• the nucleic acid can be a gene, in which case the sequence SEQ ID NO. 9 can be interrupted by non-coding sequences (introns).
• the nucleic acid can optionally be derivatized.
• a derivative of the nucleic acid can be, for example, a salt or a nucleic acid derivative which, in addition to the natural nucleotides, also contains modified nucleotides or is composed entirely of modified nucleotides.
• the invention also relates to methods for producing a nucleic acid which codes for the angiotensin receptor and which has the sequence SEQ ID NO. 9 contains.
• a nucleic acid can be amplified in a PCR in which oligonucleotides, the sequences of which correspond to homologous regions (conserved regions) in the nucleotide sequences of the angiotensin receptors AT1 and AT2, as primers and cDNA, which are preferably from a tissue (or cells), on which angiotensin 1-7 or a derivative thereof has an effect and / or is produced from tissue (or cells) in which AT1 and / or AT2 are not expressed.
• degenerate primers are used, the sequences of which are derived from the homologous regions of the sequences of AT1 and AT2.
• the primers Ang3A and Ang3B are preferably used.
• cDNA made from endothelial cells can be used as a template.
• cDNA made from HUVECs Human Umbilical Vein Endothelial Cells
• the invention also relates to methods for producing the new receptor.
• the new angiotensin receptor can be produced by a nucleic acid, the sequence of which codes for the angiotensin receptor and which preferably has the sequence SEQ ID NO. 9 contains, in a suitable vector integrated, the recombinant vector is introduced into a cell and the angiotensin receptor is expressed in this cell.
• the invention further relates to the use of a nucleic acid which codes for the new angiotensin receptor and which preferably has the sequence SEQ ID NO. 9, for example in a method for producing a recombinant angiotensin receptor, the nucleic acid, which is under the control of a suitable promoter, being introduced into a cell and expressed.
• the invention further relates to the use of a nucleic acid which codes for the new angiotensin receptor and which preferably has the sequence SEQ ID NO. 9 contains for the production of a recombinant cell in which the angiotensin receptor can be expressed.
• the invention relates to a recombinant cell which has an angiotensin receptor with the sequence SEQ ID NO. 10 expressed.
• the invention further relates to the use of a nucleic acid which codes for the angiotensin receptor and which preferably has the sequence SEQ ID NO. 9 contains, for the manufacture of a medicament for the treatment and prevention of diseases which are associated with an incorrect regulation of the cardiovascular system, the electrolyte balance or the fluid balance.
• the invention relates to a medicament that a nucleic acid with the sequence ID NO. 9 contains.
• the invention further relates to the use of a nucleic acid which codes for the angiotensin receptor and which preferably has the sequence SEQ ID NO. 9 contains, for the production of a transgenic animal which does not contain the corresponding angiotensin receptor gene or a transgenic animal which overexpresses the corresponding angiotensin receptor gene.
• transgenic animals can be used to characterize the specificity and biochemical function of the angiotensin receptor.
• the invention relates to a non-human transgenic animal that expresses the angiotensin receptor.
• the invention further relates to the use of a nucleic acid which codes for the angiotensin receptor and which preferably has the sequence SEQ ID NO.
• the invention contains, in methods for the identification and characterization of substances which can be used as antagonists and agonists of the angiotensin receptor and / or in methods in which substances are identified which inhibit or activate the functional expression of the encoded angiotensin receptor.
• the invention relates to a method for identifying and characterizing agonists or antagonists of the angiotensin receptor, wherein a nucleic acid which has the sequence SEQ ID NO. 9, introduced into a cell and the angiotensin receptor is expressed in this cell, this cell is incubated with a substance to be examined and the effect of this substance on the angiotensin receptor is determined.
• the invention also relates to the use of a nucleic acid which codes for the angiotensin receptor and which preferably has the sequence SEQ ID NO. 9 contains, in gene therapy or for the manufacture of a medicament that can be used in gene therapy.
• the invention relates to the use of a nucleic acid which codes for the angiotensin receptor and which preferably has the SEQ ID NO. 9 contains as a tool in molecular biology (e.g. as a probe).
• the invention also relates to the use of the new angiotensin receptor.
• the angiotensin receptor can be used in methods for identifying and characterizing substances that can be used as agonists or antagonists of the receptor (e.g. binding assays and functional assays for screening).
• angiotensin receptor or parts thereof can be, for example, an epitope which is identified, for example, by the amino acid sequence SEQ ID NO. 10 or a part the same is specified, for the production of antibodies by known methods (Harlow and Lane, "Antibodies-A Laboratory Manual", Cold Spring Harbor Laboratory, ISBN-0-87969-314-2) are used. In this way, antibodies are produced which are specific bind to the new angiotensin receptor can be used for the detection of the new receptor, eg in diagnostic methods and / or for further characterization of the receptor
• oligonucleotides were synthesized whose sequences correspond to conserved regions of the sequences coding for AT1 / AT2 or which are directed against these conserved regions (Ang3A and Ang3B ). These oligonucleotides were used as a template in PCR reactions with cDNA from human HUVECs (Human Umbilical Vein Endothelial Cell). This cell line is derived from endothelial cells that line the blood vessels. These cells do not express AT1 or AT2 receptors.
