INTRODUCTION Technical Field The technical field of this invention concerns the identification and cloning of an angiotensin π type 2 receptor, a peptide hormone receptor involved in growth and differentiation, and the use of this novel receptor in the diagnosis and treatment of cardiovascular disease.

Background

Angiotensin π is an octapeptide hormone that induces a variety of physiological responses including vasoconstriction in cardiovascular tissues, reduced glomerular filtration rate in the kidneys, catecholamine and aldosterone secretion from adrenal gland, dipsogenia in the brain, and the induction of salt appetite and drinking behavior. Accordingly, there is a profound medical interest in regulating and exploiting host responsiveness to this molecule. For over two decades, investigators have attempted to identify and characterize angiotensin π receptors. The physiological response diversity and studies with analogs of angiotensin π led researchers to speculate that multiple heterogeneous receptor types exist. Studies with nonpeptide angiotensin II receptor antagonists such as DuP 753 and PD 123177/123319 have classified receptor binding sites as type 1 (ATi, which bind Dup 753) or type 2 (AT ₂ , which bind PD 123319). Recently, cDNAs encoding a rat and a bovine ATi receptor were reported.

The abundant expression of AT ₂ receptors in fetal tissues, immature brain, skin wound and atretic ovarian follicles suggest a role in growth and development. Like ATi receptors, AT ₂ receptors also appear to be involved in neointima

iυimauυn aiier vascular injury. Unfortunately, the structure anu cuaiauiri υi uic AT ₂ receptors remain unknown. Also unknown are the clinical implications of therapeutic use of AT _r specific antagonists on AT ₂ -mediated pathways, either directly or indirectly through altered serum angiotensin II levels. Thus, for both the development of new therapeutics and the optimization of existing treatments, especially relating to hypertension, there is an urgent need to define the AT ₂ structure.

Using AT ₂ receptor-specific antagonists, several studies have attempted to characterize AT ₂ receptor structure and function. Despite these efforts and in part because of the receptor's instability and poor immunogenicity, AT ₂ receptors have defied definitive identification, characterization and cloning. Without a source of recombinant AT ₂ receptor, it has not been possible to assemble a defined assay to screen for modulators of AT ₂ receptor-mediated growth/differentiation. Cloning and characterizing an AT ₂ receptor would provide the pharmaceutical industry with the critical, missing ingredient for drug development relating to AT ₂ receptor targeting, including cancer, cardiovascular disease, reproductive medicine, etc.; for refining existing therapeutic use of I^ receptor antagonists; and for developing specific cellular and model animal systems for the development of cardiovascular disease therapy.

Relevant Literature

For background on the angiotensin π receptors, see: Whitebread, S., Mele, M., Kamber, B. & de Gasparo, M. Biochem. biophys. Res. Commun. 163, 284- 291 (1989); Chiu, A.T. et al. Biochem. biophys. Res. Commun. 165, 196-203 (1989); Millan, M.A. et al. Science 244, 1340-1342 (1989); Tsutsumi, K., Stromberg, C, Viswanathan, M. & Saavedra, J.M. Endocrinology 129, 1075- 1082 (1991); Millan, M.A., Jacobowitz, D.M., Aguilera, G. & Catt, K.J. Proc. natn. Acad. Sci. U.S.A. 88, 11440-11444 (1991); Pucell, A.G., Hodges, J.C., Sen, I., Bumpus, F.M. & Husain, A. Endocrinology 128, 1947-1959 (1991); Dudley, D.T., Hubbell, S.E. & Summerfelt, R.M. Mol. Pharmacol. 40, 360-367 (1991); Yamano, Y., Ohyama, K., Chaki, S., Guo, D.F. & Inagami, T. Biochem. biophys. Res. Commun. 187, 1426-1431 (1992); Ohyama, K., Yamano, Y., Chaki, S., Kondo, T. & Inagami, T. Biochem. biophys. Res. Commun. 189, 677-683 (1992); Janiak, Pillon, Prost and Vilaine Hypertension 20, 737-745 (1992).

For the cloning of the type 1 receptor, see, Sasaki, K. et al. Nature 351, 230-233 (1991); Sasamura, H., Hein, L., Krieger, J.E., Pratt, R.E., Kobilka, B.K. & Dzau, V.J. Biochem. biophys. Res. Commun. 185, 253-259 (1992); Murphy, TJ., Alexander, R.W., Griendling K.K., Runge, M.S. & Bernstein, K.E. Nature 351, 233-236 (1991); Iwai, N. & Inagami, T. FEBS Lett. 298, 257- 260 (1992).

For a description of other related proteins, see: Yamada, Y. et al. Proc. natn. Acad. Sci. U.S.A. 89, 251-255 (1992); Rens-Domiano, S. et al. Mol. Pharmacol. 42, 28-34 (1992); Sokoloff, P., Giros, B., Martres, M.P., Bouthenet, M.L. & Schwartz, J.C. Nature 347, 146-151 (1990); McEachern, A.E. et al. Proc. natn. Acad. Sci. U.S.A. 88, 7724-7728 (1991); Sreedharan, S.P., Robichon, A., Peterson, K.E. & Goetzl, E.J. Proc. natn. Acad. Sci. U.S.A. 88, 4986-4990 (1991); O'Dowd, B.F., Lefkowitz, R.J. & Caron, M.G. A. Rev. Neurosci. 12, 67-83 (1989); Gressens, P., Hill, J.M., Gozes, I., Fridkin, M. & Brenneman, D.E. Nature 362, 155-158 (1993).

SUMMARY OF THE INVENTION Angiotensin π type 2 receptors (AT ₂ receptor), structurally related cell receptors, nucleic acids encoding such AT ₂ receptors, AT ₂ receptor-related peptides, and methods of using AT ₂ receptors and AT ₂ receptor-encoding nucleic acids are provided. In particular, the present invention provides for the cloning of a class of unique seven-transmembrane receptors fulfilling the criteria of AT ₂ receptors. AT ₂ receptor-specific binding compounds are disclosed including antibodies to AT ₂ receptor epitopes. The invention provides AT ₂ receptor-based pharmaceutical compositions and AT ₂ receptor-based methods for screening chemical libraries for regulators of cell growth/differentiation. In particular, the invention provides methods for identifying agonists and antagonists of the disclosed AT ₂ receptors. Such compositions find broad utility in the treatment of cardiovascular disease, cancer, reproductive disease, etc. Additionally, the invention provides specific cellular and animal systems for the development of cardiovascular disease therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Binding characteristics of [ ¹²⁵ I]CGP42112A to membranes prepared from COS-7 cells transfected with the mouse AT ₂ receptor cDNA. Saturation isotherm of the specific binding of [ ¹²⁵ I]CGP42112A to membranes from COS-7 cells expressing the mouse AT ₂ receptor. Inset shows a Scatchard plot of the same data. The estimated maximal binding, B _^ , and K,, values were 0.58 pmol per mg protein and 0.12 nM, respectively.

FIG. 2. Displacement of specific [ ¹²⁵ -QCGP42112A binding in the mouse AT ₂ receptor cDNA-transfected COS-7 cell membranes by unlabeled CGP42112A (•), [Sar ¹ , -αe ⁸ ]-Ang H (O), Ang H (A), Ang I (□), PD123319 (■) and DuP753 (x).

FIG. 3. Restriction map (α) and nucleotide (SEQ ID NO:05) and deduced amino- acid sequence (SEQ ID NO:03) (b) of the rat AT ₂ receptor clone ρMRAT2. The open bar shows the coding region. Putative transmembrane domains I-Vπ are indicated above the sequence. (♦), potential N-glycosylation sites; (#), potential phosphorylation sites; two possible polyadenylation signals are underlined; selective mRΝA destabilizing signals are indicated by boxes, c, Sequence comparison between rat AT ₂ (SEQ ID ΝO:03) and AT (SEQ ID NO:04) receptors. Boxes, identical amino-acid residues; bars, conservative substitutions. 6 conserved cysteine residues are outlined. Bold letters show consensus residues in the G protein-coupled receptor superfamily. d, Comparison in the third intracellular loop of rat AT ₂ (SEQ ID NO:06), mouse SSTR1 (SEQ ID NO:07) and rat D ₃ (SEQ ID NO:08) receptors (148 amino acids are not shown), and of the Drosophila cell polarity gene product Fz (SEQ NO:09) and two rat homologues, Fz-1 (SEQ ID NO: 10) and Fz-2 (SEQ ID NO: 11). Boxes indicate conserved residues.

DESCRIPTION OF SPECIFIC EMBODIMENTS The present invention discloses a class of unique seven-transmembrane receptors fulfilling the criteria of AT ₂ receptors: 1) ligand binding specificity, 2) effect of dithiothreitol (DTT) on binding characteristics, 3) lack of effect of guanylnucleotide analogues, 4) lack of phosphoinositide or calcium signalling, and 5) tissue distribution and developmental pattern of expression. In one

embodiement, the invention provides isolated portions of the receptors, the AT ₂ genes, and transcripts thereof.

As used herein, a "unique" portion of the disclosed AT ₂ receptors or a AT ₂ peptide is a peptide with a sequence unique to that disclosed in that it is not found in any previously known proteins. Thus a unique portion has an amino acid sequence length at least long enough to define a novel polypeptide. An AT ₂ receptor portion is an at least about five, preferably at least about ten, more preferably at least about twenty amino acid sequence of the disclosed AT ₂ receptor. Unique portions of the disclosed AT receptor are readily identified by comparing the disclosed sequence with known protein sequence data bases. Particularly preferred unique portions are disclosed herein.

The disclosed full length AT ₂ receptor sequences are about 363 amino acids. A substantially fiill-length sequence comprises or encodes at least about 240, preferably at least about 300, more preferably at least about 340 amino acids of the disclosed AT ₂ receptor sequence; and typically includes at least 3, preferably about seven of the disclosed AT ₂ receptor transmembrane regions.

An "angiotensin π type-2 binding specificity" means the subject polypeptide retains a molecular conformation specific to one of the disclosed AT ₂ receptors that is specifically recognizable by a ligand, agonist, antibody, etc. of the AT ₂ receptor. As such, AT ₂ receptor binding specificities include AT ₂ receptor-specific catalytic and signal transducing activity, immunological epitopes, lectin binding sites, and preferably AT ₂ angiotensin π, agonist or antagonist binding sites.

