United States Patent (19) 11 Patent Number: 5,958,710 Kuiper Et Al

USOO595871 OA United States Patent (19) 11 Patent Number: 5,958,710 Kuiper et al. (45) Date of Patent: Sep. 28, 1999

54) ORPHAN RECEPTOR Debuire et al., Sequencing the erbA Gene of Avian Eryth roblastosis Virus Reveals a New Type of Oncogene, Science, 75 Inventors: Georg Kuiper, Huddinge; Eva L. K. vol. 224, Jun. 1984. Enmark, Tullinge; Jan-Ake Weinberger et al., “Domain Structure of human glucocorti Gustafsson, Stockholm, all of Sweden coid receptor and its relationship to the v-erb-A oncogene product”, Nature, vol. 318, Dec. 1985. 73 Assignee: Karo Bio AB, Huddinge, Sweden Green et al., Human oestrogen receptor cDNA: Sequence, 21 Appl. No.: 08/836,620 expression and homology to v-erb-A, Nature, vol. 320, Mar. 1986. 22 PCT Filed: Sep. 9, 1996 Greene et al., “Sequence and Expression of Human Estrogen 86 PCT No.: PCT/EP96/03933 Receptor Complementary DNA”, Science, vol. 231, Mar. 1986. S371 Date: May 8, 1997 Bishop, “Oncogenes as hormone receptors', Nature, Vol. S 102(e) Date: May 8, 1997 321, May 1986. Latham et al., “Solubilized Nuclear Receptors for Thyroid 87 PCT Pub. No.: WO97/09348 Hormones”,Journal of Biological Chemistry, vol. 251, No. 23, Dec. 1976, pp. 7388-7397. PCT Pub. Date: Mar. 13, 1997 Silva et al., “Partial Purification of Triiodothyronine Recep 30 Foreign Application Priority Data tor from Rat Liver Nuclei’, Journal of Biological Chemistry, Sep. 8, 1995 GB United Kingdom ...... 9518272 vol. 252, No. 19, Oct. 1977, pp. 6799–6805. Mar. 15, 1996 GB United Kingdom ...... 96.OSSSO Nikodem et al., “Affinity labeling of rat liver thyroid hor Apr. 11, 1996 GB United Kingdom ...... 9607S32 mone nuclear receptor, Proc. Natl. Acad. Sci. USA, Vol. 77, May 8, 1996 GB United Kingdom ...... 96.O9576 No. 12, Dec. 1980, pp. 7064–7068. Aprilett et al., “Affinity Chromatography of Thyroid Hor 51) Int. Cl...... G01N 33/53; C12N 15/12; mone Receptors”,Journal of Biological Chemistry, vol. 256, CO7K 14/705 No. 23, Dec. 1981, pp. 12094-12101. 52 U.S. Cl...... 435/7.21; 536/23.5; 530/350 Casanova et al., “5'-Flanking DNA of the Rat Growth 58 Field of Search ...... 536/23.5; 530/350; Hormone Gene Mediates Regulated Expression by Thyroid 435/69.1, 6, 7.2, 7.1, 325, 320.1, 7.21 Hormone”, Journal of Biological Chemistry, vol. 260, No. 56) References Cited 21, Sep. 1985, pp. 11744–11748. Cattini et al., “The Human Growth Hormone Gene Is U.S. PATENT DOCUMENTS Negatively Regulated by Triiodothyronine When Trans 5,071,773 12/1991 Evans et al.. ferred into Rat Pituitary Tumor Cells”,Journal of Biological 5,217.867 6/1993 Evans et al.. Chemistry, vol. 261, No. 28, Oct. 1986, pp. 13367-13372. 5,262,300 11/1993 Evans et al.. Spurret al., “Chromosomal localisation of the human homo 5,298.429 3/1994 Evans et al.. logues to the oncogenes erbA and B, EMBO Journal, vol. 5,310,662 5/1994 Evans et al.. 3, No. 1, 1984, pp. 159–163. 5,312,732 5/1994 Evans et al.. 5,438,126 8/1995 DeGroot et al.. Jhanwar et al., “Germ-Line Chromosomal Localization of 5,534,418 7/1996 Evans et al.. Human C-Erb-A Oncogene”, Somatic Cell and Molecular 5,597.693 1/1997 Evans et al.. Genetics, vol. 11, No. 1, 1985, pp. 99-102. 5,597,705 1/1997 Evans et al.. 5,599,904 2/1997 Evans et al.. (List continued on next page.) 5,602,009 2/1997 Evans et al.. 5,639,616 6/1997 Liao et al.. Primary Examiner Marianne P. Allen 5,712,372 1/1998 DeGroot et al.. Attorney, Agent, or Firm Todd E. Garabedian; Wiggin & FOREIGN PATENT DOCUMENTS Dana 0 733705 A1 9/1996 European Pat. Off.. 57 ABSTRACT 0 798378 a2 10/1997 European Pat. Off.. This invention relates to a novel estrogen receptor-related OTHER PUBLICATIONS nuclear receptor, hereinafter termed “ERB having the amino acid sequence of FIGS. 1, 13A or 14A or substantially Toresani et al., “Partial Purification And Characterization the same ambino acid Sequence as the amino acid Sequence Of Nuclear Triiodothyronine Binding Proteins’ Biochemical shown in FIGS. 1, 13A or 13B or an amino acid sequence And Biophysical Research Communications, vol. 81, No. 1, functionally similar to that Sequence. The invention also 1978. relates to DNA sequences encoding the receptor. The recep Mosselman et al., “ERB: identification and characterization tor may be useful in isolating molecules for the treatment of of a novel human estrogen receptor, FEBS Letters 392, disorderS Such as prostate cancer, benign prostatic 1996, pp. 49-53. hyperplasia, Osteoporosis or cardiovascular disorders and in Vennstrom et al., “Isolation and Characterization of Chicken the testing of Substances for estrogenic and other hormonal DNA Homologous to the Two Putative Oncogenes of Avian effects. Erythroblastosis Virus', Cell, vol. 28, Jan. 1982, pp. 135-143. 7 Claims, 17 Drawing Sheets 5,958,710 Page 2

