WO1993005073A1 - RECEPTEURS D'ANGIOTENSINE IIcAMP/VASOPRESSINEV2 ET MOLECULES ET PROCEDES ASSOCIES - Google Patents

RECEPTEURS D'ANGIOTENSINE IIcAMP/VASOPRESSINEV2 ET MOLECULES ET PROCEDES ASSOCIES Download PDF

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WO1993005073A1
WO1993005073A1 PCT/US1992/007786 US9207786W WO9305073A1 WO 1993005073 A1 WO1993005073 A1 WO 1993005073A1 US 9207786 W US9207786 W US 9207786W WO 9305073 A1 WO9305073 A1 WO 9305073A1
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avp
receptor
aii
polypeptide
cell
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PCT/US1992/007786
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Nelson Ruiz-Opazo
Victoria L. M. Herrera
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The Trustees Of Boston University
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    • 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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to receptors, particularly angiotensin 11/ vasopressin receptors.
  • Angiotensin II (All) and vasopressin (arginine- vasopressin, AVP) receptors are both G protein-coupled receptors with diverse physiological roles (Crane et al., J. Biol . Chem . 257:4959. 1982; Rogers et al., J. Pharmacol . Exp. Ther. 236:438 1986; Douglas, Am . J. Physiol . 253:F1, 1987; Jard, Curr. Top. Mem. Transp. .18:255, 1983; Jard, Adv. Nephrol . 16:1, Physiol . Rev. 52:313, 1977; Capponi et al., in Biochemical Regulation of Blood Pressure , R.L.
  • AVP receptors respond to a nonapeptide hormone, arginine- vasopressin, affecting vasoconstriction and vasodilation; positive and negative cardiac chronotropy; regulation of the secretion of corticotropin by the adenohypophysis and increased firing rate of specific neuronol groups in the brain; induction of hepatocyte glycogenolysis and gluconeogenesis; and increased water reabsorption by collecting ducts and increased solute transport by ascending limb of Henle's loop in the kidney (Jard, 1983, suprar Jard, 1987, supra) .
  • isoreceptors have been described for both All and AVP based on differing coupling/effector pathways and affinity profiles to various agonist and antagonists.
  • All receptors two non-correlated classifications have been described, each with two subtypes.
  • type A is functionally coupled to the cAMP mobilizing effector pathways
  • type B is negatively coupled to the adenylate cyclase pathways (Douglas, 1987, supra) .
  • types 1 and 2 have been described, based on differential anatomical localization of nonpeptide ligand binding (Chiu et al. , Biochem. Biophys. Res . Comm. 165:196. 1989).
  • type 1 functionally coupled to calcium mobilizing effector pathways
  • type 2 type 2
  • antidiuretic type coupled to the adenylate cyclase system and found in kidney (Jard, 1983, supra) .
  • the invention features recombinant angiotensin II cAMP /vasopressin v2 (i.e., AII/AVP V2 ) receptor polypeptide, preferably, including an amino acid sequence substantially identical to the amino acid sequence shown in Fig. 1 (SEQ ID NO: 1) .
  • the invention also features a substantially pure polypeptide which is a fragment or analog of an AII/AVP V2 receptor and which includes a domain capable of binding angiotensin II (All) or arginine-vasopressin (AVP) (see below) .
  • the receptor is derived from a mammal, preferably, a human or a rat.
  • the invention further features a polypeptide including an All-binding portion of an AII/AVP V2 receptor, preferably, including amino acids 392 to 399 of Fig. 1 (SEQ ID NO: 1); a polypeptide including an AVP-binding portion of an AII/AVP V2 receptor, preferably, including amino acids 342 to 350 of Fig. 1 (SEQ ID NO: 1) ; and a polypeptide including an extracellular domain of an AII/AVP V2 receptor or an immunogenic analog thereof, preferably, including amino acids 30-94, amino acids 151- 251, amino acids 338-390, or amino acids 437-481 of Fig.
  • polypeptide may be a recombinant polypeptide.
  • AII/AVP V2 receptor polypeptide is meant all or part of a cell surface protein which specifically binds All and AVP and signals the appropriate All- and AVP-mediated cascade of biological events (leading, for example, to an increase in intracellular cAMP) .
  • polypeptide is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation) .
  • a “substantially pure polypeptide” is one which is substantially free of other proteins, carbohydrates and lipids with which it is naturally associated.
  • substantially identical amino acid sequence is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the biological activity of the receptor.
  • Such equivalent receptors can be isolated by extraction from the tissues or cells of any animal which naturally produce such a receptor or which can be induced to do so, using the methods described below, or their equivalent; or can be isolated by chemical synthesis; or can be isolated by standard techniques of recombinant DNA technology, e.g., by isolation of cDNA or genomic DNA encoding such a receptor.
  • derived from is meant encoded by the genome of that organism and present on the surface of a subset of that organism's cells.
  • the invention features purified DNA which encodes a receptor (or fragment or analog thereof) described above.
  • the purified DNA is cDNA; is purified DNA which encodes a rat AII/AVP V2 receptor; is purified DNA which encodes a human AII/AVP V2 receptor; is included in the plasmid pSVL-Al/V9; and is included in the plasmid pMAM-DR-AII/AVP v2 .
  • purified DNA is meant a DNA molecule which encodes an AII/AVP V2 receptor (or an appropriate receptor or analog) , but which is free of the genes that, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene encoding the AII/AVP V2 receptor.
  • the invention features vectors which contain such purified DNA and are capable of directing expression of the protein encoded by the DNA in a vector-containing cell; and cells containing such purified DNA (preferably eukaryotic cells, e.g., mammalian cells, e.g., COS 1 cells or C127 cells).
  • cells containing such purified DNA preferably eukaryotic cells, e.g., mammalian cells, e.g., COS 1 cells or C127 cells.
  • the expression vectors or vector-containing cells of the invention can be used in a method of the invention to produce recombinant AII/AVP V2 receptor polypeptide and the receptor fragments and analogues described above.
  • the method involves providing a cell transformed with DNA encoding an AII/AVP V2 receptor or a fragment or analog thereof positioned for expression in the cell; culturing the transformed cell under conditions for expressing the DNA; and isolating the recombinant AII/AVP V2 receptor protein.
