CA2330229A1

CA2330229A1 - Novel angiotensin receptor, production and use thereof

Info

Publication number: CA2330229A1
Application number: CA002330229A
Authority: CA
Inventors: Holger Heitsch; Jorg-Peter Kleim; Gunther Riess; Gertrud Sibenhorn
Original assignee: Individual
Current assignee: Sanofi Aventis Deutschland GmbH
Priority date: 1998-06-08
Filing date: 1999-05-12
Publication date: 1999-12-16
Also published as: HUP0102540A3; JP2002517237A; KR20010052703A; BR9912183A; WO1999064585A2; ID27566A; AR020086A1; EP1086217A2; CN1304449A; HUP0102540A2; TR200003662T2; AU4361699A; PL345949A1; WO1999064585A3; DE19825494A1

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

Description Novel angiotensin receptor, its preparation and use The invention relates to a novel angiotensin receptor, its preparation and use.
The renin angiotensin system plays an important role in the regulation of the cardiovascular system and of electrolyte and fluid balance. The physiologically active effector hormones, e.g. angiotensin II (Angll) and angiotensin 1-7 (Ang1-7) are produced by proteolytic cleavage of the precursor angiotensinogen, which is synthesized in the liver, and, respectively, of angiotensin I, which is formed from the angiotensinogen. A
large number of proteases are involved in these cleavages (e.g. renin and angiotensin converting enzyme (ACE)).
Two human angiotensin receptors, which have been designated AT1 and AT2, have been described to date. Both receptors are angiotensin II
(Ang II) receptors, i.e. these receptors preferably bind 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 possess seven hydrophobic regions which constitute potential transmembrane domains. Because of this characteristic domain structure, it is assumed that angiotensin receptors AT1 and AT2 belong to the family of 7-transmembrane receptors, which mediate the effect of the ligand angiotensin II by way of intracellular G protein-coupled signal transduction cascades.
Because of the pharmacological effects which are mediated by other angiotensins differing from Angll and/or of the peptides (endogenously formed fragments of Angl and Angll) which are derived from angiotensin I
and angiotensin II, respectively, it has been suspected that further human angiotensin receptors must exist. In this connection, a receptor has recently been demanded for the heptapeptide Ang1-7 (Ferrario, CM et al. (1997) Hypertension 30, 535-541 ).

s The present invention relates to a novel angiotensin receptor which possesses at least one subunit which contains the amino acid sequence SEQ ID NO. 10, with this amino acid sequence possibly possessing further amino acids at the C-terminal end.
The angiotensin receptor is a transmembrane receptor. In contrast to the previously described angiotensin receptors, it preferably possesses fewer than 7 transmembrane domains. The subunit which contains the sequence SEQ ID NO. 10 preferably possesses one transmembrane domain. This transmembrane domain is preferably more than 6, preferably 8 or 9, particularly preferably 7, amino acids in length. The transmembrane domain preferably has the sequence "MSIVIPL" (written in the single letter code for amino acids).
The angiotensin receptor can possess one or more subunits. For example, the receptor can be present either as a) a monomer (one subunit which contains the sequence SEQ ID NO. 10), or as b) a dimer, containing an identical subunit or a different subunit, or as c) a tetramer, containing three further identical subunits or containing one identical subunit and two different subunits.
However, the angiotensin receptor can also be present as a monomer (one subunit) or as a dimer (two different subunits) until the ligand has been bound and only change into a dimeric or tetrameric form, respectively, as a result of binding the ligand.
Receptors which possess a similar structure to that of the novel angiotensin receptor are, inter alia, 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, hematopoietic cytokine receptors (e.g. IL1, IL5), and adhesion molecules (integrins and selectins).
Where appropriate, the novel angiotensin receptor can be modified post-translationally; for example, the receptor can be glycosylated and/or phosphorylated.