"Specific binding" or recognition is empirically determined by contacting, for example the disclosed AT ₂ receptor, with a mixture of components and identifying those components that preferentially bind the receptor. Specific binding is most conveniently shown by displacement of labeled ligand using recombinant AT ₂ receptor cellular expression systems disclosed herein. Alternatively, solubilized, e.g. truncated or detergent treated, receptor may be used. Generally, specific binding of the subject AT ₂ receptor has binding affinity of IQr ⁶ M, preferably 10 ^"8 M, more preferably 10 ^"10 M, under optimized in vitro conditions or conditions as exemplified below.

"Xenogeneic" AT ₂ receptor analogs are nonhuman-derived proteins with substantial sequence identity to the disclosed AT ₂ receptor. Nonhuman sources of

AT ₂ receptors analogs include animal sources, such as Drosophila. Spodoptera. and preferably mammalian sources. Of particular interest are rodents, primates, and livestock animals including bovine, ovine, equine and avian species

"Substantial sequence similarity" means at least about 80%, more preferably at least about 90%, and most preferably at least about 95% sequence identity. Where substantially similar sequences diverge, the differences are preferably conservative, i.e. an acidic for an acidic amino acid substitution, a hydrophobic for a hydrophobic amino acid. Dissimilar sequences are typically aggregated within regions, especially within regions identified herein as transmembrane regions, rather than being distributed evenly over the polymer, and conserved (identical) sequences are typically aggregated within regions identified herein as other than transmembrane regions.

An "isolated" peptide or polypeptide (poly/peptide) is unaccompanied by at least some of the material with which it is associated in its natural state. Generally, an isolated poly/peptide constitutes at least about 1 %, preferably at least about 10%, and more preferably at least about 50% by weight of the total peptide and protein in a given sample. Included in the poly/peptide weight are alternative forms such as differentially glycosylated or phosphorylated or otherwise post- translationally modified forms. A stained band of the polypeptide is readily identified by Coomassie staining when the polypeptide, in isolated form, is subjected to electrophoresis according to the method of Laemmli, U.K. (1970) Nature 227, 680-685.

A composition comprising substantially pure poly/peptide is at least about 10%, preferably at least about 30%, more preferably at least about 70% by weight total peptide and protein. By pure polypeptide is intended at least about 90% , preferably at least 95%, and more preferably at least about 99% by weight of protein. Protein peptide weight percentages are determined by dividing the weight of AT ₂ receptor or fragments thereof, including alternative forms and analogs such as alternatively spliced, differentially phosphorylated or glycosylated, or otherwise post-translationally modified AT ₂ receptors, present in a fraction by the total protein/peptide weight present.

The invention provides polypeptides containing unique peptides (and sequence homologs) of the disclosed AT ₂ receptor having an AT ₂ -specific binding

property. AT ₂ receptor peptides find use in defining functional domains of AT ₂ receptors, identifying compounds that associate with AT ₂ receptors, designing compounds capable of modifying AT ₂ -receptor mediated cell signalling, for example, by binding or modulating an epitope or exploiting structural features of AT ₂ receptors to directly modify signal transmission or transduction, as immunogens, and the like. Therapeutic AT ₂ peptides are provided that are capable of interfering with AT ₂ receptor-ligand binding or AT ₂ receptor-mediated signal transmission or transduction. Typically, such peptides are effective by competitively inhibiting AT ₂ receptor association with another compound, typically an AT ₂ receptor ligand/agonist. Preferred peptides are selected for binding angiotensin, agonist or antagonist binding affinity using an AT ₂ receptor cellular expression system such as disclosed herein. Particularly useful peptides include the ligand binding site (e.g. including lysine at position 183 such as peptide 164-184 and peptide 182-202), regions involved in ligand selectivity (e.g. surrounding His273) regions of low hydrophobicity (e.g. N- or C-terminal peptides and peptides spanning the regions between the transmembrane domains), and regions of low homology to ATj (e.g. peptide 103-120 and 219-239), including fragments thereof. Other binding and associational domains of AT ₂ receptors are ascertainable by those skilled in the art using the methods and compositions disclosed herein. For example, AT ₂ receptor mutants, including deletion mutants are generated from the disclosed AT ₂ receptor sequence and used to identify regions important for specific protein-ligand or protein-protein interactions, for example, by assaying for the ability to mediate AT ₂ signal transmission/transduction. Further, x-ray crystallographic data of the disclosed protein are used to rationally design binding molecules of determined structure or complementarity.

Selected peptides, preferably derived from recombinant products, are also readily modified through physical, chemical, and molecular techniques disclosed or cited herein or otherwise known to those skilled in the relevant art. For example, site directed mutagenesis of nucleotides in the DNA encoding the disclosed AT ₂ receptor or for portions less than about 60 amino acids, in vitro peptide synthesis. Typically, amino acid insertions of about 1 to 10 amino acids, substitutions of single residues, and deletions of about 1 to 60 amino acids are made. Generally, mutatations of the disclosed AT ₂ receptors are located in the non-transmembrane

regions. Preferred mutations include:

Deletions: Insertions: Substitutions:

Lys215 215-Glu-Ser-Ala Lys215:Arg

Aspl41-Tyrl43 Asp90:Glu

(G-protein coupling Arg235-Lys256: region) Trp219-Arg240

Arg235-Lys240 His273:Xxx

Asp90

The subject peptides are subject to post-translational/sythesis modification to further modulate binding specificity/affinity by for examples, selection of the appropriate expression host, chemical/enzymatic intervention, etc. In particular, many of the disclosed AT ₂ receptor peptides sequence contain serine and threonine residues which are phosphorylated or dephosphorylated. Preferred targets for phosphorylation are serine 348, 351, 352, 353 and threonine 346. See e.g. methods disclosed in Roberts et al. (1991) Science 253, 1022-1026 and in Wegner et al. (1992) Science 256, 370-373. Many of the disclosed AT ₂ receptor peptides also contain glycosylation patterns which may modified, e.g. by enzymes like glycosidases or used to purify/identify the receptor, e.g. with lectins. Thus, mutagenesis is used to eliminate the N or O-linked glycosylation sites of the disclosed AT ₂ receptor, e.g. Asn residues at 156, 242, 247, 314, 323 are sequentially deleted or substituted for by another basic amino acid such as Lys or His for N-linked glycosylation alterations, or deletions or polar substitutions are introduced at the forementioned Ser and Thr residues for modulating O-linked glycosylation. Glycosylation variants are also produced by selecting appropriate host cells, e.g. yeast, insect, or various mammalian cells, or by in vitro methods such as neuaminidase digestion. Useful expression systems include COS-7, 293, BHK, CHO, TM4, CV1, VERO-76, HELA, MDCK, BRL 3A, W138, Hep G2, MMT 060562, TRI cells, for examples. Other covalent modifications of the disclosed AT receptor may be introduced by reacting the targeted amino acid residues with an organic derivatizing (e.g. methyl-3-[(p-azido-phenyl)dithio]

propioimidate) or crosslinking agent (e.g. l,l-bis(diazoacetyl)-2-phenylethane) capable of reacting with selected side chains or termini. For therapeutic and diagnostic localization, the receptor and peptides thereof may be labeled directly (radioisotopes, fluorescers, etc.) or indirectly with an agent capable of providing a detectable signal, for example, a heart muscle kinase labeling site. Especially useful modifications (e.g. fatty acid-acylation, proteolysis, and site-directed mutations) alter receptor solubility, membrane transportability, stability, binding specificity and affinity, and signal transduction capability.

Compounds that specifically bind a portion of the disclosed AT ₂ receptor are obtained using immunologic, chromatographic or synthetic methods available to those skilled in the art. Such compounds include specifically binding oligopeptides or oligonucleotides and specific antibodies that can be modified to a monovalent form, such as Fab, Fab', or Fv, and most preferably, small molecular weight organic angiotensin H agonists and antagonists. Anti-idiotypic antibody, especially internal imaging anti-ids are also prepared using the disclosures herein. Exemplary methods for producing AT ₂ receptor-specific polyclonal or monoclonal antibodies are disclosed in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

AT ₂ -specific compounds find a wide variety of uses including therapeutic intervention in disease; for instance, AT ₂ receptor-associated proteins are isolated by affinity immobilization or immunoprecipitation of AT ₂ receptor complexes from cell lysates or membrane fractions. AT ₂ receptor-derived peptides can be synthesized in pure form and also find use in diagnosis and therapy, for example, to modulate native AT ₂ receptor interaction with native ligand. The oligopeptides will generally be more than six and fewer than about 60 amino acids, more usually fewer than about 30 amino acids, although large oligopeptides may be employed. A portion of the AT ₂ receptor may be used in purified form, generally greater than about 90%, usually greater than about 95% pure. Methods for purifying such peptides to such purities include various forms of chromatographic, chemical, and electrophoretic separations disclosed herein or otherwise known to those skilled in the art.

The invention provides isolated nucleic acid sequences encoding an AT ₂ receptor or unique portion thereof, including sequences substantially identical or

homologous to sequences encoding such a receptor or epitope thereof. An "isolated" nucleic acid sequence is present as other than a naturally occurring chromosome or transcript in its natural state and typically is removed from at least some of the nucleotide sequences with which it is normally associated with on a natural chromosome. A complementary sequence hybridizes to a unique portion of the disclosed AT ₂ receptor sequence under low stringency conditions, for example, at 50°C and SSC (0.9 M saline/0.09 M sodium citrate) and that remains bound when subject to washing at 55 °C with SSC. Regions of non-identity of complementary nucleic acids are preferebly or in the case of homologous nucleic acids, a nucleotide change providing a redundant codon. A partially pure nucleotide sequence constitutes at least about 5%, preferably at least about 30%, and more preferably at least about 90% by weight of total nucleic acid present in a given fraction.

Unique portions of the disclosed nucleic acid sequence are of length sufficient to distinguish previously known nucleic acid sequences. Thus, a unique portion has a nucleotide sequence at least long enough to define a novel oligonucleotide. Preferred nucleic acid portions encode a unique AT ₂ receptor peptide. The nucleic acids of the invention and portions thereof, other than those used as PCR primers, are usually at least about 60 bp and usually less than about 6 kb in length. PCR primers are generally between about 15 and 100 nucleotides in length.