OTHER PUBLICATIONS West et al., “Interaction of a Tissue-Specific Factor with an Zabel et al., “Cellular homologs of the avian erythroblastosis Essential Rat Growth Hormone Gene Promoter Element', Virus erb-A and erb-B genes are Syntenic in mouse but Molecular and Cellular Biology, vol. 7, No. 3, Mar. 1987, asyntenic in man’, Proc. Natl. Acad. Sci. USA, Vol. 81, Aug. pp. 1193–1197. 1984, pp. 4874–4878. Druege et al., “Introduction of estrogen-responsiveness into Pascual et al., “Photoaffinity Labeling of Thyroid Hormone mammalian cell lines', Nucleic Acids Research, vol. 14, No. Nuclear Receptors in Intact Cells”, Journal of Biological 23, 1986. Chemistry, vol. 257, No. 16, Aug. 1982, pp. 9640–9647. Underwood et al., “A thyromimetic that drecreases plasma Casanova et al., “Photoaffinity Labeling of Thyroid Hor cholesterol levels without increasing cardiac activity”, mone Nuclear Receptors”, Journal of Biological Chemistry, Nature, vol. 324, Dec. 1986. vol. 289, No. 19, Oct. 1984, pp. 12084-12091. Hitpab et al., “An Estrogen-Responsive Element Derived Latham et al., “Interaction of Amiodarone and DeSethyla from the 5' Flanking Region of the Xenopus Vitellogenin A2 miodarone With Solubilized Nuclear Thyroid Hormone Gene Functions in Transfected Human Cells’, Cell, vol. 46, Receptors”, JACC, vol. 9, No. 4, 1987, pp. 872-876. Sep. 1986. Giguere et al., “Functional Domains of the Human Gluco Bolger et al., “Molecular Interactions between Thyroid corticoid Receptor', Cell, vol. 46, Aug. 1986, pp. 646-652. Hormone Analogs and the Rat Liver Nuclear Receptor', Journal of Biological Chemistry, vol. 255, No. 21, Nov. Weinberger et al., “Human Steroid Receptors and erb A 1980, pp. 10271-10278. Proto-oncogene Products: Members of a New Superfamily Weinberger et al., “The c-erb gene encodes a thyroid of Enhancer Binding Proteins', appearing at Cold Spring hormone receptor', Nature, vol. 324, Dec. 1986. Harbor Symposia on Quantitative Biology, vol. LI, 1986. Sap et al., “The c-erb A protein is a high-affinity receptor for Angier, N., “New Respect for Estrogen's Influence”, The thyroid hormone”, Nature, vol. 324, Dec. 1986. New York Times, Jun. 24, 1997. Thompson et al., “Identification of a Novel Thyroid Hor mone Receptor Expressed in the Mammalian Central Ner Pennisi, E., “Differing Roles Found for Estrogen's Two vous System”, Science, vol. 327, Sep. 1987. Receptors”, Science, vol. 277, Sep. 1997, p. 1439. Damm et al., “A Single point mutation in erbA restores the Parker, M., “Nuclear receptor Superfamily reunion”, Trends erythroid transforming potential of a mutant avian erythro in Genetics, vol. 12, No. 7, Jul. 1996. pp. 277–278. blastosis virus (AEV) defective in both erbA and erbB oncogenes”, EMBO Journal, vol. 6, No. 2, 1987, pp. “Novel Estrogen Receptor Discovered”, Environmental 375-382. Health Perspectives, vol. 104, No. 12, Dec. 1996, pp. Ichikawa et al., “Purification and characterization of rat liver 1273-1274. nuclear thyroid hormone receptors”, Proc. Natl. Acad. Sci. Katzenellenbogen, B. et al., “A New Actor in the Estrogen USA, vol. 84, May 1987, pp. 3420–3424. Receptor Drama ; Enter ER-B”, Endocrinology 186: Koenig et al., “Thyroid hormone receptor binds to a Site in 861–862 (1997). the rat growth hormone', Proc. Natl. Acad. Sci. USA, Vol. 84, Aug. 1987, pp. 5670–5674. Enmark, E. et al., “Human Estrogen Receptor B-Gene Wight et al., “Discrete Positive and Negative Thyroid Hor Structure, Chromosomal Localization, and Expression Pat mone-responsive Transcription Regulatory Elements of the tern”, Journal of Clinical Endocrinology and Metabolism, Rat Growth Hormone Gene”, Journal of Biological Chem vol. 82, No. 12, 1997. istry, vol. 262, Apr. 1987. Kaike, S. et al., NAR, 15(6): 2499-2513, 1987.