The angiotensin receptor can bind its ligand(s), and can mediate the effect of this/these ligand(s) intrac~llularly. 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 which are derived from Ang1-7, e.g. degradation products of 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 possesses or contains the amino acid sequence shown in Table 2, SEQ ID NO. 10. The angiotensin receptor, or the subunit which contains the sequence SEO ID NO. 10, is at least 175 amino acids in length.
The invention furthermore relates to a nucleic acid which possesses a sequence which encodes the novel angiotensin receptor ("receptor"). For example, the receptor can be encoded, entirely or partially, by a nucleic acid which possesses the sequence SEQ ID NO. 9. A nucleic acid which encodes the receptor or a subunit of the receptor can contain the sequence SEQ ID NO. 9 and, possibly, possess further nucleotides, which belong to the coding sequence, at the 3' end.
The nucleic acid may, for example, be a DNA, a cDNA or an RNA. The nucleic acid may be a gene, with it then being possible for the sequence SEQ ID NO. 9 to be interrupted by noncoding sequences (introns).
Where appropriate, the nucleic acid can be derivatized. A derivative of the nucleic acid can, for example, be a salt or a nucleic acid derivative which contains modified nucleotides in addition to the natural nucleotides or which is composed completely of modified nucleotides.
The invention also relates to a process for preparing a nucleic acid which encodes the angiotensin receptor and which contains the sequence SEQ
ID NO. 9. For example, such a nucleic acid can be amplified in a PCR
which uses oligonucleotides, as primers, whose sequences correspond to homologous regions (conserved regions) in the nucleotide sequences of the angiotensin receptors AT1 and AT2 and cDNA which is preferably prepared from a tissue (or cells) on 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. In a particular embodiment of the process, use is made of degenerate primers whose sequences are derived from the homologous regions of the sequences of AT1 and AT2. Preference is given to using the primers Ang3A and Ang3B for this purpose. cDNA which has been prepared from endothelial cells can, for example, be used as the template.
For example, cDNA prepared from HUVECs (human umbilical vein endeothelial cells) can be used as a template.
The invention also relates to a process for preparing the novel receptor. For example, the novel angiotensin receptor can be prepared by integrating a nucleic acid, whose sequence encodes the angiotensin receptor and which preferably contains the sequence SEQ ID NO. 9, into a suitable vector, with the recombinant vector being introduced into a cell and the angiotensin receptor being expressed in this cell.
The invention furthermore relates to the use of a nucleic acid, which encodes the novel angiotensin receptor and which preferably contains the sequence SEQ ID N0.9, for example in a process for preparing a recombinant angiotensin receptor, with the nucleic acid, which is under the control of a suitable promoter, being introduced into a cell and expressed in this cell.
The invention furthermore relates to the use of a nucleic acid, which encodes the novel angiotensin receptor and which preferably contains the sequence SEQ ID NO. 9, for preparing a recombinant cell in which the angiotensin receptor can be expressed. The invention relates to a recombinant cell which expresses an angiotensin receptor having the sequence SEQ ID NO. 10. The invention furthermore relates to the use of a nucleic acid, which encodes the angiotensin receptor and which preferably contains the sequence SEQ ID N0.9, for producing a drug for the treatment and prevention of diseases which are accompanied by faulty regulation of the cardiovascular system, of electrolyte balance or of fluid balance. The invention relates to a drug that comprises a nucleic acid having the sequence [lacuna] ID NO. 9.
The invention furthermore relates to the use of a nucleic acid, which encodes the angiotensin receptor and which preferably contains the sequence SEQ ID NO. 9, for producing a transgenic animal which does not contain the corresponding angiotensin receptor gene, or for producing a transgenic animal which overexpresses the corresponding angiotensin receptor gene. Transgenic animals can be used, for example, for characterizing the specificity and biochemical function of the angiotensin 5 receptor. The invention relates to a nonhuman transgenic animal which expresses the angiotensin receptor.