The nucleotide (cDNA) sequence encoding a full length AT ₂ receptor is included herein as Fig 3. The disclosure also provides for the disclosed sequence modified by transitions, transversions, deletions, insertions, or other modifications such as alternative splicing and also provides for genomic AT ₂ receptor sequences, and gene flanking sequences, including regulatory sequences; included are DNA and RNA sequences, sense and antisense. Preferred DNA sequence portions include portions encoding the preferred amino acid sequence portions disclosed above, portions between about 6 and about 36 amino acids including Lys215, or Asp90, the peptide Aspl41-Tτyl43 or the peptide Arg235-Lys240. For antisense applications where the inhibition of AT ₂ receptor expression is indicated, especially useful oligonucleotides are between about 10 and 30 nucleotides in length and include sequences surrounding the disclosed ATG start site, especially the

oligonucleotides defined by the disclosed sequence beginning about 5 nucleotides before the start site and ending abo t 10 nucleotides after the disclosed start site. Other especially useful AT ₂ receptor mutants involve deletion or substitution modifications of the disclosed cytoplasmic C-terminus. Accordingly, AT ₂ receptor mutants with angiotensin II binding affinities but with altered intracellular signal transductions capacities are produced. Preferred C-terminus deletion mutants with impair transduction ability include AT ₂ receptor with deleted amino acids 314-363 or 326-363.

For modified AT ₂ receptor-encoding sequences or related sequences encoding proteins with AT ₂ receptor-like functions, there will generally be substantial sequence identity between at least a segment thereof and a segment encoding at least a portion of the disclosed AT ₂ receptor sequence, preferably at least about 60%, more preferably at least 80%, most preferably at least 90% identity. Homologous segments are particularly within regulatory regions and regions encoding protein domains involved in protein-protein or protein-ligand interactions, particularly AT ₂ receptor-angiotensin II interactions and differences within such segments are particularly conservative substitutions,

Typically, the invention's AT ₂ receptor encoding polynucleotides are associated with heterologous sequences. Examples of such heterologous sequences include regulatory sequences such as promoters, enhancers, response elements, signal sequences, polyadenylation sequences, etc., introns, 5' and 3' noncoding regions, etc. Other useful heterologous sequences are known to those skilled in the art or otherwise disclosed references cited herein. According to a particular embodiment of the invention, portions of the AT ₂ receptors encoding sequence are spliced with heterologous sequences to produce fusion proteins. For example, most of the AT ₂ receptor peptides are expressed as soluble, secreted fusion proteins using appropriate signal sequences and optionally, a fusion partner such as β-Gal.

The disclosed sequences are also used to identify and isolate natural AT ₂ receptors analogs, including xenogeneic analogs. Further, as the present disclosure documents a unique class of seven-transmembrane receptors for which G protein coupling has not been demonstrated, the disclosed compositions and methods are used to identify, characterize, isolate, and purify other members of this class. In particular, the disclosed nucleic acid sequences are used as

hybridization probes under low-stringency or PCR primers, e.g. oligonucleotides encoding functional AT ₂ receptor domains are ³² P-labeled and used to screen λcDNA libraries at low stringency to identify similar cDNAs that encode proteins with related functional domains. Additionally, nucleic acids encoding at least a portion of the disclosed AT ₂ receptor are used to characterize tissue specific expression of AT ₂ receptor as well as changes of expression over time, particularly during organismal development or cellular differentiation.

The AT ₂ receptor encoding nucleic acids can be subject to alternative purification, synthesis, modification, sequencing, expression, transfection, administration or other use by methods disclosed in standard manuals such as Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), Current Protocols in Molecular Biology (Eds. Aufiibel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY, NY, 1992) or that are otherwise known in the art. For example, the nucleic acids can be modified to alter stability, solubility, binding affinity and specificity, etc. AT ₂ receptor-encoding sequences can be selectively methylated, etc. The nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescers, biotinylation, etc. The invention also provides vectors comprising nucleic acids encoding AT ₂ receptor or analogs. A large number of vectors, including plasmid and viral vectors, have been described for expression in a variety of eukaryotic and prokaryotic hosts. Advantageously, vectors may also include a promoter operably linked to the AT ₂ receptor-encoding portion. Vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. The inserted AT ₂ receptor coding sequences may be synthesized, isolated from natural sources, prepared as hybrids, etc. Suitable host cells may be transformed/transfected/infected by any suitable method including electroporation, CaCl ₂ mediated DNA uptake, viral infection, micromjection, microprojectile, or other methods.

Appropriate host cells include bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. Of particular

interest are E. coli. B. subtilis. Saccharomyces cerevisiae. SF9 cells, C129 cells, 293 cells, Neurospora, and CHO, COS, HeLa cells, immortalized mammalian myeloid and lymphoid cell lines, and pluripotent cells, especially mammalian ES cells and zygotes. Preferred replication systems include M13, ColEl, SV40, baculovirus, lambda, adenovirus, AAV, BPV, etc. A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced AT ₂ receptor or analogs.

For the production of stably transformed cells and transgenic animals, nucleic acids encoding the disclosed AT ₂ receptor may be integrated into a host genome by recombination events. For example, such a sequence can be micromjected into a cell, and thereby effect homologous recombination at the site of an endogenous gene, an analog or pseudogene thereof, or a sequence with substantial identity to an AT ₂ receptor-encoding gene. Other recombination-based methods such as nonhomologous recombinations, deletion of endogenous gene by homologous recombination, especially in pluripotent cells, etc., provide additional applications. Preferred transgenics and stable transformants over-express the disclosed receptor gene and find use in drug development and as a disease model. Alternatively, knock-out cells and animals find use in development and functional studies. Methods for making transgenic animals, usually rodents, from ES cells or zygotes are known to those skilled in the art. The compositions and methods disclosed herein may be used to effect gene therapy. See, e.g. Zhu et al. (1993) Science 261, 209-211; Gutierrez et al. (1992) Lancet 339, 715-721. For example, cells are transfected with AT ₂ receptor sequences operably linked to gene regulatory sequences capable of effecting altered AT ₂ receptor expression or regulation. To modulate AT ₂ receptor translation, cells may be transfected with complementary antisense polynucleotides.

For gene therapy involving the transfusion of AT ₂ receptor transfected cells, administration will depend on a number of variables that are ascertained empirically. For example, the number of cells will vary depending on the stability

of the transfused cells. Transfusions media is typically a buffered saline solution or other pharmacologically acceptable solution. Similarly the amount of other administered compositions, e.g. transfected nucleic acid, protein, etc., will depend on the manner of administration, purpose of the therapy, and the like. The invention provides methods and compositions for identifying agents useful in modulating host angiotensin π responsiveness. Such agents find use in the diagnosis or treatment of disease, particularly cardiovascular disease and cancer.

Typically, prospective agents are screened from large libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.

Useful agents are identified with a range of assays employing an AT ₂ receptor or encoding nucleic acids. Particularly, cells transfected with AT ₂ receptor-encoding DNA is used in in vitro binding assays with prospective agonists/antagonists/ligands. For instance, the effect of prospective agents on AT ₂ receptor-angiotensin π binding may be assayed. Preferred transfects encode deletion mutants, especially C-terminal deletion mutants, of the disclosed receptor sequence. Alternatively, the intracellular C-terminal domain is substituted with a sequence encoding a oligopeptide or polypeptide domain that provides an intracellular signal upon ligand binding different from the natural receptor. More preferably, such signal is more easily detected as a direct indicia of specific receptor-ligand binding to the host cell surface. Useful intracellular domains include those of the human insulin receptor and the TCR, especially domains with kinase activity and domains capable of triggering calcium influx which is conveniently detected by fluorimetry by preloading the host cells with Fura-2. Preferred assays are amenable to scaled-up, high throughput usage suitable for volume drug screening. Such screening will typically require the screening of at least about 10, preferably at least about 100, and more preferably at least about 1000 agents per week. A particular exemplary screen assays displacement of

radiolabled CGP42112A from 293 cells stably transfected with AT ₂ receptor- encoding nucleic acids.

Useful agents are typically those that bind to the AT ₂ receptor or disrupt the association of AT ₂ receptor with angiotensin II. Preferred agents are AT ₂ receptor- specific and to not cross react with any ATj receptor nor affect ATj receptor- angiotensin π interactions. Useful agents may be found within numerous chemical classes, though typically they are organic compounds; preferably small organic compounds. Small organic compounds have a molecular weight of more than 100 yet less than about 4,500, preferably less than about 1500, more preferably, less than about 500. Exemplary classes include peptides, saccharides, steroids, heterocyclics, polycyclics, substituted aromatic compounds, and the like.

Selected agents may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways to enhance their proteolytic stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxyl terminus, e.g., for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like. Other methods of stabilization may include encapsulation, for example, in liposomes, etc.

Agents may be prepared in a variety of ways known to those skilled in the art. For example, peptides under about 60 amino acids can be readily synthesized today using conventional commercially available automatic synthesizers. Alternatively, DNA sequences may be prepared encoding the desired peptide and inserted into an appropriate expression vector for expression in a prokaryotic or eukaryotic host. A wide variety of expression vectors are available today and may be used in conventional ways for transformation of a competent host for expression and isolation. If desired, the open reading frame encoding the desired peptide may be joined to a signal sequence for secretion, so as to permit isolation from the culture medium. Methods for preparing the desired sequence, inserting the sequence into an expression vector, transforming a competent host, and growing the host in culture for production of the product may be found in U.S. Patent Nos. 4,710,473, 4,711,843 and 4,713,339.