  • transformed cell is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding an AII/AVP V2 receptor (or a fragment or analog, thereof) .
  • a DNA molecule is "positioned for expression” meaning that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of the AII/AVP V2 receptor protein, or fragment or analog, thereof) .
  • the invention features purified antibody which binds preferentially to an AII/AVP V2 receptor (or a fragment or analog thereof) .
  • purified antibody is meant one which is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally associated to permit therapeutic administration.
  • Such an antibody "preferentially binds" to an AII/AVP V2 receptor (or fragment or analog, thereof) , i.e., does not substantially recognize and bind to other antigenically-unrelated molecules.
  • the antibody neutralizes in vivo the protein to which it binds.
  • neutralize is meant to partially or completely block receptor-ligand binding.
  • the invention further features a method of testing a candidate compound for the ability to inhibit binding of All or AVP to an AII/AVP V2 receptor.
  • the method involves: a) contacting the candidate compound with a recombinant AII/AVP V2 receptor (or All- or AVP-binding fragment or analog) , preferably expressed on the surface of a recombinant cell, and with All or AVP; b) measuring binding of All or AVP to the receptor (or receptor fragment or analog) ; and c) identifying antagonist compounds as those which decrease such binding.
  • Preferred antagonists are those which also reduce the All- or AVP-mediated increase in the intracellular cAMP concentration of a cell bearing the recombinant receptor or receptor fragment or analog on its surface.
  • an “antagonist” is meant a molecule which also inhibits a particular activity, in this case, inhibition of the ability of All or AVP to bind an AII/AVP V2 receptor and, preferably which inhibits the biological events normally resulting from such binding (e.g., an increase in intracellular cAMP concentration) .
  • the antagonists i.e., the polypeptides or antibodies described above
  • the active ingredient may be formulated with a physiologically-acceptable carrier or anchored within the membrane of a cell.
  • the therapeutic compositions are used in a method of treating All- or AVP-mediated disorders, including increased contraction of blood vessels leading to hypertension.
  • the method involves administering the therapeutic composition to a mammal in a dosage effective to inhibit binding of All or AVP to an AII/AVP V2 receptor.
  • the proteins of the invention are involved in mediating the effects of angiotensin II and vasopressin (All and AVP, respectively) ; cells bearing AII/AVP V2 receptors derive (without limitation) from the kidney, the liver, the central nervous system, the heart, and the vasculature.
  • the diverse processes likely regulated by the proteins of the invention include water reabsorption and solute transport in the kidney; chronotropy and inotropy of the heart; stimulation of thirst and salt appetite centers in the brain; induction of the absorption of sodium and water in the intestine; and, of particular interest in the instant invention, modulation of blood vessel contraction.
  • Such proteins are therefore useful to treat or, alternatively, to develop therapeutics to treat hypertension and, generally, AII- or AVP-mediated disorders of the vascular system (e.g., stroke triggered, at least in part, by hypertension) .
  • Preferred therapeutics include antagonists e.g., peptide fragments, antibodies, or drugs, which block All or AVP ligand or AII/AVP V2 receptor function by interfering with the All or AVP: receptor interaction.
  • the instant invention provides a simple and rapid approach to the identification of useful therapeutics. Such an approach was previously difficult because of the presence on the surface of AII/AVP v2 receptor-bearing cells (e.g., vascular cells) of related receptors. Isolation of the AII/AVP V2 receptor gene (as cDNA) allows its expression in a cell type remote from those cells on whose surface the receptor normally resides, effectively providing a system for assaying an All:receptor or AVP:receptor interaction without interference caused by ligand interaction with related receptors.
  • AII/AVP V2 receptor gene as cDNA
  • a peptide- or antibody-based therapeutic may be produced, in large quantity and inexpensively, using recombinant and molecular biological techniques.
  • Fig. 1 is the nucleotide sequence and deduced amino acid sequence of the AII/AVP V2 receptor (SEQ ID NO: 1).
  • Fig. 2 is a tabular representation of the effect of AVP on cAMP accumulation is Xenopus laevis oocytes which were microinjected with A1/V9 mRNA.
  • Fig. 3 is a graphical representation of AII- induced and AVP-induced accumulation of cAMP in Cos 1 cells (A) and Cos A1/V9 cells (B) .
  • Fig. 4 A and B are bar graphs showing the effects of various putative ligands and antagonists on cAMP accumulation is Cos A1/V9 cells.
  • Fig. 5 is a graphical representation of cAMP accumulation in Cos A1/V9 cells as a function of All concentration (A) or AVP concentration (B) .
  • Fig. 6 is a tabular representation of the pharmacologic parameters of the AII/AVP V2 receptor.
  • Fig. 7A is a graphical representation of a dissociation analysis of All binding to Cos A1/V9 cells;
  • Fig. 7B is a Scatchard plot of the results of Fig. 7A.
  • Fig. 8A is a graphical representation of a saturation analysis of AVP binding to Cos A1/V9 cells;
  • Fig. 8B is a Scatchard plot of the results of Fig. 8A.
  • Fig. 9 is a graphical representation of a competition binding analysis of various All and/or AVP agonists or antagonists.
  • Fig. 10 is a hydropathy analysis of the AII/AVP V2 receptor.
  • Fig. 11 is the putative structure of the AII/AVP V2 receptor.
  • Fig. 12 A and B are graphical representations of (A) AVP-induced or (B) All-induced cAMP accumulation in cells expressing either wild-type or mutant AII/AVP V2 receptors.
  • Fig. 13 is a tabular representation of the effect of NaCl on AVP-dependent and All-dependent cAMP accumulation.
  • Polypeptides according to the invention include the entire human AII/AVP V2 receptor and the entire rat AII/AVP V2 receptor (as described in Fig. 1; SEQ ID NO: 1) . These polypeptides are used, e.g., to screen for antagonists which disrupt an interaction between All or AVP and the receptor (see below) . Polypeptides of the invention also include any analog or fragment of the human AII/AVP V2 receptor or the rat AII/AVP V2 receptor capable of interacting with All or AVP. Such analogues and fragments may also be used to screen for AII/AVP V2 receptor antagonists.
  • subset of receptor fragments or analogues which bind All or AVP and are, preferably, soluble (or insoluble and formulated in a lipid vesicle) may be used as antagonists to reduce AII/AVP V2 receptor-mediated disorders, e.g., those described herein.