The invention furthermore relates to the use of a nucleic acid, which encodes the angiotensin receptor and which preferably contains the sequence SEQ ID NO. 9, in processes for identifying and characterizing substances which can be employed as antagonists and agonists of the angiotensin receptor and/or in processes in which substances which inhibit or activate the functional expression of the encoded angiotensin receptor are identified. The invention relates to a process for identifying and characterizing agonists or antagonists of the angiotensin receptor, with a nucleic acid which possesses the sequence SEQ ID N0.9 being introduced into a cell and the angiotensin receptor being expressed in the cell, this cell being incubated with a substance to be investigated and the effect of this substance on the angiotensin receptor being determined.
The invention also relates the use of a nucleic acid, which encodes the angiotensin receptor and which preferably contains the sequence SEQ ID
NO. 9, in gene therapy or for producing a drug which can be employed in gene therapy.
In addition to this, the invention relates to the use of a nucleic acid, which encodes the angiotensin receptor and which preferably contains the [lacuna] SEQ ID NO. 9, as a tool in molecular biology (e.g. as a probe).
The invention also relates to the use of the novel angiotensin receptor. For example, the angiotensin receptor can be used in processes for identifying and characterizing substances which can be employed as agonists or antagonists of the receptor (e.g. binding assays and functional assays for screening).
Furthermore, the angiotensin receptor, or parts thereof, for example an epitope which is predetermined, for example, by the amino acid sequence SEQ ID NO. 10 or a part thereof, can be used for preparing antibodies in accordance with known methods (Harlow and Lane, "Antibodies-A
Laboratory Manual", Cold Spring Harbor Laboratory, ISBN-0-87969-314-2).
Antibodies which have been prepared in this way, and which bind specifically to the novel angiotensin receptor, can be used for detecting the novel receptor, e.g. in diagnostic methods and/or for subjecting the receptor to further characterization.
Detailed description of the invention In order to find the novel receptor, use was made of the known sequences which encode the angiotensin II receptors AT1 and AT2 for synthesizing oligonucleotides whose sequences correspond to conserved regions of the AT1/AT2 coding sequences or which are directed against these conserved regions (Ang3A and Ang3B). These oligonucleotides were employed in PCR reactions using cDNA from human HUVECs (human umbilical _vein endothelial cell) as a template. This cell line is derived from endothelial cells which line the blood vessels. These cells do not express any AT1 or AT2 receptors.
A DNA fragment, having an open reading frame and a nucleotide sequence (SEQ ID NO. 3) which had not previously been described, was amplified and identified. This nucleotide sequence did not exhibit any concordance with sequences in the EMBL or Genbank~ databases. Comparison of its sequence with sequences in the Lifeseq~ database using the "Blast" and "Assembly" program modules (GGC program package) showed that SEQ
ID NO. 3 overlapped with the Lifeseq~ clones Nos. 1458429, 3341232, 3440195, 1250818, 2836520, 1310286 and 1362205 (ESTs of unknown function). The theoretical 0.5 kbp DNA fragment which could be derived from this virtual (3) contig was confirmed experimentally (Example 2, PCR
using the primer pair Ang4A and Ang4B). The sequence of the DNA
fragment, together with the open reading frame contained therein, was elucidated (SEQ ID NO. 9) by 5' extension (Example 3) into the vector sequence. The sequence SEQ ID NO. 9 also contains the 5'-untranslated region or a part thereof.
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 was located at position -3 and cytosine nucleotides were located at positions -1 and -4. The location of these nucleotides at positions -1, -3 and -4 corresponds to the eukaryotic consensus sequence for the start point for initiating mRNA translation.