U.S. Patent Sep. 28, 1999 Sheet 2 of 17 5,958,710

rat ERO mOUSe ERO

human ERO. F. G. 2A pig ERC chick ERO

Zebra finch ERO xenopus ERO killfish ER

O. aureuSER

rainbow trout ER rat ERB

mouse ER3 human ER3

japanese eel ER

human ERR1 human ERR2 rat GR

ALIGNMENT OF ER8 TO OTHER F. G. 2B ESTRO GEN RECEPTORS A/B 55% o ziz rat ER3 79.6985 85.6 916 human ERB 95.6 mouse ER8

16s 697 15.6 3.2 human ERR1

13.7 72.7 human ERR2

U.S. Patent Sep. 28, 1999 Sheet 4 of 17 5,958,710

U.S. Patent Sep. 28, 1999 Sheet 5 of 17 5,958,710

G. 3A

U.S. Patent Sep. 28, 1999 Sheet 6 of 17 5,958,710

SCATCHARO PLOT OF ER-BETA 0.5 F.G. 5A

O, 1

O,O O 100 2OO 300 BOUND E2 (pM)

----a- - - CORTICO. - A - 3 BETA-AD ----A -- 3 ALPHA-AD O1 1 1 10 1OO 1OOO FOLD COMPETTOR U.S. Patent Sep. 28, 1999 Sheet 7 of 17 5,958,710

Ol. E2 BNDING BY ER-BETA FIG. 5C -O- TOTAL BOUND -- BOUND -o- EXCESS COLD E2 O3

O2 i O1 O, 0 0,0 10 2,0 3.0 l,0 5,0 6,0 H-E2 (nM)

TRANSACTIVATION BY ER BETA F.G. 6 30 O ERE-REPORTER ONLY ER-BETA ER-BETA - 100 nM E2 ER-BETA + 100 nM E2/1000 nM. Tan s 19.6 U.S. Patent Sep. 28, 1999 Sheet 8 of 17 5,958,710 FG.7

E2 ST MULATED TRANSACTIVATION F.G. 7A

10 ESTRADIOL nM) U.S. Patent Sep. 28, 1999 Sheet 9 of 17 5,958,710

DEECIE FRAF ER EXPRESSEN BY R-PCR.

ER32 ERet be

,! å a F.G. 8

U.S. Patent Sep. 28, 1999 Sheet 10 0f 17 5,958,710

U.S. Patent Sep. 28, 1999 Sheet 11 of 17 5,958,710

FG.1OA HILL PLOT COMPARING hERO AND reR(3.

Kd58412 (.085, al 1.279 0.082865 O 100 200 300 400 500 600 700 800 FREE 1751-E2 (pM)

FIG.1OB SCATCHARD PLOT COMPARING hERo AND reR6.

H------rR3: Kid = 0. l.2nM

O 2 4 6 8 10 12 1, 16 SPECIFIC BOUND 751-E2 (pM) U.S. Patent Sep. 28, 1999 Sheet 12 of 17 5,958,710

F G.11 A PLOT OF RELATIVE BNONG AFFINITY (RBA) DERVED FROM IC50- VALUES, COMPARING hERO AND rR3. 350 300

250 (Hexes tr. C 200 s C-164384 150 s 17B-E2 100 0 16 a-BrE2

O 50 100 150 200 250 300 350 RBA (IC50) hERO.

FG.11B PLOT OF RELATIVE BINDING AFF NITY (RBA), MAGNIFEO OETAL OF FG 12a 50

3 O

H-E2 10 E Clom 5a-AD33-diol0. Moxe

O 10 2O 30 l.0 50 RBA (C50) hERO.

U.S. Patent Sep. 28, 1999 Sheet 14 of 17 5,958,710

1 MTFYSPAVMN YSIPSNVTNL EGGPGROTTS PNVLWPTPGH LSPLVVHRQL 51 SHLYAEPQKS PWCEARSLEH TLPVNRETLK RKVSGNRCAS PVTGPGSKRD 101 AHFCAVCSDY ASGYHYGVWS CEGCKAFFKR SIQGHNDYIC PATNOCTIDK 151 NRRKSCQACR LRKCYEVGMV KCGSRRERCG YRLVRRQRSA DEQLHCAGKA 201 KRSGGHAPRV RELLLDALSP EQLVLTLLEA EPPHVLISRP SAPFTEASMM 251 MSLTKLADKE LVHMISWAKK IPGFVELSLF DOVRLLESCW MEVLMMGLMW 301 RSIDHPGKLI FAPDLVLDRD EGKCVEGILE IFDMLLATTS RFRELKLQHK 351 EYLCVKAMIL LNSSMYPLVT ATODADSSRK LAHLLNAVTD ALVWVIAKSG 401 ISSQQQSMRL ANLLMLLSHV RHASNKGMEH LLNMKCKNVV PVYDLLLEML 451 NAHVLRGCKS SITGSECSPA EDSKSKEGSO NLOSO FIG. 13A