For therapeutic uses, the compositions and agents disclosed herein may be administered by any convenient way, preferably parenterally, conveniently in a physiologically acceptable carrier, e.g., phosphate buffered saline, saline, deionized water, or the like. Typically, the compositions are added to a retained physiological fluid such as blood or synovial fluid. Generally, the amount administered will be empirically determined, typically in the range of about 10 to 1000 /ig/kg of the recipient. For peptide agents, the concentration will generally be in the range of about 100 to 500 μg/ml in the dose administered. Other additives may be included, such as stabilizers, bactericides, etc. These additives will be present in conventional amounts.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1. Cloning and Analysis of rat AT ₂ receptor. cDNA prepared from size-fractionated (> 1 kb) poly (A) ⁺ RNA (Sprague- Dawley whole rat fetus, 18 days postcoitum) was ligated into an expression vector pcDNAI (Invitrogen), and amplified in 900 pools of 1,000 clones. Plasmid DNA prepared from every 8 pools by the alkali lysis method was transfected over 2-3 h using DEAE-dextran/chloroquine into COS-7 cells grown on glass chamber slides (Nunc) (Mathews, L.S. & Vale, W.W. Cell 65, 973-982 (1991)). 48-60 h after transfection, cells were washed with 50 mM Tris-HCl, pH 7.4, containing 0.1% BSA and incubated with 200 pM ^U5 I-[Sar ^I ,Ile']-Aιιg π (2,200 Ci umol ¹ , NEN) with or without 1 mM DuP753 for 2 h at room temperature. Cells were then washed four times with cold 50 mM Tris-HCl, pH 7.4, fixed with 0.1% glutaraldehyde at 4 C and subjected to emulsion autoradiography (Mathews, L.S. & Vale, W.W. Cell 65, 973-982 (1991)). As a positive control, the mouse AT _lb receptor clone (Sasamura, H., Hein, L., Krieger, J.E., Pratt, R.E., Kobilka, B.K. & Dzau, V.J. Biochem. biophys. Res. Commun. 185, 253-259 (1992)) was transfected similarly, and constantly resulted in a positive staining over 20% of cells that was completely abolished by 1 uM DuP753. Positive pool (RF904) which gave 20 DuP753-resistant positive cells was divided into subpools and

rescreened, and after three rounds of screening a single clone pMRAT2 was isolated. Sequencing of both strands of clone pMRAT2 was done by the dideoxy chain termination method with Sequenase (USB).

Radioligand binding to membranes from pMRAT2-transfected COS-7 cells indicates that the expressed protein has pharmacology indistinguishable from that of AT ₂ receptor. Binding assays were performed with crude membranes isolated from COS-7 cells (Sasamura, H., Hein, L., Krieger, J.E., Pratt, R.E., Kobilka, B.K. & Dzau, VJ. Biochem. biophys. Res. Commun. 185, 253-259 (1992)) transiently transfected with pMRAT2. Reactions were performed in 100 ml of 20 mM Tris- HCl, pH 7.4, containing 0.25 % BSA and 0.1 uM PMSF, ¹²⁵ I-CGP42112A (2, 176 Ci mmol ^"1 , Peptide Radioiodination Center, Washington State Univ.), 5 ug membranes and various concentrations of unlabelled ligands. 2 h after incubation at room temperature, bound and free ligands were separated with GF/C filters (Whatman). 150 pM of radioligand was used for displacement experiments. Nonspecific binding was defined in the presence of 1 uM unlabelled CGP42112A and was 5% of total binding. Each experiment was carried out at least twice in duplicate, and the results shown here represent one experiment.

Single-component, saturable, high-affinity binding sites for an AT ₂ -selective ligand ¹²⁵ I-CGP42112A with a dissociation constant (Kj) of 0.17 nM were observed in pMRAT2-transfected cells but not untransfected cells or cells transfected with the vector DNA. Competition profiles yielded an order of inhibition potency of [SarSfle^-Ang II = CGP42112A >_ Ang H > PD123319 ¹⁰ - ¹⁵ > Ang I. DuP753, an ATi-selective antagonist, has no effect. As with previous reports, DTT increased binding affinity for both Ang π and CGP42112A by 50-100%. Fig. 3 reveals a 2,884-nucleotide cDNA (SEQ ID NO:05) encoding a deduced 363-amino-acid AT ₂ receptor (SEQ ID NO:03) with a molecular mass of 41,330 attributable to its amino acid sequence. Hydropathy analysis indicates seven putative transmembrane domains. Two recently reported ATj receptor clones have the closest homology (34% identical and 53% similar amino acids), followed by receptors for somatostatin (both SSTR1 and SSTR2), bradykinin and vasoactive intestinal peptide (VIP)(30-32% sequence identity). There are 5 consensus N-glycosylation sites in the Ν-terminus, and several serine and threonine residues in the cytoplasmic domains - preferred phosphorylation sites.

AT ₂ receptor has a transmembrane topology similar to AT _X receptor with consensus residues found in other seven-transmembrane receptors. Similarity is highest in the second and fifth transmembrane domains with 2 stretches of 6- amino-acid identity that may be involved in ligand binding. Indeed, Lys-199 in the AT _X receptor shown to be essential for Ang π binding is conserved. 6 cysteine residues are conserved. AT ₂ receptor has 8 additional cysteine residues which may play a role in its binding characteristics in response to DTT.

Northern blot analysis of poly (A) ⁺ RNA from rat tissues revealed a 3.5-kb hybridizing band. For Northern blot analysis of mRNA from fetal rat tissues, poly (A) ⁺ NA (5 ug per lane) was electrophoresed and RNA blot hybridization analysis was carried out as described in Naftilan, A.J., Pratt, R.E. & Dzau, VJ. J. din. Invest. 83, 1419-1424 (1989) using the 2.4-kb Hinάm-Sacl fragment excised from clone pMRAT2 and the 0.78-kb Pstl-Xbal fragment of a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) clone (ATCC), as probes. It was found that the mRNA is abundant in whole fetus but decreases rapidly after birth. In adult, it is highly expressed in the adrenal, present in the brain and uterus but undetectable in the heart. This tissue distribution and the developmental regulation of the mRNA differ from AT _X receptor mRNA but is identical with AT ₂ receptor as reported by ligand binding. In spite of structural similarity of this receptor with ATj and other

'classical' G protein-coupled receptors, its signal transduction mechanism is clearly unique. Unlike the cloned AT _X receptor, when transiently expressed in COS-7 cells, stimulation of cloned AT ₂ receptor failed to increase IP ₃ or intracellular calcium. For the IP ₃ study, COS-7 cells 48 h after transfection with either a control vector, an AT _t receptor clone, or an AT ₂ receptor clone were suspended in DMEM and stimulated with 1 uM Ang π for 15 sec at 37 C. Intracellular IP ₃ levels were measured using radioreceptor assay (NEN). For calcium measurements, COS-7 cells on glass coverslips 48 h after transfection were incubated with 10 uM fura-2 AM (Molecular Probes) for 20 min at 37 C and stimulated with 1 uM Ang π. Cell fluorescence was measured with excitation at 340 nm and 380 nm. Ionomycin (10 uM) and EGTA (10 mM) were used for calibration. cAMP and cGMP levels and phosphatase activity revealed no apparent

effects. Furthermore, the ligand displacement curve of this receptor was not affected by guanylnucleotide analogues. These results are consistent with previous observations on the endogenously expressed AT ₂ receptor, suggesting either the lack of G protein coupling or coupling to a unique G protein with a distinct signalling mechanism, or inefficient G protein coupling to the overexpressed receptor in COS-7 cells.

Mutagenesis demonstrated the importance of the third intracellular loop in G protein coupling of the ATj receptor. A comparison of the three intracellular loops in the AT _t and AT ₂ receptors reveals the lowest homology in this third loop. A comparison with the third loop of other receptors without demonstrable G protein coupling (D _3> SSTR1 and ATj) reveals a conserved, 5-amino-acid motif (Fig. 3d). This motif is poorly conserved in ATj (1/5 identical), D _x (2/5 identical) or SSTR2 receptor (0/5 identical). The present results indicate that AT ₂ , SSTR1 and D ₃ receptors belong to a unique class of receptors, and that this motif is responsible for the apparent lack of tight coupling to 'classical' G proteins and may be involved in coupling to an as yet unidentified G protein(s), or may mediate an unknown signalling mechanism without G protein coupling. With regards to function, both SSTR1 and AT ₂ receptors (plus an uncloned VIP receptor that is GTP-insensitive) are associated with development and growth. Moreover, these receptors (AT ₂ , SSTR1, D ₃ and VIP) all exhibit fetal/neonatal and/or neuronal- specific expression.

The Drosophila frizzled protein, Fz, whose expression is developmentally regulated and is essential for normal epidermal morphogenesis and hair polarity (Vinson, C.R., Conover, S. & Adler, P.N. Nature 338, 263-264 (1989)) has a seven-transmembrane structure with a strikingly similar third loop to the AT ₂ receptor including this motif (64% overall similarity in loop 3 to the AT ₂ receptor; 3/5 identical, 5/5 similar in the motif) (Fig. 3d). Furthermore, its rat homologues (Fz-1 and Fz-2) have well conserved third loop and are highly expressed in neonatal tissues and in adult uterus, ovary, and brain (Chan, S.D.H. et al. J. biol. Chem. 267, 25202-25207 (1992)). The similar tissue distribution of the AT ₂ receptor suggests a role similar to that of Fz in cell morphogenesis and related events in growth and development. Alternatively, these receptors might function as ligands for other unknown receptors. Indeed, another Drosophila seven-

transmembrane protein with developmental cues, boss gene product (Kramer, H., Cagan, R.L. & Zipursky, L. Nature 352, 207-212 (1991)), acts as a ligand for sevenless tyrosine-kinase receptor during eye development.

Example 2. Murine AT ₂ Receptor: Cloning and Functional Analysis. Materials. Ang π, Ang I, and a non-selective Ang π receptor antagonist, [Sar^Ile^-Ang π, were purchased from Sigma (St. Louis, MO). An AT ₂ -selective ligand CGP42112A was a gift from Ciba-Geigy (Basel, Switzerland); [ ¹²⁵ rjCGP42112A from Peptide Radioiodination Center (Washington State Univ., WA). Another AT ₂ -selective ligand, PD 123319, was a gift from Parke-Davis (Ann Arbor, MI).