• a DNA fragment with an open reading frame and a previously not described nucleotide sequence (SEQ ID NO. 3) was amplified and identified. This nucleotide sequence showed no agreement with sequences in the databases EMBL or Genbank ® .
• a sequence comparison with sequences in the Lifeseq® database using the program modules "Blast” and “Assembly” (GGC program package) revealed an overlap of SEQ ID NO. 3 with Lifeseq® clones No. 1458429, 3341232, 3440195, 1250818, 2836520, 1310286 and 1362205 (ESTs with no known function).
• the theoretical 0.5 kbp DNA fragment to be derived from this virtual Contig- (3) was experimentally confirmed (Example 2, PCR with the pair of primers Ang4A and Ang4B).
• the sequence of the DNA fragment with the open reading frame contained therein was extended by 5 ' (Example 3) to into the vector sequence (SEQ ID NO. 9).
• the SEQ ID NO. 9 also contains or part of the 5 ' untranslated region.
• SEQ ID NO. 9 encodes a protein with at least 175 amino acids (SEQ ID NO. 10).
• a hydrophobicity analysis of this amino acid sequence using the GCG program showed that the sequence at the N-terminus has a hydrophobic domain with a length of about 7 amino acids. This hydrophobic domain can represent a transmembrane domain with which this new receptor is anchored in the membrane. In contrast, the sequence has a more or less hydrophilic domain at the C-terminus.
• the oligonucleotide primers Ang3A and Ang3B were used, which include a 0.19 kbp DNA fragment - based on the AT1 / 2 receptor genes.
• CDNA from a commercially available HUVEC cDNA bank (Stratagene Catalog No. 937223, Stratagene GmbH, Heidelberg) was used as template DNA.
• PCR reaction 1 ⁇ g cDNA from the HUVEC Bank with recombinant Thermus aquaticus (Taq) DNA polymerase (Boehringer Mannheim, Germany) and 10 pmol primer Ang3A and Ang 3B each per 50 ⁇ l reaction mixture in a thermocycler (Perkin Elmer 2400 thermocycler, Perkin Elmer Applied Biosystems, Rothstadt).
• the PCR reaction was carried out with the following temperature profile: 20 " at 96 ° C, 10 " at 38 ° C and 20 " at 72 ° C (60 cycles). A 130 base pair (bp) long DNA fragment was amplified.
• This 130 bp DNA fragment was ligated into the vector pCR 2.1 TOPO® (Invitrogen BV, NV Leek, Netherlands) and transformed into E. coli ToplOF ' .
• the sequence of the 130 bp DNA fragment present in pCR2.1 was determined using a capillary electrophoresis sequencer (ABI 310, Applied Biosystems, Rothstadt) (SEQ ID NO. 3).
• sequence SEQ ID NO. 3 was created using the FASTA and TFASTA program modules (software package from the University of Wisconsin Genetics Computer Group (GCG), USA) with the sequences found in the public domain Databases (eg EMBL, GenBank ® ) are compared. In addition, the sequence was used for a corresponding homology comparison with the sequences which are recorded in the Lifeseq® database from Incyte (Palo Alto, USA).
• PCR reactions were carried out with 0.1 ⁇ g cDNA from the HUVEC Bank as a template and 10 pmol primers Ang4A and Ang4B per 50 ⁇ l reaction mixture, using the Advantage-cDNA polymerase mix (Clontech, Heidelberg). For the PCR reactions, the following temperature profile was used: 30 "94 ° C and 2 '30" 60 ° C (60 cycles). A 500 bp DNA fragment was obtained. This was cloned and sequenced as described in Example 1 (SEQ ID NO. 6). The sequence has an open reading frame (ORF).
• the nucleotide sequence of Ang5A and Ang5B each corresponds to a part of the sequence of the 500bp DNA fragment. These two primers are used in further PCRs with the primers M13 Reverse and M13 Forward (-20).
• the primers M13 Reverse and M13 Forward (-20) correspond to sequences of the Uni-ZAP bacteriophage vector used to produce the HUVEC cDNA library.
• PCR reactions were carried out with 0.1 ⁇ g cDNA from the HUVEC Bank as a template and 10 pmol primer Ang5A or Ang5B and M13 Reverse or M13 Forward (-20) (from the TOPO-TA cloning kit (Clontech)) and Taq Polymerase (Boehringer Mannheim) and Taq Start TM Antibody (Clontech).
• the PCR reactions were carried out with the following temperature profile: 30 " 96 ° C, 30 " 50 ° C, and 45 " 72 ° C (70 cycles).
• the DNA fragment has the sequence SEQ ID NO. 9 on.
• the sequence SEQ ID NO. 9 contains the entire 5 'coding area.

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• 1998
  • 1998-06-08 DE DE19825494A patent/DE19825494A1/de not_active Withdrawn
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