MAFYSPAVMNYSVPSSTGNLEGGPVROTASPNVLWPTSGH 40 LSPLATHCQSSLLYAEPQKSPWCEARSLEHTLPVNRETLK 80 RKLGGSGCASPVTSPSTKRDAHFCAVCSDYASGYHYGVWS 120 CEGCKAFFKRSIQGHNDYICPATNQCTIDKNRRKNCQACR 160 LRKCYEVGMVKCGSRRERCGYRIVRRQRSASEOVHCLNKA 200 KRTSGHTPRVKELLLNSLSPEQLVLTLLEAEPPNVLVSRP 240 SMPFTEASMMMSLTKLADKELVHMGWAKKIPGFVELSLL 280 DOVRLLESCWMEVLMVGLMWRSIDHPGKLIFAPDLVLDRD 320 EGKCVEGILEIFDMLLATTARFRELKLQHKEYLCVKAMIL 360 LNSSMYHLATASQEAESSRKLTHLLNAVTDALVWVISKSR 400 ISSQQOSVRLANLLMLLSHVRHISNKGMEHLLSMKCKNVV 440 PVYDLLLEMLNAHTLRGYKSSISGSGCCSTEDSKSKEGSO 480 NLOSQ. 486 FIG. 1AA U.S. Patent Sep. 28, 1999 Sheet 15 0f 17 5,958,710

CTATGACATT CTACAGTCCT GCTGTGATGA ATTACAGCAT TCCCAGCAAT 51 GTCACTAACT TGGAAGGTGG GCCTGGTCGG CAGACCACAA GCCCAAATGT 101 GTTGTGGCCA ACACCTGGGC ACCTTTCTCC TITAGGGTC CATCGCCAGT 151 TATCACATCT GTATGCGGAA CCTCAAAAGA GTCCCTGGTG TGAAGCAAGA 20 TCGCTAGAAC ACACCTTACC TGTAAACAGA GAGACACTGA AAAGGAAGGT 251 TAGTGGGAAC CGTTGCGCCA GCCCTGTTAC TGGTCCAGGT TCAAAGAGGG 3 O ATGCTCACTT CTGCGCTGTC TGCAGCGATT ACGCATCGGG ATATCACTAI 351 GGAGTCTGGT CGTGTGAAGG ATGTAAGGCC TTTTTTAAAA GAAGCATTCA 401 AGGACATAAT GATTATATTT GTCCAGCTAC AAATCAGTGT ACAATCGATA 451. AAAACCGGCG CAAGAGCTGC CAGGCCTGCC GACTTCGGAA GGTTACGAA 5O1 GTGGGAATGG TGAAGTGTGG CTCCCGGAGA GAGAGATGTG GGTACCGCCT 551 TGTGCGGAGA CAGAGAAGTG CCGACGAGCA GCTGCACTGT GCCGGCAAGG 60 CCAAGAGAAG TGGCGGCCAC GCGCCCCGAG GCGGGAGCT GCTGCGGAC 65. GCCCTGAGCC CCGAGCAGCT AGTGCTCACC CTCCGGAGG CTGAGCCGCC 7 O1 CCATGTGCTG ATCAGCCGCC CCAGTGCGCC CTTCACCGAG GCCTCCAGA 7 S1 TGATGTCCCT GACCAAGTTG GCCGACAAGG. AGTGGTACA CATGACAGC 8O TGGGCCAAGA AGATTCCCGG CTTTGTGGAG CTCAGCCTGT TCGACCAAGT 85 GCGGCTCTTG GAGAGCTGTT GGATGGAGGT GTTAATGATG GGGCTGATGT 901 GGCGCTCAAT TGACCACCCC GGCAAGCTCA TCTTIGCTCC AGATCTTGIT 951 CTGGACAGGG ATGAGGGGAA ATGCGTAGAA GGAATTCTGG AAATCTIGA 1001 CATGCTCCTG GCAACTACTT CAAGGTTTCG AGAGTTAAAA CTCCAACACA 105. AAGAATATCT CTGTGTCAAG GCCATGATCC TGCTCAATTC CAGTATGTAC 1101 CCTCTGGTCA CAGCGACCCA GGATGCTGAC AGCAGCCGGA AGCTGGCTCA 115 CTTGCTGAAC GCCGTGACCG AIGCTTTGGT TTGGGTGATT GCCAAGAGCG 1201. GCATCTCCTC CCAGCAGCAA TCCATGCGCC TGGCTAACC CCTGATGCTC 1251 CTGTCCCACG TCAGGCATGC GAGTAACAAG GGCATGGAAC ATCTGCICAA 13 O1 CATGAAGTGC AAAAATGTGG TCCCAGTGTA TGACCTGCTG CTGGAGAGC 1351 TGAATGCCCA CGTGCTTCGC GGGTGCAAGT CCTCCAICAC GGGGTCCGAG 14 O1 TGCAGCCCGG CAGAGGACAG TAAAAGCAAA GAGGGCTCCC AGAACCTACA 1451 GTCTCAGTGA FIG. 13B U.S. Patent Sep. 28, 1999 Sheet 16 of 17 5,958,710