Molecular Cloning by Plaque Hybridization. Total RNA was prepared from 16- 18- day whole Balb/c mouse fetus using RNAzol (Tel-Test, Friendswood, TX). Poly(A) ⁺ RNA was isolated using PolyATtract mRNA Isolation Systems (Promega, Madison, WI). A cDNA library carrying cDNAs larger than 2.0 kilobase pairs (kb) was prepared in IZAPII vector (Stratagene, La Jolla, CA). A HindJWSacI fragment of rat AT ₂ receptor cDNA was used as a probe (herein). Clones (4.3 x 10 ⁵ ) derived from the cDNA library were transferred to nylon membranes (Colony/Plaque Screen Hybridization Transfer Membrane, Du Pont-NEN, Boston, MA) and screened by hybridization to rat AT ₂ receptor cDNA. Hybridization was carried out at 42°C in 2 x SSC containing 5 x Denhardt's solution, 1% SDS, 30% formamide, 10% dextran sulphate, 100 mg/ml heat-denatured salmon sperm DNA (Sigma) and the radiolabeled probe (10 ⁶ cpm/ml) for 12 h. Filters were washed twice at 42°C in 2 x SSC containing 1 % SDS for 40 min and in 0.2 x SSC containing 1% SDS for 40 min. Eight positive clones were picked up and converted to plasmids via in vivo excision method and sequenced. One of the clones was revealed to be a full-length clone (MC5).

Expression in COS-7 Cells and Ligand Binding Assay. The EcoKLIXhol insert of MC5 was subcloned into pcDNA I (Invitrogen, San Diego, CA), and the plasmid was transfected into COS-7 cells by modified DEAE-dextran method (Sussman, D.J., and Milman, G. (1984) Mol. Cell. Biol. 4, 1641-1643; and

herein). The transfected cells were harvested and homogenized. The 100,000 x g pellet of this homogenate was suspended and used as a membrane fraction. [ ¹²⁵ rjCGP42112A binding in the membrane was determined as described herein. Displacement experiment was carried out with 0.2 nM [ ¹²⁵ I]CGP42112A in the presence of various concentrations of compounds.

Northern Blot Analysis. Poly (A) ⁺ RNA from (5 ug per lane), isolated from the heart (Ht), whole brain (Br), liver (Li) and whole fetus at 16-18 days of gestation (Fet) was separated by electrophoresis on a 1 % agarose gel, and transferred onto a nylon membrane (GeneScreen, Du Pont-NEN). Hybridization was carried out as described previously (herein; Naftilan, A.J., Pratt, R.E., and Dzau, VJ. (1989) J. Clin. Invest. 83, 1419-1424) using a ³² P-labeled HindUL-NsiJ fragment excised from clone MC5 for the AT ₂ receptor, or GAPDH as a control. To assess equal loading of the lanes, the blots were similarly hybridized with a 0.78-kb Pstl-Xbal fragment of a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) clone (American Type Culture Collection, Rockville, MD). In the lane comprising the Fet sample, a band corresponding to 3.2kb was visible in addition to the control GAPDH band. Northern blot analysis showed that the AT ₂ receptor mRNA is abundant in the whole mouse fetus but undetectable in the heart and liver.

Genomic Southern Blot Analysis. The copy number of the mouse AT ₂ receptor gene in the mouse genome was determined by Southern blot hybridization analysis Genomic DNA (10 ug) from Balb/c mouse liver (Clontech, Palo Alto, CA) was digested with restriction endonucleases EcøRI or BamH , electrophoresed on a 1% agarose gel, and blotted to a nylon membrane (GeneScreen). The membrane was hybridized with the ³² P-labeled fiill-length mouse AT ₂ receptor cDNA probe (EcoΕI/XhoI fragment excised from clone MC5). Hybridization was carried out at 42°C in 5 x SSPE containing 0.5% SDS, 50% formamide, 10% dextran sulphate, 100 mg/ml heat-denatured salmon sperm DNA and the radiolabeled probe (10 ⁶ cpm/ml) for 24 h. The filter was washed twice at 56°C in 0.2 x SSC containing 0.1% SDS for 30 min. In the EcøRI lane, a band appeared in the region between 4.4 and 6.6. In the BamH lane, a band appeared in the region between the 9.4 and 23.1.

The full-length mouse AT ₂ receptor cDNA was used to probe the mouse genomic DNA digested with two restriction endonucleases, EcoΕL and BamHL. Each enzyme generated a single fragment that hybridized to the probe, indicating that the mouse AT ₂ receptor locus exists as a single copy in the mouse genome. Many G protein-coupled receptors, like b-adrenergic receptor (Kobilka, B.K., et al., (1987) J. Biol. Chem. 264, 7321-7327) and AT _X receptor (Iwai, N., and Inagami, T. (1992) FEBS Lett. 298, 257-260), lack introns in the coding region. However, a number of exceptions are known such as dopamine receptors (Sibley, D. R., and Monsma, F.J.(1992) Trends Pharmacol. Sci. 13, 61-69), tachykinin receptors (Takahashi, K., et al. (1992) Eur. J. Biochem. 204, 1025-1033; Gerard, N.P., et al. (1990) J. Biol. Chem. 265, 20455-20462; Hershey, A.D., et al. (1991) J. Biol. Chem. 266, 4366-4374) and endothelin receptors (Hosoda, K., et al. (1992) J. Biol. Chem. 267, 18797-18804;Arai, H., et al.. (1993) J. Biol. Chem. 268, 3463-3470).

Polymerase Chain Reaction (PCR). For reverse transcription-PCR (RT-PCR), first strand cDNA was synthesized from the mRNA using random primers and Molony Murine Leukemia Virus reverse transcriptase (GeneAmp PCR Reagent Kit, Peririn Elmer Cetus, Norwalk, CT). PCR primers for AT ₂ receptor are as foUows: 5'-ATTCCTGTTCTCTACTAC-3' (primer 1) (SEQ ID NO: 12), 5'- GTAACACGTTGCTATGAA-3' (primer 2) (SEQ ID NO: 13), 5'- CGGAACTGAAAGCTTACT-3' (primer 3)(SEQ ID NO: 14), 5'- AAGGACAACTTCAGTTTT-3' (primer 4) (SEQ ID NO: 15), and 5'- AGACACAAAGGTG TCCAT-3' (primer 5) (SEQ ID NO: 16). These primers are designed from mouse AT ₂ receptor sequence and they are specific for the AT ₂ receptor and quite different from the ATi receptor. PCR primers for mouse GAPDH were purchased from Clontech.

Mouse liver DNA (Clontech) was used for the analysis of the gene encoding the AT ₂ receptor. Reaction was carried out with 30 cycles of 1 min of denaturation at 94°C, 1 min of annealing at 50°C and 3 min of extension at 72°C. Poly(A) ⁺ RNA from heart (Ht), whole brain (Br), liver (Li) and whole fetus at 16- 18 days of gestation (Fet) was analyzed by RT-PCR using the primers 1 and 2. In all four lanes, a band was visible corresponding to GAPDH. An additional band

which was 554 b was visible above the GAPDH band in the Fet lane. RT-PCR analysis demonstrates that the AT ₂ receptor mRNA is expressed abundantly in fetus but at a much lower level in the adult brain. This tissue distribution is consistent with the known distribution of the AT ₂ receptor as reported by ligand binding data of mouse and rat tissues and by Northern blot analysis of rat tissues (Whitebread, S., et al.(1989) supra; Chiu, et al. (1989) supra; Millan, M.A., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11440-11444).

Mouse genomic DNA and mouse AT ₂ receptor cDNA clone (MC5) were analyzed by PCR to examine the presence of introns in the coding region. Mouse AT ₂ receptor cDNA clone (MC5) and mouse genomic DNA were analyzed by PCR using the primers 3 and 5, as well as the primers 4 and 5. Both DNA samples yielded PCR products of the same size in either reaction. (1220b and 1086 b fragments for both lanes). PCR analysis of the gene for mouse AT ₂ receptor showed that this gene does not contain introns in the coding region. The nucleotide (SEQ ID NO:01) and deduced amino acid (SEQ ID NO:02) sequences of the mouse AT ₂ receptor cDNA clone, MC5, are provided in SEQ ID NOS: 1 and 2, respectively. MC5 contains the 2.8-kb insert which has an open reading frame of 1089 base pairs. The deduced amino acid sequence consists of 363 amino acids with a relative molecular mass of 41373. Hydropathy analysis of the deduced amino acid sequence reveals putative seven transmembrane domains. There are 5 consensus sites for N-glycosylation (Hubbard, S.C., and Ivatt, R.J. (1981) Annu. Rev. Biochem. 50, 555-583) in a relatively long Ν-terminal extracelluar domain and 5 serine and 1 threonine residues in the cytoplasmic domains for possible phosphorylation (Kemp, B.E., and Person, R.B. (1990) Trends Biochem. Sci. 15, 342-346) identical to the rat AT ₂ receptor.

Membranes of COS-7 cells transfected with the cDΝA showed specific binding to [ ¹²⁵ I]CGP42112A, a radioligand of the AT ₂ receptor (Whitebread, S., et al. (1989) Biochem. Biophys. Res. Commun. 163, 284-291). The Scatchard plot of the binding showed K,, and B, _^ of 0.12 nM and 0.57 pmol/mg protein, respectively Fig. 1. No specific binding was detectable in untransfected COS-7 cells. Displacement of this binding is shown in Fig. 2. The binding of [ ¹²⁵ rjCGP42112A was competed by unlabeled angiotensin analogs and AT ₂ receptor ligands in the following rank order: [Sar^Ile^-Ang π = CGP42112A >_ Ang π >

PD 123319 > Ang I. DuP753, an AT selective antagonist (Chiu, et al. (1989) Biochem. Biophys. Res. Commun. 165, 196-203; Sasaki, K., et al. (1991) Nature 351, 230-233; Murphy, T.J., et al. (1991) Nature 351, 233-236; Sasamura, H., et al. (1992) Biochem. Biophys. Res. Commun. 185, 253-259; Iwai, N., and Inagami, T. (1992) FEBS Lett. 298, 257-260.), was virtually inactive at a concentration up to 10" ⁵ M. These binding characteristics are in good agreement with those observed in the membranes from whole rat fetus and the cloned rat AT ₂ receptor.

