DE19825494A1 - New angiotensin receptor, production and use of the same - Google Patents

Abstract

The invention relates to a novel angiotensin receptor and to the production and use thereof. The novel angiotensin receptor hat at least one subunit containing the amino acid sequence SEQ ID NO. 10.

Description

The invention relates to a new angiotensin receptor, the production and Use of the same.

The Renin Angiotensin System is essential for the regulation of the cardiac Circulatory system, electrolyte and fluid balance involved. The physiologically effective effector hormones such. B. Angiotensin II (AngII) and Angiotensin 1-7 (Ang1-7) are obtained by proteolytic cleavage of the in the Liver synthesized precursors angiotensinogen or the derived therefrom formed angiotensin I. There are a multitude of these divisions of proteases (e.g. Renin, Angiotensin Converting Enzyme (ACE)).

Two human angiotensin receptors have been described so far were designated with AT1 and AT2. Both receptors are among the Arigiotensin II (Ang II) receptors, i.e. H. bind these receptors preferably Ang 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 seven hydrophobic areas, the potential transmembrane domains represent. Because of this characteristic domain structure assumed that the angiotensin receptors AT1 and AT2 belong to the family of 7-transmembrane receptors include the effects of their ligand Angiotensin II coupled via intracellular G protein Mediate signal transformation cascades.  

Due to the pharmacological effects caused by others from AngII various angiotensins or those derived from angiotensin I or II Peptides (endogenously formed fragments of Angl and Ang / l) mediated , it has been suggested that other human angiotensin receptors exist. In this context, a receptor for the Heptapeptide Ang1-7 required (Ferrario, cm et al. (1997) Hypertension 30, 535-541).

The present invention relates to a new angiotensin receptor, which has at least one subunit that has the amino acid sequence SEQ ID NO. Contains 10, this amino acid sequence optionally at the C- terminal end has further amino acids.

The angiotensin receptor is a transmembrane receptor. He points out in Contrary to the angiotensin receptors described so far preferably less than 7 transmembrane domains. The subunit, which have the sequence SEQ ID NO. 10 contains, preferably has one Transmembrane domain. This transmembrane domain is preferred a length of more than 6, preferably 8 or 9, particularly preferably 7 Amino acids. Preferably the transmembrane domain has the sequence "MSIVIPL" (specified in the one-letter code for amino acids).

The angiotensin receptor can have one or more subunits. For example, the receptor can either be as

• a) Monomer (a subunit containing sequence SEQ ID NO.10) or as
• b) Dimer with an identical or another subunit or as
• c) Tetramer with three other identical subunits or with one identical and two other subunits.

The angiotensin receptor can also be used up to the binding of the ligand Monomer (one subunit) or as a dimer (two different  Subunits) are present and only by binding the ligand in a pass dimeric or tetrameric form.

Receptors with a structure similar to that of the new angiotensin Receptor, including the insulin receptor, are 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 (e.g. IL1, IL5), and Adhesion molecules (integrins, selectins).

The new angiotensin receptor may be post-translational modified, for example the receptor can be glycosylated and / or be phosphorylated.

The angiotensin receptor can bind its ligand and the Mediate the effect of this ligand or these ligands intracellularly. Of the Angiotensin receptor can be angiotensin 1-7 and / or a derivative of Bind angiotensin 1-7. Derivatives of angiotensin 1-7 are preferred truncated peptides derived from Ang1-7, e.g. B. degradation products from Ang1-7. Examples of derivatives of Ang1-7 are angiotensin III and Angiotensin IV. The receptor preferably binds Ang1-7.

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 the sequence SEQ ID NO. 10 contains one At least 175 amino acids in length.

The invention further relates to a nucleic acid which has a sequence which encodes the new angiotensin receptor ("receptor"). For example, the receptor can be completely or partially by a Nucleic acid that has the sequence SEQ ID NO. 9 has to be encoded. A Nucleic acid, which is responsible for the receptor or a subunit of the receptor the sequence SEQ ID NO. 9 received and at the 3 'end  optionally have further nucleotides that form the coding sequence belong.

The nucleic acid can be, for example, a DNA, a cDNA or an RNA be. 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 For example, nucleic acid can be a salt or a nucleic acid Derivative, which besides the natural nucleotides also modified Contains nucleotides or consists entirely of modified nucleotides is.