5,958,710 1 2 ORPHAN RECEPTOR reproductive organs and the immune and nervous Systems. On the basis of possible parallels between actual wildlife This invention relates to cellular nuclear receptors and effects, seen for example in birds and Seals living in highly their uses. polluted areas, and proposed effects in humans, in combi A large family of nuclear receptors which confer cells 5 nation with documented human reproductive effects caused with responsiveness to molecules Such as retinoid acid, by prenatal exposure to the pharmaceutical estrogen, diethyl Vitamin D, Steroid hormones and thyroid hormones has been stilbestrol (DES), “estrogenic" chemicals have been pro identified. Extensive studies have shown that the members posed to threaten the reproductive capability of both animals of this Superfamily of nuclear receptors activate and/or and humans. Among the chemicals known or Suspected to repress gene transcription through direct binding to discrete act as estrogen mimics on the human body, or in other ways cis-acting elements termed "hormone response elements' disturb the human endocrine System, there are Several which (HRE). It has been shown that these HRE’s comprise repeats have already been identified as environmental hazards. of consensus palindromic hexanucleotide DNA motifs. The Among the chemicals that have been mentioned as potential specificity of the HRE’s is determined by the orientation of, causes of disruption of reproductive function in animals and and spacing between, halfsites (i.e. half a palindromic 15 humans are chlorinated organic compounds Such dieldrin, sequence)(Umenesono K., et al., 1991 Cell 65, 1255-1266). endoSulfans, chlordanes, endrins, aldrin, DDT and Some Specific DNA binding is mediated by a strongly PCBs, plastics such as Bisphenol A, phthalates and conserved DNA binding domain, containing two Zinc nonylphenol, and aromatic hydrocarbons. Some of the pro fingers, which is conserved among all thus discovered posed effects on humans have been Suggested to be due to nuclear receptors. Three amino acids at the C-terminal base an increasing exposure to environmental estrogens-in fact, of the first zinc finger (known as the "P-box”) are important exposure to chemical compounds to which higher organisms for the recognition of the half Site nucleotide Sequence. during the foetal period react in a way that is Similar to when Members of the nuclear receptor Superfamily have been they are exposed to high dosages of estrogens. The effects classified into different groups on the basis of the amino acid are manifested by for example perturbations of the SeX sequence within the P box. 25 characteristics and impaired reproductive potential. In All members of the nuclear receptor Superfamily also humans, elevated risks of breast cancer and other hormone contain a hyperVariable N-terminal domain and a ligand related disease has also been discussed as possible effects. In binding domain containing Some “patches of conserved addition, to the documented “estrogcnic effects, it has sequence. One of these is called the “Ti-domain”. recently been demonstrated that environmental pollutants Molecules which are thought to be nuclear receptors, as may also act on hormonal pathways other than the estro they are structurally related to cliaracterised receptors, but genic pathway-it has been shown that p.p'-DDE the main for which no ligand has been found, are termed “orphan metabolite of DDT (also in humans) is a fairly anti receptors. Many Such orphan receptors have been identified androgenic agent (Kelce W. R. et al Nature 1995 (see for example Evans R. M. (1988) science 240,889-895 375:581-585). Epidemiological studies on these issues are, and O'Malley, B. (1990) Mol. Endocrinol. 4 363-369) 35 however, presently difficult to interpret. Nevertheless, there We have now unexpectedly identified, initially in rat a is a growing opinion against these potentially hormone new orphan receptor, which is related to the known estrogen disrupting chemicals, and very palpable public and environ receptor ERC, and which we have designated “ERB' mental demand for the governmental agencies and industry (specifically “reRB” in rat). In this specification “ERf8” will to act. In view of the similarities between the receptor of the be used to refer to the receptors hERB or rBRB or related 40 present invention, ERB and the classical estrogen receptor, receptors. The nucleotide and amino acid Sequences of rERB ERB may be used in the testing of chemicals for estrogenic have now been determined and are shown in FIG. 1. We effect. have also identified a human ERB-"hERB’, the amino acid An amino acid Sequence functionally-Similar to the DNA and sequences of which are shown in FIG. 13A and sequence shown in FIGS. 1, 13A or 14A may be from a 13B respectively. 