  • the efficacy of a receptor analog or fragment is dependent upon its ability to interact with All or AVP; such an interaction may be readily assayed using any of a number of standard in vitro binding methods and AII/AVP V2 receptor functional assays (e.g., those described below) .
  • Specific receptor analogues of interest include full-length or partial (see below) receptor proteins including an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the receptors' ability to bind All or AVP (e.g., as assayed below) .
  • conservative amino acid substitutions for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the receptors' ability to bind All or AVP (e.g., as assayed below) .
  • Specific receptor fragments of interest include any portions of the AII/AVP V2 receptor which are capable of interaction with All or AVP V2 .
  • Such a portion preferably includes amino acids 392-399 or 342-350 of Fig. 1 (SEQ ID NO: 1) or an All or AVP-binding portion (respectively) , thereof.
  • Such fragments may be useful as antagonists (as described above) .
  • the extracellular domains i.e., amino acids 30 to 94; amino acids 151 to 251; amino acids 338 to 390; and amino acids 437 to 481) or fragments thereof (preferably, amino acids 193-200) are also useful as a source of immunogens for producing antibodies, e.g., those which neutralize the activity of the AII/AVP V2 receptor in vivo (e.g., by interfering with the interaction between the receptor and All or AVP) .
  • the secondary protein structure and, therefore, the extracellular domain regions may be deduced semi-empirically using a hydrophobicity/hydrophilicity calculation such as the Chou-Fasman method (see, e.g., Chou and Fasman, Ann. Rev. Biochem. 47.:251, 1978).
  • Hydrophilic domains, particularly ones surrounded by hydrophobic stretches e.g., transmembrane domains
  • extracellular domains may be identified experimentally using standard enzymatic digest analysis, e.g., tryptic digest analysis.
  • Candidate fragments are tested for interaction with All or AVP by the assays described herein. Such fragments are also tested for their ability to antagonize the interaction between All or AVP and its endogenous receptor using the assays described herein.
  • Analogues of useful receptor fragments (as described above) may also be produced and tested for efficacy as screening components or antagonists (using the assays described herein) ; such analogues are also considered to be useful in the invention.
  • Oligonucleotides were designed based on the complementary mRNA sequence of the rat AVP ligand and the rat All ligand (Ohkubo et al., Proc. Natl . Acad. Sci . USA .80:2196, 1983; Ivell and Richter, Proc. Natl . Acad. Sci . USA 81:2006, 1984). These oligonucleotides, of 24 and 26 bp in length, respectively were obtained from Research Genetics (Huntsville, AL) and were of sequence:
  • the oligonucleotide probe was 32 P end-labelled as described in Sambrook et al. (Molecular Cloning : A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1989) and used to screen an adult rat kidney cDNA library obtained from Clontech (Palo Alto, CA) .
  • Hybridization was carried out using 10 6 cpm/ml probe and the hybridization buffer: 6X SSPE [i.e., IM NaCl, 60mM NaH 2 P0 4 (pH 7.4), 6mM EDTA (pH 7.4)], 100 ug/ml denatured calf thymus DNA, 0.1% sodium pyrophosphate, 1% sodium dodecyl sulfate (SDS) , and 200 ug/ml polyadenylic acid. Filters were washed 3 times in 2X SSPE, 0.1% pyrophosphate, 0.1% SDS at 40°C; each wash was carried out for 15 minutes.
  • 6X SSPE i.e., IM NaCl, 60mM NaH 2 P0 4 (pH 7.4), 6mM EDTA (pH 7.4)
  • 100 ug/ml denatured calf thymus DNA 0.1% sodium pyrophosphate, 1% sodium dodecyl s
  • A1/V9 receptor cDNA Functionality of the A1/V9 receptor cDNA was also investigated; specifically, the A1/V9 cDNA was expressed in either Xenopus laevis oocytes or mammalian cells, and activation or inhibition of the adenylate cyclase system by AVP and All was determined as follows.
  • the 2.25 kb A1/V9 cDNA was subcloned, in both orientations, into the EcoRI site of the transcription vector, pSP73 (Promega Corp, Madison, WI) , and the clones were arbitrarily designated, Al/V9(+) and Al/V9(-).
  • RNA concentrations were ascertained by RNA blot analysis and densitometric quantitation of the autoradiographic signal (as carried out by standard techniques) ; 75 ng of each RNA was injected in 50 nl water; prior to microinjection, each oocyte was checked for the complete removal of its vitelline membrane, and vitelline membrane remnants were mechanically removed. Oocyte membranes were isolated as described in Colman (1984, supra) . and the adenylate cyclase assay was conducted as described in Murayama and Ui (J. Biol . Chem.
  • the Al/V9(-) RNA microinjected oocyte membranes did not show any increase in cAMP levels as compared to the basal levels but did have a 3-fold increase in cAMP accumulation upon the addition of 10 mM sodium fluoride (equal to that observed for the Al/V9(+) RNA- icroinjected oocyte membranes) , demonstrating that the adenylate cyclase system in the A1/V9(-) RNA- microinjected oocyte membranes were functionally active. From this experiment, it was concluded that A1/V9(+) contained the 2.25 kb A1/V9 cDNA inserted in the sense orientation.
  • the A1/V9 cDNA was then expressed by insertion (in the sense orientation) into the pSVL expression vector (PHARMACIA, Piscataway, NJ) and transfection into Cos 1 tissue culture cells. Specifically, the 2.25 kb A1/V9 cDNA was excised by digestion with Xhol and EcoRV and subcloned directionally (5' to 3') into the Xhol-Smal sites of the pSVL expression vector, to create plasmid pSVL-Al/V9. pSVL-Al/V9 was co-transfected into Cos 1 cells (i.e.. Green monkey kidney cells, ATCC Accession No.
  • Cos 1 cells i.e.. Green monkey kidney cells, ATCC Accession No.
  • Cos 1 cells were cotransfected with an unrelated cDNA-pSVL expression plasmid and pSV2neo in a 20:1 ratio, and an identical selection in G418 was performed. Transfectants were analyzed for the presence of the A1/V9 cDNA sequences. Southern blot analysis of genomic DNA obtained from Cos A1/V9 cells showed multiple copies of high molecular weight integrated (>8kb) and non-integrated (7kb) pSVL-Al/V9 sequences. As expected, at stringent hybridization conditions, no Al/V9-specific sequences were noted in the control mock-transfected Cos 1 cells.