A comparison of deduced amino acid sequences of the mouse AT ₂ (SEQ ID NO:02), rat AT ₂ , (SEQ ID NO:03) and mouse AT _ta (SEQ ID NO:04) receptors was made. The mouse AT ₂ receptor amino acid sequence is 99% identical to that of the rat AT ₂ receptor. However, there is only a 34% identity between the mouse AT ₂ and the mouse AT _U receptor. Hydropathy analysis shows that the mouse AT ₂ receptor sequence exhibits a conserved transmembrane topology with consensus residues that are found in other seven-transmembrane, G protein-coupled receptors (O'Dowd, B.F., et al. (1989) Annu. Rev. Neurosci. 12, 67-83).

The above results demonstrate that the structures of mouse and rat AT ₂ receptors are highly conserved. Mouse AT ₂ receptor is expressed at high levels during development but is expressed at lower levels in selective tissues in the adult. The mouse AT ₂ receptor locus exists as a single copy in the mouse genome and the gene for the receptor dose not contain introns in the coding region. In contrast to the ATi receptor, the actions and the signaling mechanism of the AT ₂ receptor are not clearly defined. The successful cloning of mouse AT ₂ receptor cDNA and analysis of its gene described herein enable transgenic studies to further elucidate the functions and the signal mechanisms of the AT ₂ receptor.

Example 3. Preparation of Stable Transfectants

293 cells, a human embryonic kidney cell line (ATCC), were grown in DMEM supplemented with 10% fetal calf serum. pMRAT2, a rat AT ₂ receptor full-length cDNA cloned into the expression vector pcDNA I, was cotransfected with the plasmid pSV2neo into 293 cells using the calcium phosphate precipitation method (Southern, P.J. and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341). The cells were then subcultured and selected with the antibiotic G418 (800 ug/ml,

Gibco-BRL). The antibiotic-resistant cells were cloned by isolation and assayed for the AT ₂ receptor expression by radioligand binding. The clone expressing the highest number of the AT ₂ receptor, S293RAT2, was further characterized.

SV-T2 cells, an SV40-transformed Balb/3T3 fibroblast cell line (ATCC), were similarly cotransfected with pMRAT2 and pSV2neo and were selected with 400 ug/ml G418, resulting in the establishment of SV-RAT2, the SV-T2 cell line stably transfected with the rat AT ₂ receptor cDNA.

Radioligand binding assays with crude membranes from S293RAT2 cells reveal that the cells stably overexpress saturable, single-component, high-affinity binding sites for [ ¹² TJCGP42112A characteristic of the AT ₂ receptor (K, = 0.18 nM. B _m - _x ^~ 10-8 pmol/mg protein). Competition profiles with various angiotensin receptor ligands are essentially identical to those of the AT ₂ receptor endogenously expressed or transiently overexpressed in COS-7 cells.

It is evident from the above results that one can use the methods and compositions disclosed herein for making and identifying diagnostic probes and therapeutic drugs. It will also be clear to one skilled in the art from a reading of this disclosure that advantage can be taken to effect alterations of angiotensin π responsiveness in a host.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Dzau, Victor J

Mukoya a, Masashi

(ii) TITLE OF INVENTION: TYPE-2 ANGIOTENSIN II RECEPTOR AND GENE

(iii) NUMBER OF SEQUENCES: 16

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: FLEHR, HOHBACH, TEST, ALBRITTON & HERBERT

(B) STREET: 4 Embarcadero Center, Suite 3400

(C) CITY: San Francisco

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 94111-4187

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: WO Not yet assigned

(B) FILING DATE: 19-JUL-1994

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Rowland, Bertram I

(B) REGISTRATION NUMBER: 20,015

(C) REFERENCE/DOCKET NUMBER: FP-58491-1/BIR

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (415) 781-1989

(B) TELEFAX: (415) 398-3249

(C) TELEX: 910 277299

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2862 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 132..1223

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CGGAACTGAA AGCTTACTTC AGCCTGCATT TTAAGGAGTG CATGCGGGAG CTGAGTAAGC 60

TGATTTATGA TAACTGCTTT AAACACTGGC AACTAAAAAG GTGTAAGAAT TTGGAGTTGC 120

TGCAGTTCAA T ATG AAG GAC AAC TTC AGT TTT GCT GCC ACC AGC AGA AAC 170 Met Lys Asp Asn Phe Ser Phe Ala Ala Thr Ser Arg Asn 1 5 10

ATT ACC AGC AGC CGT CCT TTT GAT AAT CTC AAC GCA ACT GGC ACC AAT 218 lie Thr Ser Ser Arg Pro Phe Asp Asn Leu Asn Ala Thr Gly Thr Asn 15 20 25

GAG TCC GCC TTT AAT TGC TCA CAC AAA CCA TCA GAT AAG CAT TTG GAA 266 Glu Ser Ala Phe Asn Cys Ser His Lys Pro Ser Asp Lys His Leu Glu 30 35 40 45

GCA ATT CCT GTT CTC TAC TAC ATG ATT TTT GTG ATT GGG TTT GCT GTT 314 Ala lie Pro Val Leu Tyr Tyr Met lie Phe Val lie Gly Phe Ala Val 50 55 60

AAT ATT GTT GTG GTC TCA CTG TTT TGT TGT CAA AAG GGC CCT AAA AAG 362 Asn lie Val Val Val Ser Leu Phe Cys Cys Gin Lys Gly Pro Lys Lys 65 70 75

GTG TCC AGC ATT TAC ATC TTC AAT CTG GCC TTG GCT GAC TTA CTC CTT 410 Val Ser Ser lie Tyr lie Phe Asn Leu Ala Leu Ala Asp Leu Leu Leu 80 85 90

TTG GCT ACC CTC CCT CTC TGG GCA ACC TAT TAC TCT TAT AGA TAT GAT 458 Leu Ala Thr Leu Pro Leu Trp Ala Thr Tyr Tyr Ser Tyr Arg Tyr Asp 95 100 105

TGG CTT TTT GGA CCT GTG ATG TGC AAA GTG TTT GGT TCT TTT CTG ACT 506 Trp Leu Phe Gly Pro Val Met Cys Lys Val Phe Gly Ser Phe Leu Thr 110 115 120 125

CTG AAC ATG TTT GCA AGC ATT TTT TTT ATT ACC TGC ATG AGT GTC GAT 554 Leu Asn Met Phe Ala Ser lie Phe Phe lie Thr Cys Met Ser Val Asp 130 135 140

AGG TAC CAA TCG GTC ATC TAC CCT TTT CTG TCT CAA AGA AGG AAT CCC 602 Arg Tyr Gin Ser Val lie Tyr Pro Phe Leu Ser Gin Arg Arg Asn Pro 145 150 155

TGG CAA GCA TCT TAT GTA GTT CCC CTT GTT TGG TGT ATG GCT TGT CTA 650 Trp Gin Ala Ser Tyr Val Val Pro Leu Val Trp Cys Met Ala Cys Leu 160 165 170

TCC TCA TTG CCA ACA TTT TAT TTC CGG GAT GTC AGA ACC ATT GAA TAC 698 Ser Ser Leu Pro Thr Phe Tyr Phe Arg Asp Val Arg Thr lie Glu Tyr 175 180 185

TTA GGT GTG AAT GCT TGT ATT ATG GCT TTC CCA CCC GAG AAA TAT GCT 746 Leu Gly Val Asn Ala Cys lie Met Ala Phe Pro Pro Glu Lys Tyr Ala 190 195 200 205

CAG TGG TCT GCT GGG ATT GCC TTA ATG AAA AAT ATT CTT GGC TTT ATT794 Gin Trp Ser Ala Gly lie Ala Leu Met Lys Asn lie Leu Gly Phe lie 210 215 220

ATT CCT TTA ATA TTC ATA GCA ACG TGT TAC TTT GGA ATC AGA AAA CAT 842 lie Pro Leu lie Phe lie Ala Thr Cys Tyr Phe Gly lie Arg Lys His 225 230 235

CTG CTG AAG ACT AAT AGC TAT GGG AAG AAC AGA ATT ACC CGT GAC CAA 890 Leu Leu Lys Thr Asn Ser Tyr Gly Lys Asn Arg lie Thr Arg Asp Gin 240 245 250

GTC CTG AAG ATG GCA GCT GCT GTT GTG TTG GCA TTC ATC ATT TGC TGG 938 Val Leu Lys Met Ala Ala Ala Val Val Leu Ala Phe lie lie Cys Trp 255 260 265

CTT CCC TTC CAT GTT CTG ACC TTC TTG GAT GCT CTG ACC TGG ATG GGT 986 Leu Pro Phe Hie Val Leu Thr Phe Leu Asp Ala Leu Thr Trp Met Gly 270 275 280 285

ATC ATT AAT AGC TGT GAA GTT ATA GCA GTC ATT GAC CTG GCA CTT CCT 1034 lie lie Asn Ser Cys Glu Val lie Ala Val lie Asp Leu Ala Leu Pro ,; 290 295 300

TTT GCC ATC CTC CTG GGA TTC ACC AAC AGC TGT GTT AAT CCC TTC CTG 1082 Phe Ala lie Leu Leu Gly Phe Thr Asn Ser Cys Val Asn Pro Phe Leu 305 310 315

TAT TGT TTT GTT GGA AAC CGC TTC CAA CAG AAG CTC CGC AGT GTG TTT 1130 Tyr Cys Phe Val Gly Asn Arg Phe Gin Gin Lys Leu Arg Ser Val Phe 320 325 330

AGA GTT CCC ATT ACT TGG CTC CAA GGC AAG AGA GAG ACT ATG TCT TGC 1178 Arg Val Pro lie Thr Trp Leu Gin Gly Lys Arg Glu Thr Met Ser Cys 335 340 345

AGA AAA GGC AGT TCT CTT AGA GAA ATG GAC ACC TTT GTG TCT TAAATCTGTT 1230 Arg Lys Gly Ser Ser Leu Arg Glu Met Asp Thr Phe Val Ser 350 355 360