45 different mammalian Species. According to one aspect of the invention there is pro An amino acid Sequence which is more than about 89%, Vided a novel estrogen receptor-related nuclear receptor, identical with the sequence shown in FIGS. 1, 13A or 14A hereinafter termed “ERB having the amino acid Sequence is Substantially the same amino acid Sequence for the of FIGS. 1, FIG. 13A or 14A or substantially the same amino purposes of the present application. Preferably, the amino acid Sequence as the amino acid Sequence shown in FIGS. 50 acid sequence is more than about 95identical with the 1, 13A or 14A or an amino acid Sequence functionally sequence shown in FIGS. 1, 13A or 14A. Similar to those Sequences. The isolated receptor may be According to another aspect of the invention there is particularly useful in the Search for molecules for use in provided a DNA sequence encoding a nuclear receptor treatment of diseases or conditions Such as cardiovascular according to the first aspect of the invention. Preferably, the diseases, central nervous System diseases or conditions or 55 DNA sequence is that given in FIGS. 1, 13A or 14A or is a Osteoporosis, prostate cancer or benign prostatic hyperpla DNA sequence encoding a protein or polypeptide having the S. functionality of ERB. The receptor of the invention may also be used in the ERB is unique in that it is extremely homologous to the testing of environmental chemicals for estrogenic activity. rat estrogen receptor, in particular in its DNA binding There has been increasing concern over the effect of various 60 domain. It appears that ERB has a very limited tissue chemicals released into the environment on the reproduction distribution. In female rats, it appears to be present only in of humans and animals. Threats to the reproductive capa the ovaries, and in male rats in the prostate and testes. AS bilities of birds, fish, reptiles, and Some mammals have these tissues are classic targets for estrogen action, it can be become evident and Similar effects in humans have been deduced that ERB may mediate some of the effects of proposed. Substantial evidence is now emerging which 65 eStrogen. shows that exposure to certain chemicals during critical The different ligand specificity of ERC. and ERB may be periods of foetal life may distort the development of the exploited to design pharmaceutical agents which are Selec 5,958,710 3 4 tive for either receptor. In particular, the differences in ligand Human ERB from testis (SEQ ID NO:15), Rat ERC. (SEQ ID Specificity may be used to develop drugs that Specifically NO:16), Human ERC. (SEQ ID NO:17), Human ERR1 (SEQ target cardiovascular disease in postmenopausal women or ID NO:18), and Human ERR2 (SEQ ID NO:19) Osteoporosis. FIG. 13 Ashows the amino acid sequence of human ERB; The nuclear receptor of the invention, ERB, a method of (SEQ ID NO:3) producing it, and tests on its functionality will now be FIG. 13B shows the DNA sequence of human Erf; (SEQ described, by way of example only, with reference to the ID NO:4) accompanying drawings. FIGS. 1 to 15 in which: FIG. 14A shows the amino acid sequence of mERB; FIG. 1 shows the amino acid sequence of ERB (SEQ ID (SEQ ID NO:5) NO:2) and the nucleotide Sequence of the gene encoding it FIG. 14B shows the DNA sequence of mouse ERB; (SEQ (SEQ ID NO:1); ID NO:6) and FIG. 2A is a phylogenetic tree showing the evolution of FIG. 15 illustrates ligand binding affinities for various ERB and other receptors; phytoestrogens by ER's of the invention. FIG. 2B shows the homology between the different domains in ERB and certain other receptors, A. CLONING OF RAT ERB FIG. 2C is an alignment of the amino acid Sequence in 15 1. PCR-Amplification and Complementary DNA Cloning the ligand binding domains of rERB, (SEQ ID NO:7) reRC, A set of degenerate primers (DBD 1,2,3 and WAK/FAK) (SEQ ID NO:8) mERC (SEQ ID NO:9) and hERC (SEQ ID were designed previously according to the most highly NO:10); conserved sequences of the DNA-binding domain (P-box) FIG. 2D is an alignment of the amino acid Sequence in and ligand binding domain (Ti-stretch) of members of the the DNA binding domains of rBRB(SEQ ID NO:11), reRC, nuclear receptor family (Enmark, E., Kainu, T., Pelto mERC. and hERC. (SEQ ID NO:12); Huikko, M., & Gustafsson, J-A (1994) Biochem. Biophys. FIG. 3A is a film autoradiograph of prostate gland Res. Commun. 204, 49-56). Single strand complementary showing Strong expression of a clone of the receptor of the DNA reverse transcribed from rat prostate total RNA was invention clone 29; employed with the primers in PCR reactions as described in FIG. 3B is a darkfield image showing prominent Signal 25 Enmark, E., Kainu, T., Pelto-Hulkko, M., & Gustafsson, J -A for clone 29 in epithelium (e) of prostatic alveoli. The (1994) Biochem. Biophys. Res. Commun. 204, 49–56. The Stroma(s) exhibit(s) weaker Signal; amplification products were separated on a 2% low melting FIG. 3C is a bipolarization image of cresyl violet coun agarose gel and DNA products between 400 and 700 bp were terstained section showing silver gains over epithelium (e) isolated from the gel and ligated to TA cloning vector whereas the Stroma(s) contain(s) less grains, (Invitrogen). As alternatives, we also used the RP-I/RP-2 The bar represents 0.7 mm for FIG.3A, 200 um for FIG. and DBD66-100/DBD210-238 primer sets in the DNA 3B and 30 um for FIG. 3C; binding domain of nuclear receptorS exactly as described by FIG. 4A shows a film autoradiograph of ovary showing Hirose T, Fijimoto, W., Yamaai, T., Kim, K. H., Matsuura, strong expression of clone 29 in follicles at different devel H., & Jetten, A. M. (1994) Mol. Endocrinol. 