  • RNA blot analysis was also performed as described in Herrera and Ruiz-Opazo (1990, supra) .
  • PCR amplification was carried out as described in PCR Protocols: A Guide to Methods and Applications (eds, Innis et al., 1990) using Al/V9-specific primers.
  • Transient and permanent Cos A1/V9 transfectants were assayed for receptor function. Because of a higher and more consistent level of expression, dissection of receptor function was subsequently carried out in the permanent Cos A1/V9 transfectants only.
  • Cells were grown in Dulbecco's modified Eagle's medium (DMEM) in 48- multiwell dishes.
  • DMEM Dulbecco's modified Eagle's medium
  • cAMP [ 1 5 I] Assay System, AMERSHAM) . Reactions were terminated by the addition of 2 volumes of 100% ethanol to the cells. To standardize the data with respect to variations of cAMP levels among independent but concordant groups of experiments, results were expressed as the percent stimulation of cAMP accumulation with respect to the zero time point.
  • Cos 1 cells Fig. 3A
  • Cos A1/V9 cells Fig. 3B
  • All ⁇
  • AVP A basal control
  • cAMP accumulation was measured from 30 seconds to 5 minutes. Each time point was performed in duplicate; the percent range of variation was 0.06-6% with a mean percent variation of 2%.
  • the Cos A1/V9 transfectants showed 60- 70% All-induced and 90-100% AVP-induced stimulation of cAMP accumulation over the control untransfected and mock-transfected Cos 1 cells.
  • the cAMP levels increased briskly reaching plateau levels at about 1 minute and the responses to AVP were consistently greater than the responses to All by a difference of approximately 30%.
  • Results from un-transfected and mock-transfected cells were identical; both exhibited minimal, if any, cAMP accumulation (Fig. 3A) .
  • the Cos A1/V9 cells did not respond to the peptide hormones, endothelin-1 or bradykinin.
  • the rank order for % stimulation of cAMP accumulation by the different angiotensin peptides was as follows: All > Al > AIII.
  • each assay point consisted of 10 6 cells cultured in P-35 dishes; binding assays were done in 1 ml of binding buffer (Rogers et al., 1986, supra) containing the appropriate concentration of ligand; cells with bound 125 I-AII were removed with 1 ml of 0.25 N NaOH, 0.25% SDS; for 3 H-AVP binding, each assay point consisted of 3 X 10 6 cells cultured in P-60 dishes; binding assays were done in 2 ml of binding buffer with the appropriate concentration of ligand; and cells with bound 3 H-AVP were removed with 2 ml of 10 mM Tris/HCl, pH 7.4, 10 mM EDTA, 3% Triton X- 100.
  • Fig. 7A shows the dissociation analysis of 125 ⁇ - AII specific binding performed on intact Cos A1/V9 cells. Each point represents the mean ⁇ range of variation (I) of three separate experiments with each point performed in duplicate. The percent variation was 0.1-6%; mean percent variation was 3.1%.
  • Fig 7B shows a Scatchard plot (LIGAND Program, McPherson) of the results of Fig. 7A. Two affinity sites are depicted. Affinity values and corresponding B max values are presented in Fig. 6. Results of the 125 I-AII displacement curve were analyzed for both All affinity sites (i.e., the low affinity site, K L , and the high affinity site, K H ) . B max values are shown in fmols/10 6 cells.
  • FIG. 8A illustrates the saturation analysis of 3 H- AVP specific binding to intact Cos A1/V9 cells. Each point represents the mean ⁇ range of variation (I) of three separate experiments with each point performed in duplicate. The percent variation was 0.8-13%; mean 0 percent variation was 5%.
  • Fig. 8B shows a Scatchard plot of the results of Fig. 8A. Affinity and B max values are * presented in Fig. 6.
  • the K H value for All obtained here was equivalent, if not 10-fold lower, than the K H values obtained in membrane binding assays done in the absence or presence of guanine nucleotides (Crane et al., 1982, supra; Rogers et al., supra 1986) and the K H value obtained in the binding assay of intact cells using radiolabeled antagonist (Rogers et al., 1986, supra) .
  • the level of expressed functional All/AVP receptors in Cos A1/V9 cells was comparable to, if not better than, the levels of All and/or AVP receptors (measured separately) in other cell lines or tissues.
  • the B max value for 125 I-AII binding in Cos A1/V9 cells of 354 fmol/mg membrane protein was at the median of the range of published B max value for All binding (i.e., 35 - 1300 fmol/mg membrane protein) obtained from rat tissues and primary cell lines (Gunther et al., Circ. Res . .42:278, 1980; Campanile et al., J. Biol . Chem . 257:4951, 1982; Rogers et al, 1986, supra: Douglas, Am. J. Physiol . 253:Fl. 1987; Bouscarel et al., J. Biol . Chem .
  • the number of A1/V9 cDNA-encoded AII/AVP V2 receptors expressed in Cos A1/V9 cells measured by either All or AVP binding ranged from 3.5 to 4.2 x 10 3 receptors per cell. This, again, was comparable to the number of expressed cDNA-encoded serotonin lc receptor in mouse fibroblast 3T3 cells (Julius et al.. Science 241:558, 1988).
  • the levels of All- and AVP- induced cAMP accumulation in Cos A1/V9 cells were within the range obtained in the analysis of AVP V2 _ type receptors in MDCK cells, (i.e., 46 pmol/mg membrane protein/min; (Friedlander and Amiel, Biochem . Biophy ⁇ . Acta 929:311. 1987); in somatic cell hybrid cells (i.e., 0.89 to 8.34 pmol/mg membrane protein/min Jans et al.
  • LLC/PK1 cells i.e., 653 pmol/mg membrane protein/ min (Jans et al. , 1990, supra) . Because of the novelty of the dual peptide ligand/single receptor system, competition by All and AVP for the other's specific binding was investigated as follows.
  • the specific Vl-type receptor antagonist [,9-mercaptol ⁇ , ⁇ cyclopenta-methylenepropionyl 1 , -0-Me-Tyr 2 , Arg 8 ]-AVP, abbreviated [d(CH2) 5 , Tyr(Me) ]-AVP, exhibited markedly less displacement, (Figs. 9 and 6) .