AGTGGGATGC ATGTAATCAG CCTAGCCATT GGTTTGGAGG CCCACACAAA TGATCTTTAA 1290

GTGGCATCAG TATAATACAG TTCTTTGCTT TATCTAATCT TTACTTACTC CCCCGAGAAC 1350

AGGAAGTCAA GTAGAACTGT AAATCTTTAT ACTCCACCAG CTTTCAGTGA TAGTGCCTTC 1410

TTTTGCTGGT CCTTTGGCAT GAGATTGTCA TATGTGAGCT AGATCTATAA TCTAGAAGTA 1470

TCTGGGGGAA TTATCCCAAC TTATAATTAA CAACAAATTA TGAGTGGTGA TTTGACATCT 1530

CAGACTTCTC CCTGGAAAAT GCTGGCATTT CTTAGTGGAG TTTTTTGTCC ATTTTCATCA 1590

GATTTCTTTT TTCTTGAACA AAGGCCAATT TAAACTTCTT ATACTATCCA ACCATATGAT 1650

ATAGCATGAG AGGTGAGCAC TAAGTTTAGC ATGATATACT CTTCTATATA TGCCATAGGT 1710

TGGTAGTGGC TTATTCAGTC TCTAGGTATA GAGTTTCTCC TTTTAAAGAA ATTGTAAGTT 1770

GTGTTCCTTT TCCATTTCAC TCAAGTATAG CTTTTGTACT TATTCTACAG CTACACACTG 1830

AGCAGATCTA GAATGTAGAT TAAATCACAC ATCTGTCTTA GCTTATTCTT GCAGTTATAG 1890

AAAGTACACT ATTTAGTAAA ACAGAACTGC AATGAAAAGT ATTTTAGTAT CCACAAAACT 1950

GAATATACAC TTTGAAAATT TTTCATCCAT TTTGACTCTT GTTTATTCTA TTCTCTTCTG 2010

ATGATTTTTG AATACAACAA CAAAACACTG TATTATGACA CTACGTAAAG GTCACTTTTT 2070

AAATTTTTAA CCTTTTGAAC ATGGTGCTTT GATATATTCA ATGATGACTT GAGTTTAATT 2130

ATTCATGCTT TTGTTCTGGG CTTCGTCCCA AAATATCTCT TTGACCCTGA AAAAGAGAGC 2190

ATTCTTTAAT TCTTTAACTT TGTAATAAAG TGCAAACTGG CATGGGAAAA GGTTATGTCA 2250

GACTGGAAGT TTGATGCCTT CTTGGGGGTA AACAGACCCA GCAAATGGCA AGTTTGGTGT 2310

CCAACAAGGA ACTTGTCAGA ACAAAGACTC CCTGGGGAGT AGTTTGAATC TGCATTTCTG 2370

GGCACAGTTC CAGAATGTAT AAGAGTCTGT GAAGGTGATT TAAAGCAAGC CCAGGTCCAC 2430

AGAACTCATT CTTAACACGA GTACATCTCT TACATTAGAG GAATATAATA CCTGAAGCTG 2490

TGTTACCTAA AGTTTACTCA AACTTCTCAA TAAATATTAA TTCAGAAGTT AAAGATGTCA 2550

TTCTCTGCCT GTCCCATATT ATACCAGGTC ACCTAAGACC TTCCTGGATT GATGCTGACC 2610

TATGAGGTAG ATTCAAAGTT CTGGGAACTT AACATTTCTG TCAGATTCCA GGCGTTTTAG 2670

GTTGAAGAAT CCTCTCATAC CCCTTCCTTG GAAAACCCTG ATTTCATGTA TTCATGTTAA 2730

TTTTTAGTAA AAACAAATAG CTAAATATGT AATCAGTTAT GACTTTGTGT TTTAAGCAAT 2790

TTTACACAAA ATCTCGTAAA ATAAAATCAT TACTGGGAAA AAAAAAAAAA AAAAAAAAAA 2850

AAAAAAAAAA AA 2862

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 363 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Lys Asp Asn Phe Ser Phe Ala Ala Thr Ser Arg Asn lie Thr Ser 1 5 10 15

Ser Arg Pro Phe Asp Asn Leu Asn Ala Thr Gly Thr Asn Glu Ser Ala 20 25 30

Phe Asn Cys Ser His Lys Pro Ser Asp Lys His Leu Glu Ala lie Pro 35 40 45

Val Leu Tyr Tyr Met lie Phe Val lie Gly Phe Ala Val Asn lie Val 50 55 60

Val Val Ser Leu Phe Cys Cys Gin Lys Gly Pro Lys Lys Val Ser Ser 65 70 75 80 lie Tyr lie Phe Asn Leu Ala Leu Ala Asp Leu Leu Leu Leu Ala Thr 85 90 95

Leu Pro Leu Trp Ala Thr Tyr Tyr Ser Tyr Arg Tyr Asp Trp Leu Phe 100 105 110

Gly Pro Val Met Cys Lys Val Phe Gly Ser Phe Leu Thr Leu Asn Met 115 120 125

Phe Ala Ser lie Phe Phe lie Thr Cys Met Ser Val Asp Arg Tyr Gin 130 135 140

Ser Val lie Tyr Pro Phe Leu Ser Gin Arg Arg Asn Pro Trp Gin Ala 145 150 155 160

Ser Tyr Val Val Pro Leu Val Trp Cys Met Ala Cys Leu Ser Ser Leu 165 170 175

Pro Thr Phe Tyr Phe Arg Asp Val Arg Thr lie Glu Tyr Leu Gly Val 180 185 190

Asn Ala Cys lie Met Ala Phe Pro Pro Glu Lys Tyr Ala Gin Trp Ser 195 200 205

Ala Gly lie Ala Leu Met Lys Asn lie Leu Gly Phe lie lie Pro Leu 210 215 220 lie Phe lie Ala Thr Cys Tyr Phe Gly lie Arg Lys His Leu Leu Lys 225 230 235 240

Thr Asn Ser Tyr Gly Lys Asn Arg lie Thr Arg Asp Gin Val Leu Lys 245 250 255

Met Ala Ala Ala Val Val Leu Ala Phe He He Cys Trp Leu Pro Phe 260 265 270

His Val Leu Thr Phe Leu Asp Ala Leu Thr Trp Met Gly He He Asn 275 280 285

Ser Cys Glu Val He Ala Val He Asp Leu Ala Leu Pro Phe Ala He 290 295 300

Leu Leu Gly Phe Thr Asn Ser Cys Val Asn Pro Phe Leu Tyr Cys Phe 305 310 315 320

Val Gly Asn Arg Phe Gin Gin Lys Leu Arg Ser Val Phe Arg Val Pro 325 330 335

He Thr Trp Leu Gin Gly Lys Arg Glu Thr Met Ser Cys Arg Lys Gly 340 345 350

Ser Ser Leu Arg Glu Met Asp Thr Phe Val Ser 355 360

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 363 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Lys Asp Asn Phe Ser Phe Ala Ala Thr Ser Arg Asn He Thr Ser 1 5 10 15

Ser Leu Pro Phe Asp Asn Leu Asn Ala Thr Gly Thr Asn Glu Ser Ala 20 25 30

Phe Asn Cys Ser His Lys Pro Ala Asp Lys His Leu Glu Ala He Pro 35 40 45

Val Leu Tyr Tyr Met He Phe Val He Gly Phe Ala Val Asn He Val 50 55 60

Val Val Ser Leu Phe Cys Cys Gin Lys Gly Pro Lys Lys Val Ser Ser 65 70 75 80

He Tyr He Phe Asn Leu Ala Val Ala Asp Leu Leu Leu Leu Ala Thr 85 90 95

Leu Pro Leu Trp Ala Thr Tyr Tyr Ser Tyr Arg Tyr Asp Trp Leu Phe 100 105 110

Gly Pro Val Met Cys Lys Val Phe Gly Ser Phe Leu Thr Leu Asn Met 115 120 125

Phe Ala Ser He Phe Phe He Thr Cys Met Ser Val Asp Arg Tyr Gin 130 135 140

Ser Val He Tyr Pro Phe Leu Ser Gin Arg Arg Asn Pro Trp Gin Ala 145 150 155 160

Ser Tyr Val Val Pro Leu Val Trp Cys Met Ala Cys Leu Ser Ser Leu 165 170 175

Pro Thr Phe Tyr Phe Arg Asp Val Arg Thr He Glu Tyr Leu Gly Val 180 185 190

Asn Ala Cys He Met Ala Phe Pro Pro Glu Lys Tyr Ala Gin Trp Ser 195 200 205

Ala Gly He Ala Leu Met Lys Asn He Leu Gly Phe He He Pro Leu 210 215 220

He Phe He Ala Thr Cys Tyr Phe Gly He Arg Lys His Leu Leu Lys 225 230 235 240

Thr Asn Ser Tyr Gly Lys Asn Arg He Thr Arg Asp Gin Val Leu Lys 245 250 255

Met Ala Ala Ala Val Val Leu Ala Phe He He Cys Trp Leu Pro Phe 260 265 270

His Val Leu Thr Phe Leu Asp Ala Leu Thr Trp Met Gly He He Asn 275 280 285

Ser Cys Glu Val He Ala Val He Asp Leu Ala Leu Pro Phe Ala He 290 295 300

Leu Leu Gly Phe Thr Asn Ser Cys Val Asn Pro Phe Leu Tyr Cys Phe 305 310 315 320

Val Gly Asn Arg Phe Gin Gin Lys Leu Arg Ser Val Phe Arg Val Pro 325 330 335

He Thr Trp Leu Gin Gly Lys Arg Glu Thr Met Ser Cys Arg Lys Ser 340 345 350

Ser Ser Leu Arg Glu Met Asp Thr Phe Val Ser 355 360

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 359 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Ala Leu Asn Ser Ser Ala Glu Asp Gly He Lys Arg lie Gin Asp 1 5 10 15