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. For example, such a nucleic acid can be used in a PCR are amplified in the oligonucleotides whose sequences are homologous Areas (conserved areas) in the nucleotide sequences of the Angiotensin receptors correspond to AT1 and AT2, as primers and cDNA, which preferably from a tissue (or cells) onto which angiotensin 1-7 or a derivative thereof has an effect and / or from tissue (or cells), in which AT1 and / or AT2 are not expressed is used become. In a special embodiment of the method degenerate primers are used, the sequences of which differ from the homologous ones Derive regions of the sequences from AT1 and AT2. Preferably be the primers Ang3A and Ang3B were used for this. Can be used as a template for example cDNA made from endothelial cells can be used. For example, cDNA made from HUVECs (Human Umbilical Vein Endothelial cells) can be used as a template.

The invention also relates to methods for producing the new receptor. For example, the new angiotensin receptor can be made by adding a nucleic acid whose sequence to the angiotensin receptor  encoded and which preferably the sequence SEQ ID NO. 9 contains, in one suitable vector, the recombinant vector integrated into a cell introduced and the angiotensin receptor is expressed in this cell.

The invention further relates to the use of a nucleic acid for encodes the new angiotensin receptor and preferably the sequence SEQ ID NO. 9 contains, for example in a method of manufacture of a recombinant angiotensin receptor, the nucleic acid which is under the control of a suitable promoter in a cell is introduced and expressed.

The invention further relates to the use of a nucleic acid for. encodes the new angiotensin receptor and preferably the sequence SEQ ID NO. 9 contains for the production of a recombinant cell in which the Angiotensin receptor can be expressed.

The invention further relates to the use of a nucleic acid for encodes the angiotensin receptor and preferably the sequence SEQ ID NO. 9 contains, for the manufacture of a medicament for treatment and Prevention of diseases associated with cardiovascular dysregulation System, electrolyte balance or fluid balance accompanied.

The invention further relates to the use of a nucleic acid which is suitable for encodes the angiotensin receptor and preferably the sequence SEQ ID NO. 9 contains, for the production of a transgenic animal which does not contain the corresponding angiotensin receptor gene or one transgenic animal which has the corresponding angiotensin receptor gene overexpressed. For example, transgenic animals can be used for characterization the specificity and biochemical function of the angiotensin receptor be used.  

The invention further relates to the use of a nucleic acid for encodes the angiotensin receptor and preferably the sequence SEQ ID NO. 9 contains, in methods for identifying and characterizing Substances that act as antagonists and agonists of the angiotensin receptor can be used and / or in processes in which substances can be identified that encode the functional expression of the Inhibit or activate angiotensin receptor.

The invention also relates to the use of a nucleic acid for Angiotensin receptor encoded and which preferably the sequence SEQ ID NO. 9 contains, in gene therapy or for the manufacture of a medicinal product, which in gene therapy can be used.

Furthermore, 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. For example, the angiotensin receptor can be used in methods for Identification and characterization of substances that act as agonists or Antagonists of the receptor can be used (e.g. binding assays and functional assays for screening).

Furthermore, the angiotensin receptor or parts of the same z. B. a Epitope which e.g. B. by the amino acid sequence SEQ ID NO. 10 or part of the same is specified for the production of antibodies known methods (Harlow and Lane, "Antibodies-A Laboratory Manual", Cold Spring Harbor Laboratory, ISBN-0-87969-314-2) can be used. On thus produced antibodies that are specific to the new angiotensin Bind receptor can be used to detect the new receptor, e.g. B. in diagnostic method and / or for further characterization of the Receptor can be used.  

Detailed description of the invention

To find the new receptor, based on the known Sequences coding for the angiotensin II receptors AT1 and AT2, Oligonucleotides synthesized, whose sequences conserved areas of the AT1 / AT2 coding sequences correspond to or against them conserved areas (Ang3A and Ang3B). This Oligonucleotides were used in PCR reactions with cDNA from human HUVECs (Human Umbilical Vein Endothelial Cell) used as a template. This Cell line is derived from endothelial cells that line the blood vessels. These cells do not express AT1 or AT2 receptors.