  • the displacement of 5 nM 3 H-AVP by the V1/V2 antagonist, [d(CH 2 ) 5 , D-Ile 2 , lie 4 ]-AVP was less effective than that exhibited by DVDAVP and slightly more effective than that by the VI- specific antagonist (Fig. 9) .
  • 100 nM of this V1/V2 antagonist completely displaced binding of InM 3 H-AVP, consistent with the amount used to block AVP-induced cAMP accumulation (Fig. 4A) .
  • the A1/V9 cDNA was sequenced as follows. Single strand M13 templates of overlapping restriction digest fragments (in both orientations) were sequenced using the dideoxy chain termination method of Sanger et al. (Proc. Natl . Acad. Sci . USA l : 5463, 1977) and Messing et al. (Nucl . Acids . Res . 9_:309, 1981). Nucleotide sequence analysis of the A1/V9 cDNA revealed a single long open reading frame encoding a protein of 481 amino acids, with a predicted molecular weight of 53,350 Kd. The nucleic acid sequence and deduced amino acid sequence are shown in Fig. 1 (SEQ ID NO:l).
  • the predicted molecular weight approximated the apparent molecular weight from photoaffinity labeling, chemical crosslinking, and ligand affinity blotting studies (Fahrenholz et al., Eur. J. Biochem. 182:589. 1985; Fahrenholz et al., J. Recep. Res. 8_:283, 1988; Marie and Roy, Mol . Pharmacol . 3_3.:432, 1988).
  • the sequence possessed a single region of high homology for each probe sequence: 4/8 amino acids (amino acids 39 ⁇ 399 ) for All, and 4/9 amino acids (amino acids 342 - 350 ) f° r AVP.
  • the regions of highest homology (58%) to the All and (78%) to the AVP oligonucleotide probes occurred only once and corresponded to the amino acid regions homologous to the All and AVP probes.
  • Regions posessing homology with the AVP cRNA oligonucleotide probe (1) and homology with the All cRNA oligonucleotide probe (2) are marked by brackets in Fig. 1 (SEQ ID NO: 1) ; identical nucleotides are dotted.
  • Fig. 10 depicts the hydropathy profile of the AII/AVP V2 receptor polypeptide.
  • the hydropathy index is noted on the y-axis and the number of the central amino acid in the 20-amino acid window is noted on the x-axis.
  • the irst putative membrane spanning region may be longer than the predicted 17 aa-long H ⁇ , however, the length of this o-helix was sufficient to span the plasma membrane (Adams and Rose, Cell 1:1007, 1985). H 2 - H 7 were also of sufficient length to span the plasma membrane (Adams and Rose, 1985, supra) .
  • Comparison of the AII/AVP V2 receptor sequence with known G protein-coupled non-peptide cationic ligand receptor sequences showed no significant homology.
  • AII/AVP V2 receptor most likely belonged to a new subclass of the superfamily of G protein coupled receptors, as expected considering the distinction that the AII/AVP V2 receptor is a small peptide ligand receptor and not a cationic agonist receptor nor a heterodimer glycoprotein hormone receptor.
  • H3 and H4 i.e., amino acids 202-213
  • H4 and H5 i.e., amino acids 282-289
  • the AII/AVP V2 receptor possessed charged amino acids in all seven predicted hydrophobic regions.
  • Helical wheel analysis of H1-H7 revealed amphipathic putative transmembrane domains consistent with a channel ⁇ like or transporter-like structure (Krupinski et al.. Science 244:1558 , 1990). This was consistent with the putative involvement of this kidney AII/AVP V2 receptor in an AVP-sensitive water channel in kidney epithelial cells.
  • serine (i.e., S) residues within protein kinase C phosphorylation consensus sequences were found to be located in the cytoplasmic loop between H4 and H5 and are circled in Fig. 1 (SEQ ID NO: 1) .
  • Fig. 11 depicts the putative structure of the AII/AVP V2 receptor. Based on the localization of the 7 hydrophobic regions (Hl-7) as putative transmembrane domains (depicted as barrels through the stippled plasma membrane) , the putative AVP binding site [with identical amino acids (•) to the antipeptide probe sequence and conservative amino acid substitutions ( ⁇ ) indicated] in the loop between H5 and H6, and the putative All binding site within the N-terminus of H6, a putative structure was determined with the N-terminus intracellularly and the C-terminus extracellularly. Potential phosphorylation sites are marked (*) .
  • a probe is designed based on the rat AII/AVP V2 gene sequence (Fig. 1; SEQ ID NO: 1) and used to probe a human kidney ⁇ gtll cDNA library (e.g., obtained from Clontech, Palo Alto, CA) ; such a probe preferably includes the entire 2.25 kb AII/AVP V2 receptor-encoding fragment.
  • Hybridization is carried out under low stringency conditions, specifically, using a hybridization buffer containing 5X SSPE, 0.1% SDS, 0.2 mg/ml calf thymus DNA, 1% bovine serum albumin, 1% polyvinyl pyrroiidine (PVP) , 1% Ficoll, and 10% formamide, at 37°C for 24 hours. Hybridization is followed by three washes in 2X SSPE and 0.1% SDS at 45°C for 15 minutes each. Hybridizing plaques are preferably purified 4 times. Probe preparation, hybridization, and plaque purification are carried out as described in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989) .
  • a putative human AII/AVP V2 receptor-encoding cDNA is verified by DNA sequencing (and comparison with the rat homologue described herein) and by expression in mammalian cells followed by receptor binding and functional assays as described herein. Identification and Isolation of an All/AVP v2 Mutant in Hypertensive Rats
  • a cDNA library was prepared from Dahl-salt sensitive hypertensive rats (DS rats) as described in Herrera and Ruiz-Opazo (Science 2 £:1023, 1990). This library was screened, using as a hybridization probe, a 1.3 kb Pstl/Bglll fragment of the A1/V9 cDNA (i.e., coding for amino acids 30 through 466 of Fig. 1; SEQ ID NO.
  • Hybridization was carried out under low stringency, specifically, in a hybridization buffer containing: 5X SSPE, 0.1% SDS, 0.2 mg/ml calf thymus DNA, 1% BSA, 1% PVP, 50% formamide, at 37°C for 24 hours. Hybridization was followed by three washes in 2X SSPE and 0.1% SDS at 45° for 15 minutes each.