Asp Cys Pro Lys Ala Gly Arg His Ser Tyr He Phe Val Met He Pro 20 25 30

Thr Leu Tyr Ser He He Phe Val Val Gly He Phe Gly Asn Ser Leu 35 40 45

Val Val He Val He Tyr Phe Tyr Met Lys Leu Lys Thr Val Ala Ser 50 55 60

Val Phe Leu Leu Asn Leu Ala Leu Ala Asp Leu Cys Phe Leu Leu Thr 65 70 75 80

Leu Pro Leu Trp Ala Val Tyr Thr Ala Met Glu Tyr Arg Trp Pro Phe 85 90 95

Gly Asn His Leu Cys Lys He Ala Ser Ala Ser Val Ser Phe Asn Leu 100 105 110

Tyr Ala Ser Val Phe Leu Leu Thr Cys Leu Ser He Asp Arg Tyr Leu 115 120 125

Ala He Val His Pro Met Lys Ser Arg Leu Arg Arg Thr Met Leu Val 130 135 140

Ala Lys Val Thr Cys He He He Trp Leu Met Ala Gly Leu Ala Ser 145 150 155 160

Leu Pro Ala Val He His Arg Asn Val Tyr Phe He Glu Asn Thr Asn 165 170 175

He Thr Val Cys Ala Phe His Tyr Glu Ser Arg Asn Ser Thr Leu Pro 180 185 190

He Gly Leu Gly Leu Thr Lys Asn He Leu Gly Phe Leu Phe Pro Phe 195 200 205

Leu He He Leu Thr Ser Tyr Thr Leu He Trp Lys Ala Leu Lys Lys 210 215 220

Ala Tyr Glu He Gin Lys Asn Lys Pro Arg Asn Asp Asp He Phe Arg 225 230 235 240

He He Met Ala He Val Leu Phe Phe Phe Phe Ser Trp Val Pro His 245 250 255

Gin He Phe Thr Phe Leu Asp Val Leu He Gin Leu Gly Val He His 260 265 270

Asp Cys Lys He Ala Asp He Val Asp Thr Ala Met Pro He Thr He 275 280 285

Cys He Ala Tyr Phe Asn Asn Cys Leu Asn Pro Leu Phe Tyr Gly Phe 290 295 300

Leu Gly Lys Lys Phe Lys Lys Tyr Phe Leu Gin Leu Leu Lys Tyr He 305 310 315 320

Pro Pro Lys Ala Lys Ser His Ser Ser Leu Ser Thr Lys Met Ser Thr 325 330 335

Leu Ser Tyr Arg Pro Ser Asp Asn Met Ser Ser Ala Ala Lys Lys Pro 340 345 350

Ala Ser Cys Ser Glu Val Glu 355

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2884 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGGGATGGAG CGAGCACAGA ATTGAAAGCT TTCTTCAGCC TGCATTTTAA GGAGTGCGTG 60 TGGGAAGCTC AGTAAGCTGA TTTATGATAA CTGCTTTAAA CACTGGCAAC TAAAAGAGTG 120

TAAGGATTGG GAGTCTCTGA CAGTTCAATA TGAAGGACAA CTTCAGTTTT GCTGCCACCA 180

GCAGAAACAT CACCAGCAGT CTTCCTTTTG ATAATCTCAA CGCAACTGGC ACCAATGAGT 240

CCGCATTTAA CTGCTCACAC AAACCGGCAG ATAAGCATTT GGAAGCAATT CCTGTTCTCT 300

ACTACATGAT TTTTGTGATT GGTTTTGCTG TTAACATTGT TGTGGTCTCA CTGTTTTGTT 360

GTCAAAAGGG CCCTAAAAAG GTGTCCAGCA TTTACATCTT CAATCTGGCT GTGGCTGACT 420

TACTCCTTTT GGCAACCCTT CCTCTCTGGG CAACCTATTA CTCTTATAGA TATGACTGGC 480

TCTTTGGACC TGTGATGTGC AAAGTGTTTG GTTCTTTTCT GACCCTGAAC ATGTTTGCAA 540

GCATTTTTTT TATTACGTGC ATGAGTGTTG ATAGGTACCA ATCGGTTATC TACCCTTTTC 600

TGTCTCAGAG AAGGAATCCC TGGCAAGCAT CTTATGTAGT TCCCCTTGTT TGGTGTATGG 660

CTTGTCTGTC CTCATTGCCA ACATTTTATT TCCGAGATGT CAGAACCATT GAATACTTAG 720

GTGTGAATGC TTGTATTATG GCTTTCCCAC CTGAGAAATA TGCTCAGTGG TCTGCTGGGA 780

TTGCCTTAAT GAAAAATATT CTTGGCTTTA TCATTCCTTT AATATTCATA GCAACGTGTT 840

ACTTTGGAAT CAGAAAACAT CTGCTGAAGA CCAATAGCTA TGGGAAGAAC AGAATTACCC 900

GTGACCAAGT CTTGAAGATG GCAGCTGCTG TTGTGTTGGC ATTCATCATT TGCTGGCTTC 960

CCTTCCATGT TCTGACCTTC TTGGATGCTC TGACCTGGAT GGGTATCATT AATAGCTGTG 1020

AAGTTATAGC AGTCATTGAC CTGGCACTTC CTTTTGCCAT CCTCCTGGGA TTCACCAACA 1080

GCTGTGTTAA TCCCTTCCTG TATTGTTTCG TTGGAAACCG CTTCCAACAG AAGCTCCGTA 1140

GTGTGTTTAG AGTTCCCATT ACTTGGCTCC AAGGCAAGAG AGAGACTATG TCTTGCCGAA 1200

AAAGCAGTTC TCTTAGAGAA ATGGACACCT TTGTGTCTTA AATCTGTTAG TGGGATGCAT 1260

GTAATCAGCC TAGCAATGGT TTGGAGGCCC ACACAAATGA TCTTTAAGTG ACATCAGTAT 1320

AATATAATTC TTTGCTTTTT CTAATCTTTA TTTACTCCCC CCAGAACAGG AAATAAGTAT 1380

AATTATAAAC CTTTATACTC CACCAGCTTT CAGTGATAGT GCCTTCTTTT TCTGGTCCGT 1440

TGGCAGGAGA TTGTCATATG TGAGCTTTAT CTATAATCTA GAAGTATCTG GGGGAATTAT 1500

CTCGACTTAT AATTAAAAAC AAATTATGAG TGATGATTTG ATGTCTCGGA TTTCTCCCTG 1560

GAAAATGCTG GCATTTCTTA GTGGAGTTTT ATGTCCATTT TCATCTGATA TTTTTTTCTC 1620

TTGAACAAGG GCCAATTTGA ACTTCTTACA CTTTCCAACC ATATGATAGA GCATGAGAGG 1680

TGGGCACTAA GGTTAGCATG CTATACCCTT CTATATATGC CATAGGTTGG TAGTGGCTTA 1740

TTCAGTCTCT AAATATATAG GTTCTCCTTT TAAAGAAATT ATAAGTTGTG TTCCTTTTCC 1800

ATTTCACTCA AGTATAGCTT TTTACTTACT ATCTAAAACC ACTGAGTAGA TCTAGAATGT 1860

GGTTTAAATC ACACTTCTCT ATTAGCTTAT CCTTGAAGTT ATAGAGCGCA CGCTATTTAG 1920

TAAAACAGAA CTACCCTGAA AAAGTATTTT ATTAACCACA AAACTGAATA TACACTTGGA 1980

AAACTTTTCA TCCATTTTGA CTATTGTTTC AAGTTTTCTA TTCTCTTCTG ATGATTTTTG 2040

AACACGACAA CAAAACACTG TATTATTAGA TGACATAAAG GTCACTTTTT ACATTTTTAA 2100

CCTTTTGAAC ATGGTGCTTT GATATATTCT ATGGTGACTT GAGTTTAATT ATTCATGCTT 2160

TTGTTCTGGG CTGCGTCCCA AAATATCTTT TTGACCCTGA AAAAGAGAGG ATTCTTTAAT 2220

TCTTTAGCTT TATAATAAAC TGCACACTGG CATAGGAAAA GGTTATGTCA GAATGGAAGT 2280

TTGATGCCTT CTTGGGAGCA AACAGACCCA GAGAAACGGC AAGTTTGGTG TCCAACAAGG 2340

AACTTGTCAG AACAAAGGCC CCTGGGGAGT ATTTTGAATC TGCATTTCTG GGCACAGTTC 2400

CAGAATATAT AAGAGTCTGT GGAGGTGATT TAAATCAAGC CCAGGTCCAC AGAGCTCATT 2460

CTCAACACGA GTACATCTCT TACATTAGAG GAATATAATC CCGGAAACTG AGTCACCTAA 2520

AGTTCACTCA AACTTCACAA TAAGTATTAA TTCAAACGTT CAAAATGCCA TTCTCTTACT 2580

GCCCCATATT ATACCAGGTC GCCTGAGACC TTTCTGGACT GATAATGACC TCTGAGGTAG 2640

ATTTAAAGTT TTGGGAACTT AACATTTCTG TCAGATTTCA GGCTTTTTTG GTTGAAGAAT 2700

CCTCTCATAC CCCTTCCTTG GAAAACCCTG ATTTCATGTA TTCATATTAT GTGTTACTAA 2760

GATCAAGTAG CTAAATATAT AATCAGTTAT GATTTTGTGT TTTAAGTAAT TTTACACAAC 2820

ATCTCATAAA AATAAAATCA TTATTGGGAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2880

AAAA 2884 (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Arg Lys His Leu Leu Lys Thr Asn Ser Tyr Gly Lys Asn Arg He Thr 1 5 10 15

Arg Asp Gin Val Leu Lys 20

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 amino acids _ '

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Arg Met Val Ala Leu Lys Ala Gly Trp Gin Gin Arg Lys Arg Ser Glu 1 5 10 15

Arg Lys He Thr Leu 20

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( ii) MOLECULE TYPE : peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 :

Arg He Tyr He Val Leu Arg Gin Arg Pro Leu Arg Glu Lys Lys Ala 1 5 10 15

Thr Gin

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Arg He Arg Thr Val Met Lys Thr Asp Gly Lys Arg Thr Asp Lys Leu 1 5 10 15

Glu Arg Leu Met Leu Arg 20

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Arg He Arg Thr He Met Lys His Asp Gly Thr Lys Thr Glu Lys Leu 1 5 10 15

Glu Lys Leu Met Val Arg 20

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Arg He Arg Thr He Met Lys His Asp Gly Thr Lys Thr Glu Lys Leu 1 5 10 15

Glu Arg Leu Met Val Arg 20

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE; cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

ATTCCTGTTC TCTACTAC 18

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GTAACACGTT GCTATGAA 18

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CGGAACTGAA AGCTTACT 18

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: AAGGACAACT TCAGTTTT 18

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AGACACAAAG GTGTCCAT 18