It was a DNA fragment with an open reading frame and one so far amplified and not described nucleotide sequence (SEQ ID NO. 3) identified. This nucleotide sequence showed no agreement with Sequences in the EMBL or Genbank® databases. A Sequence comparison with sequences in the Lifeseq® database using the Program modules "Blast" and "Assembly" (GGC program package) resulted an overlap of SEQ ID NO. 3 with the Lifeseq® clones No. 1458429, 3341232, 3440195, 1250818, 2836520, 1310286 and 1362205 (ESTs without known function). That to be derived from this virtual contig- (3) theoretical 0.5 kbp DNA fragment was experimentally confirmed (example 2, PCR with the pair of primers Ang4A and Ang4B). The sequence of the DNA Fragments with the open reading frame contained therein were Extension (example 3) up to the vector sequence was elucidated (SEQ ID NO. 9). The SEQ ID NO. 9 also contains the 5'-untranslated region or part of the same.

An analysis of the 5 'region of the nucleotide sequence of SEQ ID NO. 9 showed that upstream of the assumed start methionine triplet (ATG) a purine nucleotide at position -3 and at positions -1 and -4  Cytosine nucleotides are localized. The location of these nucleotides on the Positions -1, -3 and -4 correspond to the eukaryotic consensus sequence for the mRNA translation initiation point.

The open reading frame in SEQ ID NO. 9 codes for 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 a hydrophobic domain with a length of about 7 at the N-terminus Has amino acids. This hydrophobic domain can be a Represent transmembrane domain with which this new receptor in the Membrane is anchored. On the other hand, the sequence has one at the C terminus more or less hydrophilic domain.

Examples example 1

For the first PCR, the oligonucleotide primers Ang3A and Ang3B were used, which include a 0.19 kbp DNA fragment - based on the AT112 receptor gene. CDNA from a commercially available HUVEC cDNA bank (Stratagene Catalog No. 937223, Stratagene GmbH, Heidelberg) was used as template DNA.
Nucleotide sequence of Ang3A:
SEQ ID NO. 1: 5'-TTG TKC TKK YCT TYW TCW TTT SCT GGM TTC CC-3 '
Nucleotide sequence of Ang3B:
SEQ ID NO. 2: 5'-TTY CCM ASA AAR CMA TAM ARA ARM GGA TT -3 '
in which
M = (A / C)
Y = (C / T)
R = (A / G)
S = (G / C)
K = (G / T)
W = (A / T)
means.

For the PCR reaction, 1 µg of cDNA from the HUVEC bank were included recombinant Thermus aquaticus (Taq) DNA polymerase (Boehringer Mannheim, Germany) and 10 µmol each of primers Ang3A and Ang 3B per 50 µl reaction mixture in a thermal cycler (Perkin Elmer 2400 thermal cycler, Perkin Elmer Applied Biosystems, Weiterstadt). 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). It was a 130 base pair (bp) long DNA fragment amplified.

This 130 bp DNA fragment was in the vector pCR 2.1 TOPO® (Invitrogen BV, NV Leek, The Netherlands) ligated and in E. coli Top10F ' transformed. The sequence of the 130 bp DNA present in pCR2.1 Fragments were analyzed using a capillary electrophoresis sequencer (ABI 310, Applied Biosystems, Weiterstadt) (SEQ ID NO. 3).

Example 2

The sequence SEQ ID NO. 3 was using the program modules FASTA and TFASTA (software package from the University of Wisconsin Genetics Computer Group (GCG), USA) with the sequences published in the accessible databases (e.g. EMBL, GenBank®) are compared. In addition, the sequence was used for a corresponding homology comparison with the sequences that are in the Lifeseq® database from Incyte (Palo Alto, USA) are used.  

In the Lifeseq® database, sequences of Expressed Sequence Tags (ESTs) were identified (Lifeseq® clones No. 1458429, 3341232, 3440195, 1250818, 2836520, 1310286 and 1362205), which are identified with SEQ ID NO. 3 overlap. The primers Ang4A and Ang4B were synthesized on the basis of a sequence which is derived from an arrangement of the overlapping Expressed Sequence Tags (ESTs) according to one's own assembly.
Nucleotide sequence of Ang4A:
SEQ ID NO. 4: 5'-TGG GGG TTG ATA CAG CAG AGA C -3 '
Nucleotide sequence of Ang4B:
SEQ ID NO. 5: 5'-GCA CTG CCC TCT CTT TAT CCA AA -3 '

There were PCR reactions with 0.1 µg cDNA from the HUVEC Bank as a template and 10 µmol primer Ang4A and Ang4B each per 50 µl Reaction approach carried out, the Advantage-cDNA polymerase mix (Clontech, Heidelberg) was used. For the PCR reactions The following temperature profile is 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).