  • a cDNA encoding a full length All/AVP v2 receptor was isolated from the DS rat library, and characterized by nucleic acid sequencing (as described above).
  • This clone termed C/R 163 , possessed a nucleic acid substitution (i.e., a T for a C) at nucleotide 487 resulting in an amino acid substitution (i.e., an arginine for a cysteine) at amino acid 163.
  • a nucleic acid substitution i.e., a T for a C
  • an amino acid substitution i.e., an arginine for a cysteine
  • Both the wild-type "DR" expression plasmid i.e., pMAM-DR-AII/AVP v2
  • the mutant "DS" expression plasmid i.e., pMAM-DS-AII/AVP V 2
  • C-127 cells i.e., 60 ⁇ g plasmid DNA/10 7 cells
  • crude membranes were prepared from the cells by the method of Takuwa et al. (J " . Clin. Invest.
  • the response to AVP and All differs significantly between the wild type DR-AII/AVP V2 receptor (i.e., pMRAlV9 in Fig. 12 and DR in Fig. 13) and the mutant DS-AII/AVP V2 receptor (i.e., pMSAlV9 in Fig. 12 and DS in Fig. 13); the DS-AII (AVP V2 receptor exhibited a 2-fold and 3-fold greater response to AVP and All, respectively.
  • DR-AII/AVP V2 receptor i.e., pMRAlV9 in Fig. 12 and DR in Fig. 13
  • the mutant DS-AII/AVP V2 receptor i.e., pMSAlV9 in Fig. 12 and DS in Fig. 13
  • the DS-AII AVP V2 receptor exhibited a 2-fold and 3-fold greater response to AVP and All, respectively.
  • Isolation of a mutation in the rat All/AVP v2 receptor which correlates with hypertension facilitates a screen which is used to identify human patients who are afflicted with hypertension or who are likely to develop hypertension in the future.
  • the screen is carried out as follows.
  • DNA from a human patient is isolated from blood cells as described in Innis et al. (PCR Protocols: A Guide to Methods and Applications , Academic Press) .
  • Polymerase chain reaction (PCR) primers are obtained commercially or synthesized using a Dupont (Willmington, DE) or Applied Biosystems (Foster City, CA) oligonucleotide synthesizer and the instructions of the supplier.
  • the sequence of the oligonucleotide primers correspond to sequences flanking the Cys 163 -containing exon of the human receptor gene; this particular exon is identified by sequence homology with the rat sequence (above) .
  • the primers are annealed to the isolated human DNA and PCR carried out by the techniques of Innis et al. (above) .
  • the PCR-amplified DNA is then sequenced (as described above) , and sequences examined for those exhibiting a mutation at the human amino acid corresponding to rat AII/AVP V2 receptor amino acid 163.
  • a patient whose AII/AVP V2 receptor DNA possesses such a mutation is diagnosed either as being hypertensive or as having a propensity toward developing hypertension.
  • Polypeptide Expression Polypeptides may be produced by transformation of a suitable host cell with all or part of an All/AVP v2 receptor-encoding cDNA fragment (e.g., the cDNAs described above) in a suitable expression vehicle.
  • All/AVP v2 receptor-encoding cDNA fragment e.g., the cDNAs described above
  • suitable expression vehicle any of a wide variety of expression systems may be used to provide the recombinant receptor protein.
  • COS 1 and C127 cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockville, MD; ATCC Accession Nos. CRL 1650 and CRL 1616, respectively) .
  • the method of transfection and the choice of expression vehicle will depend on the host system selected.
  • Mammalian cell transfection methods are described, e.g., in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989) ; expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P.H. Pouwels et al., 1985, Supp. 1987).
  • pMAMneo provides: an RSV-LTR enhancer linked to a dexamethasone- inducible MMTV-LTR promotor, an SV40 origin of replication which allows replication in mammalian systems, a selectable neomycin gene, and SV40 splicing and polyadenylation sites.
  • DNA encoding the human or rat AII/AVP V2 receptor or an appropriate receptor fragment or analog (as described above) is inserted into the pMAMneo vector in an orientation designed to allow expression.
  • the recombinant receptor protein is isolated as described below.
  • Other preferable host cells which may be used in conjunction with the pMAMneo expression vehicle include COS cells, CHO cells, and C127 cells (ATCC Accession Nos. CRL 1650, CCL 61, and CRL1616, respectively) .
  • the human or rat AII/AVP V2 receptor (or receptor fragment or analog) is produced by a stably- transfected mammalian cell line.
  • cDNA encoding the receptor is cloned into an expression vector which includes the dihydrofolate reductase (DHFR) gene.
  • DHFR dihydrofolate reductase
  • the AII/AVP V2 receptor-encoding gene into the host cell chromosome is selected for by inclusion of 0.01-300 ⁇ M methotrexate in the cell culture medium (as described in Ausubel et al., supra) . This dominant selection can be accomplished in most cell types. Recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described in Ausubel et al. (supra) ; such methods generally involve extended culture in medium containing gradually increasing levels of methotrexate.
  • DHFR-containing expression vectors commonly used for this purpose include pCVSEII-DHRF and pAdD26SV(A) (described in Ausubel et al., supra) .
  • Any of the host cells described above or, preferably, a DHFR-deficient CHO cell line e.g., CHO DHFR " cells, ATCC Accession No. CRL 9096
  • a DHFR-deficient CHO cell line e.g., CHO DHFR " cells, ATCC Accession No. CRL 9096
  • All/AVP v2 receptor protein fragment or analog, thereof is expressed, it is -solated, e.g., using affinity chromatography.
  • All, AVP, or an anti-AII/AVP v2 receptor antibody e.g., produced as described below
  • AVP an anti-AII/AVP v2 receptor antibody
  • Lysis and fractionation of ptor-harboring cells prior to affinity chromatography performed by standard methods (see, e.g., Ausubel supra) .
  • the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g.. Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, eds.. Work and Burdon, Elsevier, 1980).
  • Receptors of the invention can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, IL) .
  • Useful receptor fragments or analogues in the invention are those which interact with All or AVP. Such an interaction may be detected by an in vitro binding assay (described herein) .