The open reading frame in SEO ID NO. 9 encodes a protein containing at least 175 amino acids (SEQ ID NO. 10). A hydrophobicity analysis of this amino acid sequence, carried out using the GCG program, showed that the sequence possesses a hydrophobic domain of about 7 amino acids in length at the N terminus. This hydrophobic domain may represent a transmembrane domain which is used to anchor this novel receptor in the membrane. By contrast, the sequence possesses a domain at the C terminus which is more or less hydrophilic.
Examples:
Example 1:
The oligonucleotide primers Ang3A and Ang3B, which, based on the AT1/2 receptor genes, enclose a 0.19 kbp DNA fragment, were used for the first PCR. cDNA from a commercially available HUVEC cDNA library (Stratagene catalog No. 937223, Stratagene GmbH, Heidelberg) was used as a 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' where M = (A/C) Y = (C/T) R = (A/G) S = (G/C) K = (G/T) W = (A/T).
For the PCR reaction, 1 ,ug of cDNA from the HUVEC library was incubated with recombinant Thermus aquaticus (Taq) DNA polymerase (Boehringer Mannheim, Germany) and 10 pmol each of primers Ang3A and Ang3B
per 50,u1 reaction assay in a thermocycler (Perkin Elmer 2400 thermo-cycler, Perkin Elmer Applied Biosystems, Weiterstadt). The PCR reaction was carried out using the following temperature profile: 20" at 95°C, 10" at 38°C and 20" at 72°C (60 cycles). A DNA fragment of 130 base pairs (bp) in length was amplified.
This 130 by 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 by DNA fragment present in pCR2.1 was determined using a capillary electrophoresis sequencer (AB1310, Applied Biosystems, Weiterstadt) (SEQ ID NO. 3).
Example 2:
The sequence SEQ ID NO. 3 was compared with the sequences contained in the publicly available databases (e.g. EMBL and GenBank~) using the program modules FASTA and TFASTA (University of Wisconsin Genetics Computer Group (GCG), USA, software package). In addition, the sequence was used for a corresponding homology comparison with the sequences which are included in the Incyte company (Palo Alto, USA) Lifeseq0 database.
Sequences of expressed sequence tags (ESTs) which overlap with SEQ ID
N0.3 were identified in the Lifeseq4 database (Lifeseq~ clones Nos.1458429, 3341232, 3440195, 1250818, 2836520, 1310286 and 1362205). The primers Ang4A and Ang4B were synthesized on the basis of a sequence which the inventors assembled by ordering the overlapping expressed sequence tags (ESTs).
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' PCR reactions were carried out with in each case 0.1 ,ug of cDNA from the HUVEC library, as a template, and 10 pmol each of the primers Ang4A and Ang4B per 50,u1 reaction assay and using the Advantage cDNA
polymerase mix (Clontech, Heidelberg). The following temperature profile was used for the PCR reactions: 30" 94°C and 2'30" 60°C (60 cycles). A
DNA fragment of 500 by in length was obtained. This was cloned and sequenced (SEQ ID NO. 6) as described in Example 1. The sequence possesses an open reading frame (ORF).
Example 3:
The primers AngSA and AngSB were synthesized for elucidating the sequence of the 500 by DNA fragment in the 5' region.
Nucleotide sequence of AngSA:
SEQ ID NO. 7: 5'-TGG GGA TTG ATA GGC AG-3' Nucleotide sequence of AngSB:
SEQ ID NO. 8: 5'-ATA ACT GTT GGA TTT CTC AA-3' The nucleotide sequence of AngSA and AngSB in each case corresponds to a part of the sequence of the 500 by DNA fragment. These two primers are employed in further PCRs using the M13 reverse and M13 forward (-20) primers, respectively. The M13 reverse and M13 forward (-20) primers correspond to sequences of the Uni-ZAPO bacteriophage vector used for preparing the HUVEC cDNA library. These PCR reactions were carried out using in each case 0.1 Ng of cDNA from the HUVEC library, as a template, and 10 pmol each of the primers AngSA or AngSB and, respectively, M13 reverse or M13 forward (-20) (from the Topo-TA cloning kit (Clontech)) and Taq polymerase (Boehringer Mannheim) and also Taq StartT"" antibody (Clontech). The PCR reactions were carried out using the following temperature profile: 30" 96°C, 30" 50°C, and 45"
72°C
(70 cycles).
This resulted in a DNA fragment having a total length of 660 by being obtained. This fragment was cloned and sequenced as described in Example 1. The DNA fragment has the sequence SEO ID NO. 9. The sequence SEQ ID NO. 9 contains the entire 5' coding region.
The GCG modules Assemble, Map, Translate, Plotstructure and Pileup were also used for performing the computer-assisted sequence analyses, in addition to the software modules which have already been mentioned.