Example 3

In order to clarify the sequence in the 5 'region of the 500 bp DNA fragment, the primers Ang5A and Ang5B were synthesized.
Nucleotide sequence of Ang5A:
SEQ ID NO. 7: 5'-TGG GGA TTG ATA GGC AG-3 '
Nucleotide sequence of Ang5B:
SQ ID NO. 8: 5'-ATA ACT GTT GGA TTT CTC AA -3 '

The nucleotide sequence of Ang5A and Ang5B corresponds to one part each the sequence of the 500bp DNA fragment. These two primers are used in further PCRs with the primers M13 Reverse or M13 Forward (-20) used. The primers M13 Reverse and M13 Forward (-20) correspond Sequences of the Uni-ZAP used to produce the HUVEC cDNA library Bacteriophage vector.

These PCR reactions were each with 0.1 µg cDNA from the HUVEC Bank Template and each 10 µmol 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 Antibody (Clontech) carried out. The PCR reactions were with the following temperature profile performed: 30 "96 ° C, 30" 50 ° C, and 45 "72 ° C (70 cycles).

A DNA fragment with a total length of 660 bp receive. This was cloned and sequenced as described in Example 1. The DNA fragment has the sequence SEQ ID NO. 9 on. The sequence SEQ ID NO. 9 contains the entire 5 'coding area.

In addition to the software modules already mentioned, the Implementation of the computer-aided sequence analysis of the GCG modules Assemble, Map, Translate, Plotstructure and Pileup used.

Table 1: SEQ ID NO. 9

(Partial) nucleotide sequence of the angiotensin receptor

Table 2: SEQ ID NO. 10th

(Partial) amino acid sequence of the new receptor

Table 3: SEQ ID NO. 3rd

Table 4: SEQ ID N0. 4th

Claims (13)

1. Angiotensin receptor, characterized in that it has at least one subunit that has the amino acid sequence SEQ ID NO. 10 contains. 2. Angiotensin receptor according to claim 1, wherein the amino acid sequence SEQ ID NO. 10 optionally at the C-terminal end has further amino acids. 3. Angiotensin receptor according to one or more of claims 1 and 2, the sequence SEQ ID NO. 10 a transmembrane domain. 4. Angiotensin receptor according to one or more of claims 1 to 3, the receptor angiotensin 1-7 or a derivative of Angiotensin 1-7 binds. 5. Angiotensin receptor according to one or more of claims 1 to 4, with at least one subunit of the receptor a nucleic acid with the sequence SEQ ID NO. 9 is encoded and the sequence SEQ ID NO. 9 if necessary at the 3 'end Contains nucleotides that belong to the coding sequence.   6. Method for producing a nucleic acid with the sequence SEQ ID NO. 9, being oligonucleotides whose sequences are homologous Areas in the nucleotide sequences of the angiotensin receptors AT1 and AT2 correspond as primers and cDNA made from Cells in which AT1 and AT2 receptors are not expressed, can be used as a template in a PCR reaction. 7. A method for producing an angiotensin receptor according to a or more of claims 1 to 5, wherein a nucleic acid, the the sequence SEQ ID NO. 9 contains, in a suitable vector integrated, the recombinant vector introduced into a cell and the angiotensin receptor is expressed in this cell. 8. Use of a nucleic acid which has the sequence SEQ ID NO. 9 contains, in a process for the production of a recombinant Angiotensin receptor, the nucleic acid that is under the Control of a suitable promoter is in a cell is introduced and expressed. 9. Use of a nucleic acid which has the sequence SEQ ID NO. 9 contains, for the production of a recombinant cell in which the Angiotensin receptor can be expressed.   10. Use of a nucleic acid which has the sequence SEQ ID NO. 9 for the manufacture of a medicament for treatment and / or disease prevention associated with misregulation the cardiovascular system, the electrolyte balance and / or Fluid balance. 11. Use of a nucleic acid which has the sequence SEQ ID NO. 9 contains, for the manufacture of a medicinal product that is used in gene therapy can be used. 12. Use of a nucleic acid which has the sequence SEQ ID NO. 9 contains, in a process of identification and Characterization of substances that act as agonists or Antagonists of the angiotensin receptor can be used can. 13. Use of an angiotensin receptor according to one or several of claims 1 to 5 in a method for Identification and characterization of angonists or Receptor antagonists.