  • the receptor component may also be assayed functionally, i.e., for its ability to bind All or AVP and mediate an increase in intracellular cAMP (described herein) .
  • These assays include, as components. All or AVP and a recombinant AII/AVP V2 receptor (or a suitable fragment or analog) configured to permit detection of binding.
  • All and AVP may be obtained from Sigma (St. Louis, MO) .
  • the AII/AVP V2 receptor component is produced by a cell that naturally presents substantially no receptor, e.g., by engineering such a cell to contain nucleic acid encoding the receptor component in an appropriate expression system.
  • Suitable cells are, e.g., those discussed above with respect to the production of recombinant receptor, such as COS 1 cells or C127 cells.
  • the binding assay is preferably performed by isolating membranes from recombinant cells expressing the AII/AVP V2 receptor protein and detecting specific binding of a radiolabelled ligand as label in association with the membrane preparation.
  • the assay may also be performed by fixing the recombinant cell expressing the AII/AVP V2 receptor component to a solid substrate (e.g., a test tube, a microtiter well, or a column) by means well known to those in the art (see, e.g., Ausubel et al., supra) and presenting labelled All or AVP (e.g., 3 H-labelled AVP or 125 I-labelled All) to the immobilized cells. Binding is assayed by the detection of label in association with the receptor component (and, therefore, in association with the solid substrate) .
  • a solid substrate e.g., a test tube, a microtiter well, or a column
  • the format may be any of a number of suitable formats for detecting specific binding, such as a radioimmunoassay format (see, e.g., Ausubel et al., supra) .
  • a radioimmunoassay format see, e.g., Ausubel et al., supra
  • cells transiently or stably transfected with an AII/AVP V2 receptor expression vector are immobilized on a solid substrate (e.g., the well of a microtiter plate) and reacted with All or AVP which is detectably labelled, e.g., with a radiolabel or an enzyme which can be assayed, e.g., alkaline phosphatase or horseradish peroxidase.
  • binding may be detected using a related assay.
  • All or AVP may be adhered to a solid substrate (e.g., a microtiter plate using methods similar to those for adhering antigens for an ELISA assay; Ausubel et al., supra) and the ability of labelled AII/AVP V2 receptor-expressing cells (e.g., labelled with 3 H-thymidine; Ausubel et al., supra) can be used to detect specific receptor binding to the immobilized All or AVP.
  • a solid substrate e.g., a microtiter plate using methods similar to those for adhering antigens for an ELISA assay; Ausubel et al., supra
  • AII/AVP V2 receptor-expressing cells e.g., labelled with 3 H-thymidine; Ausubel et al., supra
  • a vector expressing the AII/AVP V2 receptor is transfected into Cos 1 or C127 cells by the DEAE dextran- chloroquine method (Ausubel et al., supra) .
  • Expression of the receptor protein confers binding of detectably- labelled All or AVP to the cells. Neither All nor AVP binds significantly to untransfected host cells or cells bearing the parent vector alone; these cells are used as a "control" against which the binding assays are measured.
  • 10 cm. tissue culture dishes are seeded with AII/AVP V2 receptor-expressing Cos l or C127 cells (approximately 750,000 cells, dish) 12-18h post- transfection.
  • a recombinant receptor may also be assayed functionally for its ability to mediate an All or AVP and AII/AVP V2 receptor-dependent increase in intracellular cAMP.
  • Cells preferably Cos l cells transfected with an AII/AVP V receptor expression vector, are assayed for intracellular cAMP levels as described herein.
  • a recombinant receptor which promotes an increased level of intracellular cAMP upon All or AVP treatment are receptors useful in the invention.
  • one aspect of the invention features screening for compounds that antagonize the interaction between All or AVP and the AII/AVP V2 receptor, thereby preventing or reducing the cascade of events that are mediated by that interaction.
  • the elements of a screen to identify antagonists are All or AVP and recombinant All/AVP v2 receptor (or a suitable receptor fragment or analog, as outlined above) configured to permit detection of binding. All and AVP are publically available from Sigma (see above) . Full-length rat or human AII/AVP V2 receptor protein (or an All- or AVP- binding fragment or analog) may be produced as described herein.
  • Binding of All or AVP to its receptor may be assayed by any of the methods described above.
  • cells expressing recombinant AII/AVP V2 receptor (or a suitable AII/AVP V2 receptor fragment or analogue) are immobilized on a solid substrate (e.g., the well of a microtiter plate or a column) or membranes including recombinant protein are isolated and reacted with detectably-labelled All or AVP (as described above) . Binding is assayed by the detection label in association with the receptor component (and, therefore, in association with the solid substrate or membrane) .
  • Binding of labelled All or AVP to receptor-bearing cells is used as a "control" against which antagonist assays are measured.
  • the antagonist assays involve incubation of the AII/AVP V2 receptor-bearing cells with an appropriate amount of candidate antagonist. To this mix, an equivalent amount of labelled All or AVP is added.
  • An All or AVP antagonist useful in the invention specifically interferes with labelled All or AVP binding to the immobilized receptor-expressing cells.
  • An antagonist is then tested for its ability to interfere with AII/AVP V2 receptor function, i.e., to specifically interfere with labelled AII/AVP V2 receptor:ligand binding without resulting in the signal transduction normally mediated by the ligand.
  • These properties of useful antagonists are tested using the functional assay described herein. Specifically, Cos 1 cells expressing the recombinant receptor are reacted with All or AVP, and the intracellular cAMP levels are measured. This is considered to be a "control" level. Addition of potential antagonists along with, or just prior to addition of, All or AVP allows for the screening and identification of authentic receptor antagonists. Such an antagonist prevents the All- or AVP-mediated increase in cAMP levels.
  • Appropriate candidate antagonists include AII/AVP V2 receptor fragments, particularly fragments containing an All- or AVP-binding portion, e.g., amino acids 392-399 and amino acids 342-350 (described above) ; such fragments preferably include five or more amino acids.
  • Other candidate antagonists include analogues of All or AVP and other peptides as well as non-peptide compounds designed or derived from analysis of the receptor and anti-AII/AVP v2 receptor antibodies.
  • Human or rat AII/AVP V2 receptor may be used to raise antibodies useful in the invention.
  • receptor fragments preferred for the production of antibodies are those fragments deduced or shown experimentally to be extracellular.