Table 1: SEQ ID NO. 9 (Partial) nucleotide sequence of the angiotensin receptor t51 ATTGGGGGTT GATACAGCAG AGACGTCATA CTTGGAAATG GCTGCAGGTT

5 Table 2: SEQ ID NO. 10 (Partial) amino acid sequence of the novel receptor 51 HHSHSYiGLPYADHNYGARPPPTPPASPPPSVLISKNEVG IFTTPNFDET

151 RQHIPDTYLCERC~PRNLDKERAVL

Table 3: SEQ ID NO. 3 Table 4: SEO ID NO. 6 201 CACCTCCGGC ?l'CCCCTCCT CCATCAGTCC TTATTAGCAA AAATGAAGTA

SEQUENCE LISTING
(1 ) GENERAL INFORMATION:
(i) Applicant:
(A) NAME: Hoechst Marion Roussel Deutschland GmbH
(B) STREET: -(C) CITY: Frankfurt (D) FEDERAL STATE: -(E) COUNTRY: Germany (F) POSTAL CODE: 65926 (G) TELEPHONE:069-305-7072 (H) FAX: 069-35-7175 (I) TELEX: -(ii) TITLE OF INVENTION: Novel angiotensin receptor, its prepara-tion and use (iii) NUMBER OF SEQUENCES: 10 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFfVIIARE: Patentln Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
REPLACEMENT SHEET (RULE 26) (ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION:1..32 (D) MISCELLANEOUS INFORMATION:/note =
"M = (A/C); Y = (C/T); R = (A/G); S = (G/C); K = (G/T);
W=(~-l.)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION:1..29 (D) MISCELLANEOUS INFORMATION:/note =
"M = (A/C); Y = (C/T); R = (A/G); S = (G/C); K = (G/T);
W=(~)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 94 base pairs (B) TYPE: Nucleotide REPLACEMENT SHEET (RULE 26) (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION:1..94 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION:1..22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

REPLACEMENT SHEET (RULE 26) (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION:1..23 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 510 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION:1..510 REPLACEMENT SHEET (RULE 26) (xi) SEQUENC E DESCRIPTION:
SEQ ID NO: 6:

ATCCGTAGAA GCTAGCCCT.GTGGTAGTTGA GAAATCCAACAGTTATCCCCACCAGTTATA120 TGGTG~~CGT CCTCC"TCCGACACCTCCGGC TTCCCCTCCTCCATCAGTCCTTRTTAGCAA240 CAGCACATCT GAGGATGGAAG:TATGG:AC TGATGTAACCAGGTGCATATGTGGTTTTAC360 ACATGATGAT GGATACATGATCTGTTGTGA CAAATGCAGCGT'-'TGGCAACATATTGACTG420 CATGG~vGATT GRTAGGCAGCATATTCCTGR TACATATCTATGTGAACGTTGTCAGCCTAG480 GAATTTGu74T AAAGAGAGGGCAGTGCTACT 510 (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION:1..17 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
TGGGGATTGA TAGGCAG 1~
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
REPLACEMENT SHEET (RULE 26) (ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION:1..20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 660 base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: exon (B) LOCATION:1..660 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
TCGTGTGTGrIACATCAGAGGGTTTGTGGATGCACTTAGAT GTTTGCAATGAGCACTGTGG60 CATTGGTTTGCCCTATGCGGACCATAATTATGGTGCTCGT CCTC~TCCGACRCCTCCGGC360 TTCCCCTCC.TCCATCAGTCCTTATTAGCAAAAATGAAGTA GGCATATTTACCACTCCTRA420 REPLACEMENT SHEET (RULE 26) (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 175 amino acids (B) TYPE: Amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Protein (ix) FEATURE:
(A) NAME/KEY: Peptide (B) LOCATION:1..175 (xi)SEQUENCE
DESCRIPTION:
SEQ
ID
NO:

10:

MetSer IleVal IlePro LeuGly ValAsp ThrAlaGlu ThrSer Tyr LeuGlu MetAla AlaGly SerGlu ProGlu SerValGlu AlaSer Pro ValVal ValGlu LysSer AsnSer TyrPro HisGlnLeu TyrThr Ser SezSer HisH=s SerHis SerTyr IleGly LeuProTyr AlaAsp His AsnTyr GlyAla ArgPro ProPro ThrPro ProAlaSer ProPro Pro 05 70 ~5 80 SerVal LeuIle SerLys AsnGlu ValGly IlePheThr ThrPro Asn PheAsp GluThr SerSer RlaThr TheIle 5erThrSer GluRsp Gly 100 105 lI0 SeeTyr GlyThr AspVal ThrArg CysZle CysGlyPhe ThrHis Asp AspGly TyrMet IleCys CysAsp LysCys SerValTrp GlnHis Zle AspCys MetGly IleAsp ArgGln HisIle ProAspThr TyrLeu Cys GluArg~CysGln ProArg AsnLeu AspLys GluArgAla ValLeu REPLACEMENT SHEET (RULE 26)

Claims

Claims:

1. An angiotensin receptor which possesses at least one subunit which contains the amino acid sequence SEQ ID NO. 10.

2. The angiotensin receptor as claimed in claim 1, wherein the amino acid sequence SEQ ID NO. 10 may possibly possess further amino acids at the C-terminal end.
3. The angiotensin receptor as claimed in one or more of claims claim 1 and 2, wherein the subunit which contains the sequence SEQ ID
NO. 10 possesses a transmembrane domain.
4. The angiotensin receptor as claimed in one or more of claims 1 to 3, wherein the receptor binds angiotensin 1-7 or a derivative of angiotensin 1-7.
5. The angiotensin receptor as claimed in one or more of claims 1 to 4, wherein at least one subunit of the receptor is encoded by a nucleic acid having the sequence SEQ ID NO. 9, and wherein the sequence SEQ ID NO. 9 possibly contains further nucleotides, which belong to the coding sequence, at the 3' end.
6. A process for preparing a nucleic acid having the sequence SEQ ID
N0.9, wherein, in a PCR reaction, oligonucleotides whose sequences correspond to homologous regions in the nucleotide sequences of the angiotensin receptors AT1 and AT2 are employed as primers and cDNA which is prepared from cells in which AT1 and AT2 receptors are not expressed is employed as a template.
7. A process for preparing an angiotensin receptor as claimed in one or more of claims 1 to 5, wherein a nucleic acid which contains the sequence SEQ ID NO. 9 is integrated into a suitable vector, the recombinant vector is introduced into a cell and the angiotensin receptor is expressed in this cell.
8. The use of a nucleic acid which contains the sequence SEQ ID
NO.9 in a process for preparing a recombinant angiotensin receptor, with the nucleic acid, which is under the control of a suitable promoter, being introduced into a cell and expressed in this cell.
9. The use of a nucleic acid which contains the sequence SEQ ID
NO. 9 for preparing a recombinant cell in which the angiotensin receptor can be expressed.
10. The use of a nucleic acid which possesses the sequence SEQ ID
NO. 9 for producing a drug for treating and/or preventing diseases which are accompanied by faulty regulation of the cardiovascular system, of electrolyte balance and/or of fluid balance.
11. The use of a nucleic acid which contains the sequence SEQ ID
NO. 9 for producing a drug which can be used in gene therapy.
12. The use of a nucleic acid which contains the sequence SEQ ID
NO. 9 in a process for identifying and characterizing substances which can be used as agonists or antagonists of the angiotensin receptor.

3. The use of an angiotensin receptor as claimed in one or more of claims 1 to 5 in a process for identifying and characterizing agonists or antagonists of the angiotensin receptor.

4. A process for identifying and characterizing agonists or antagonists of the angiotensin receptor, wherein a nucleic acid which possesses the sequence SEO ID NO.9 is introduced into a cell and the angiotensin receptor is expressed in this cell, this cell is incubated with a substance to be investigated and the effect of this substance on the angiotensin receptor is determined.

5. A drug which comprises a nucleic acid which possesses the sequence SEQ ID NO. 9.

6. A recombinant cell which is prepared by a nucleic acid having the sequence SEQ ID NO.9 being introduced into this cell and expressed in the cell.