8, 1667–1677 opmental stages (Some are indicated by arrows). The inter 35 and Chang, C., Lopes Da Silva, S., Ideta, R., Lee, Y., Yeh, Stitial tissue (arrowheads) shows low signal; S., & Burbach, J. P. H. (1994) Proc. Natl. Acad. Sci. 91, FIG. 4B shows a darkfield image showing high expres 6040–6044 respectively. Clone number 29 (obtained with Sion of clone 29 in granular cells of primary (1) Secondary the DBD-WAK/FAK set) with a length of 462 bp showed (2), tertiary (3) and mature (4) follicles. Low Signal is high homology (65%) with the rat estrogen receptor cDNA present in interstitial tissue (it); 40 (65%), which was previously cloned from rat uterus (Koike. FIG. 4C is a bipolarization image of ovary a showing S., Sakai, M., & Muramatsu, M. (1987) Nucleic Acids Res Strong Signal in granular cells (gc), whereas the oocyte (oc) 15, 2499-2513). The amino acid residues predicted by clone and the cainterna (ti) are devoid of clear Signal; 29 DNA sequences suggested that this DNA fragment The bar represents 0.9 mm for FIG. 4A, 140 um for FIG. encoded part of the DNA-binding domain, hinge region and 4B and 50 um for FIG. 4C; 45 the beginning of the ligand binding domain of a novel FIG. 5A illustrates the results of Saturation ligand bind member of the nuclear receptor family. Two PCR primers ing analysis of cloned ERB, (FIG. 1) were used to generate a probe of 204 bp consisting FIG. 5B illustrates the specificity of ligand binding by of the hinge region of the novel receptor, which was used to cloned ERB; screen a rat prostate cDNA library (Clontech gt10) under FIG. 5C illustrates E2 binding by ERB; 50 Stringent conditions resulting in four Strongly positive FIG. 6 illustrates the activation of transcription by cloned clones with a size of 0.9 kb, 18 kb, 2.5 kb and 5-6 kb ERB; respectively. The clone of 2.5 kb was sequenced and FIG. 1 FIGS. 7 and 7A illustrates stimulation by various ligands shows the nucleotide Sequence determined in the core facil by cloned ERB; ity (CyberGene AB) by cycle sequencing using fluorescent FIG. 8 illustrates the results of RT-PCR experiments on 55 terminators (Applied BioSystems) on both Strands, with a the expression on rat estrogen receptors: Series of internal primers and deduced amino acid Sequence FIG. 9 illustrates the results of RT-PCR experiments on of clone 29. Two in frame ATG codons are located at the expression of human ERB (hERB); nucleotide 424 and nucleotide 448, preceding by an in-frame FIG. 10A is a Hill plot comparing binding of 'I-E2 by Stop codon at nucleotide 319, which Suggests that they are hERC. and rERB, 60 possible start codons. The open reading frame encodes a FIG. 10B is a Scatchard plot comparing binding of protein of 485 amino acid residues (counted from the first 'I-E2 by hERC. and reRB; methionine) with a calculated molecular weight of 54.2 kDa. FIG. 11A illustrates the relative binding affinity of hERC. Analysis of the proteins by Synthesized by in-vitro transla and rBRB for various ligands, tion from the clone 29 cRNA in rabbit reticulocyte lysate FIG. 11B is a detail of FIG. 12A; 65 revealed a doublet protein band nigrating at approximately FIG. 12 is an alignment of various estrogen receptors, 57 kDa on SDS-PAGEgels (data not shown), confirming the Rat ERB (SEQ ID NO:13), Mouse ERB (SEQ ID NO:14), open reading frame. The doublet protein band is probably 5,958,710 S 6 caused by the use of both ATG codons for initiation of 24-33 and Le Goff, P., Montano, M. M., Schodin, D. J., & protein Synthesis. The amino acid Sequence of clone 29 Katzeiiellenbogen, B. S (1994) J Biol. Chem. 269, protein shows the characteristic zinc module DNA-binding 4458–4466) are conserved in clone 29 protein (Ser 30 and domain, hinge region and a putative ligand binding domain, 42, Tyr 443). Clone 29 protein consists of 485 amino acid which are the characteristic features of members of the residues while ERCS from human, mouse and rat consist of nuclear receptor family (Tsai, M-J., & O'Malley, B.W 590-600 amino acid residues. The main difference is a much (1994) Ann. Re: Biochem. 63, 451-486; Hard, T., & shorter N-terminal domain in clone 29 protein i.e. 103 amino Gustafsson, J-A (1993) Acc. Chem. Res. 26, 644–650; acid residues as compared to 185-190 amino acid residues Laudet, V., Hanni, C., Coli, J., Catzeflis, F., & Stehelin, D in the other receptor proteins. Also the non-conserved (1992) EMBL J. 11, 1003–1012). So-called F-domain at the C-terminal end of ERCS is 15 Protein Sequence comparison with Several representative amino acid residues shorter in clone 29 protein. The cDNA members of the nuclear receptor family (FIG. 2) showed the insert of a positive clone of 2.6 kb was subcloned into the clone 29 protein is most related to the rat estrogen receptor EcoRI site of pBluescript (trademark) (Stratagene). The (ERC), cloned from uterus (Koike, S., Sakai, M., & complete DNA sequence of clone 29 was determined Muramatsu, M. (1987) Nucleic. Acids Res. 15, 2499-2513), 15 (CyberGene AB) by cycle Sequencing using fluorescent with 