  • Antibodies directed to AII/AVP V2 receptor peptides are produced as follows. Peptides corresponding to the All- or AVP-binding portion (e.g., amino acids 392-399 and 342-350, respectively) or to all or part of a putative extracellular domain (i.e., amino acids 30 to 94, amino acids 151 to 251, amino acids 338 to 390, and amino acids 437 to 481, and preferably, amino acids 193- 200 of Fig. 1; SEQ ID NO: 1) are produced using a peptide synthesizer, by standard techniques (see, e.g.. Solid Phase Peptide Synthesis, supra; Ausubel et al. , supra) or by recombinant means (Ausubel et al.
  • the peptides may be coupled to a carrier protein, such as KLH as described in Ausubel et al, supra.
  • KLH-peptide is mixed with Freund's adjuvant and injected into guinea pigs, rats, or preferably rabbits.
  • Antibodies are purified by peptide antigen affinity chromatography. Once produced, antibodies are tested for specific All/AVP v2 receptor recognition by Western blot or immunoprecipitation analysis (by the methods described in Ausubel et al., supra) .
  • Antibodies which specifically recognize the AII/AVP V2 receptor are considered to be candidates for useful antagonists; such candidates are further tested for their ability to specifically interfere with the interaction between All or AVP and the AII/AVP V2 receptor (as described above) or AII/AVP V2 receptor function (as described above) .
  • Antibodies which antagonize AII:AII/AVP V2 receptor binding or AVP:AII/AVP V2 receptor binding or AII/AVP V2 receptor function are considered to be useful as antagonists in the invention.
  • Therapy Therapeutics for the treatment of hypertension are the soluble antagonist receptor fragments described above formulated in an appropriate buffer such as physiological saline.
  • the fragment may include a sufficient number of adjacent transmembrane residues.
  • the fragment may be associated with an appropriate lipid fraction (e.g., in lipid vesicles or attached to fragments obtained by disrupting a cell membrane) .
  • anti- AII/AVP V2 receptor antibodies produced as described above may be used as a therapeutic. Again, the antibodies would be administered in a pharmaceutically-acceptable buffer (e.g., physiological saline). If appropriate, the antibody preparation may be combined with a suitable adjuvant.
  • the therapeutic preparation is administered in accordance with the condition to be treated. Ordinarily, it will be administered intravenously, at a dosage that provides suitable competition for All or AVP binding. Alternatively, it may be convenient to administer the therapeutic orally, nasally, or topically, e.g., as a liquid or a spray. Again, the dosages are as described above. Treatment may be repeated as necessary for alleviation of disease symptoms. Antagonists may also be administered to prevent (as well as treat) hypertension; the antagonist is administered as described above.
  • AII/AVP V2 receptor antagonists can be used to treat or prevent disorders such as hypertension and related illness (e.g., stroke triggered by hypertension).
  • the methods of the invention may be used to reduce the disorders described herein in any mammal, for example, humans, domestic pets, or livestock. Where a non-human mammal is treated, the AII/AVP V2 receptor or receptor fragment or analog or the antibody employed is preferably specific for that species.
  • GAG GGC ATC CTG AAG CAT CAA GCA CAG TTC TCA GAA AAG GAC CTG GAG 2 Glu Gly lie Leu Lys His Gin Ala Gin Phe Ser Glu Lys Asp Leu Glu 50 55 60
  • CTCAGCCCCA TAACCGCCAA TACCTCCCTT TCTGGGCCCA CCAATCTGTC CCTTGAAGAT 1884

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Abstract

L'invention concerne des ADNc codant des récepteurs d'angiotensine II/vasopressineV2 (AII/AVPV2), les protéines recombinées exprimées à partir desdits ADNc, ainsi que des anticorps spécifiques auxdites protéines. On utilise le récepteur ainsi que des analogues du récepteur recombiné dans des procédés de triage de composés potentiels intéressants dans leur capacité à s'opposer à une interaction entre AII ou AVP et un récepteur de AII/AVPV2; les antagonistes sont utilisées comme agents thérapeutiques dans le traitement de l'hypertension. On utilise les ADNc ainsi que le récepteur protéique et des analogues de récepteurs protéiques afin de dépister l'hypertension chez des individus présentant une propension à l'hypertension.
PCT/US1992/007786 1991-09-11 1992-09-11 RECEPTEURS D'ANGIOTENSINE IIcAMP/VASOPRESSINEV2 ET MOLECULES ET PROCEDES ASSOCIES WO1993005073A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999046290A1 (fr) * 1998-03-12 1999-09-16 Shanghai Second Medical University Gene humain de type recepteur d'angiotensine ii/vasopreessine (aii/avp) (cbdakd01)
WO2001014564A2 (fr) * 1999-08-20 2001-03-01 Curagen Corporation Nouveaux polynucleotides exprimes dans les lymphocytes t actives et proteines codees par ces polynucleotides
US7183379B2 (en) 2000-12-22 2007-02-27 Bristol-Myers Squibb Company Human leucine-rich repeat containing protein expressed predominately in small intestine, HLRRSI1

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NATURE, Vol. 351, issued 16 May 1991, MURPHY et al., "Isolation of a cDNA Encoding the Vascular Type-1 Angiotensin II Receptor", pp. 233-236. *
NATURE, Vol. 357, issued 28 May 1992, LOLAIT et al., "Cloning and Characterization of a Vasopressin V2 Receptor and Possible Link to Nephrogenic Diabetes Insipidus", pp. 336-339. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999046290A1 (fr) * 1998-03-12 1999-09-16 Shanghai Second Medical University Gene humain de type recepteur d'angiotensine ii/vasopreessine (aii/avp) (cbdakd01)
WO2001014564A2 (fr) * 1999-08-20 2001-03-01 Curagen Corporation Nouveaux polynucleotides exprimes dans les lymphocytes t actives et proteines codees par ces polynucleotides
WO2001014564A3 (fr) * 1999-08-20 2001-11-22 Curagen Corp Nouveaux polynucleotides exprimes dans les lymphocytes t actives et proteines codees par ces polynucleotides
US7183379B2 (en) 2000-12-22 2007-02-27 Bristol-Myers Squibb Company Human leucine-rich repeat containing protein expressed predominately in small intestine, HLRRSI1

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