95% identity in the DNA-binding domain (amino acid terminators (Applied BioSystems) on both Strands, with a residues 103-167) (Griffiths, K., Davies, P., Eaton C. I., Series uf internal primers. Harper, M. E., Turkes, A., & Peeling, W. B. (1991) in FIGS. 2C and 2D respectively compare the ligand and Endocrine Dependent Tumours, eds. Voigt, K-D. & Knabbe, DNA binding domain of ERC. compared to rat, mouse and C. (Raven Press), pp. 83-125). A number of functional human Eros. characteristics have been identified within the DNA 2. Saturation Ligand Binding Analysis and Ligand Compe binding, domain of nuclear receptors (Hard, T., & tition Studies Gustafsson, J-A. (1993) Acc. Chem. Res 26 644-650 and Clone 29 cDNA was subcloned in p3luescript down Zilliacus, J., Carlstedt-Duke, J., Gustafsson, j-A., & Wright, stream of the T7 promoter to give p29-T7. Clone 29 protein A. P. H. (1994) Proc. Natl. Acad. Sci. USA 91,4175-4179). 25 was Synthesized in vitro using the TnT-coupled reticulocyte The So-called P-box Specifies nucleotide Sequence recogni lysate System (Promega). Translation reaction mixtures were tion of the core half-site within the response element, while diluted five times with TEDGMo buffer (40 mm Tris/HCl, the D-box mediates dimerization between receptor mono pH 7.4, 1 mM EDTA, 10% (v/v) glycerol, 10 mM NaMoO, mers. The clone 29 protein P-box and D-box sequences of 10 mM DTT) and 0.1 ml aliquots were incubated for 16 h EGCKA and PATNO, respectively, are identical to the at 8° C. with 0.3–6.2 nM (2,4,6,7-H-17B-estradiol (NEN corresponding boxes in ERC. (Hard, T., & Gustafsson. J-A. Dupont, specific radioactivity 85 Ci/mmol) in the presence (1993) Acc. Chem. Res 26, 644-650 and Koike, S., Sakai, or absence of a 200-fold excess of unlabelled E2. M., & Muramatsu, M. (1987) Nucleic Acids Res. 15, FIG. 5A illustrates the results of a Saturation ligand 2499-2513), thus predicting that clone 29 protein binds to analysis of clone 29 protein. Reticulocyte lysate containing “estrongen response element” (ERE) sequences. 35 clone 29 protein was incubated with 6 concentrations of The putative ligand binding domain (LBD) of clone 29 HE2 between 0.3 and 6.0 nM. Parallel tubes contained an protein (amino acid residues 259-457) shows closest homol additional 200 fold of non-radioactive E2. Bound and free ogy to the LBD of the rat ERC. (FIG.2), while the homology ligand were separated with a dextran-coated charcoal assay. with the human ERR1 and ERR2 proteins (Giguere, V., The Kd (0.6 nM) was calculated from the slope of the line Yang, N., Segui. P., & Evans R. M. (1988) Nature 331, 40 in the Scatchard plot shown (r=0.93), and the number of 91-94) is considerably less. With the human, mouse and binding sites was extrapolated from the intercept on the Xenopus estrogen receptors the homology in the LBD is also abscissa (Bmax=1400 fmol/ml undiluted translation around 55%, while the homology with the LBD of other mixture). Steroid receptors is not signiticant (FIG. 2). Cysteine residue For ligand competition Studies diluted reticulocyte lysate 530 in human ERC. has been identified as the covalent 45 was incubated with 5 nM 2.4.6.7-H-17B-estradiol in the attachment site of an estrogenic affinity label (Harlow, K. presence of either 0, 5, 50, 500 or 5,000 nM of non W., Smith D. N., Katzenellenbogen, J. A., Greene, G. L., & radioactive E2, estrone, estriol, testosterone, progesterone, Katzenellenbogen, B. S. (1989).J. Biol. Chem. 264, corticosterone, 5C-androstane-3?,173-diol, 5C-androstane 17476-17485). Interestingly, clone 29 protein (Cys-436) as 3C, 17 B-diol and diethylstilbestrol (DCES) for 16 h at 8° C. well as the mouse, rat and Xenopus ERCS have a cysteine 50 Bound and unbound Steroids were Separated with a dextran residue at the corresponding position. Also, two other amino coated charcoal assay (Ekman, P., Barrack, E. R., Greene, G. acid residues described to be close to or part of the ligand L., Jensen, E. V., & Walsh, P. C. (1983).J. Clin. Endocrinol binding pocket of the human ERC-LBD (Asp 426 and Gly Metab. 57, 166–176). 521) are conserved in the LBD of clone 29 protein (Asp 333 FIG. 5B illustrates the specificity of ligand binding by and Gly 427) and in the LBD of EROS from various species 55 clone 29 protein. Reticulocyte lysate containing clone 29 (20.21). The ligand-dependent transactivation function protein was equilibrated for 16 h with 5 nM HE2 and the TAF-2 identified in ERC. (Danielian, P. S., White, R., Lees, indicated fold excess of competitors. Data represent (HE2 J. A., & Parker, M. G. (1992) EMBO J. 11, 1025-1033), bound in the presence of unlabelled E2, testosterone (T), which is believed to be involved in contacting other tran progesterone (prog), corticosterone (cortico), estrone (E1), Scription factors and thereby influencing activation of tran 60 diethylstilbestrol (DES), 5o-androstane-3C, 17B-diol (3C.- Scription of tarteg genes, is almost completely conserved in AD), 5o-androstane-3,3,17B-diol (3B-AD) and estriol (E3). clone 29 protein (amino acid residues 441-457). Steroid HE2 binding in the absence of competitor was set at hormone receptors are phosphoproteins (Kuiper, G. & 100%. Brinkmann, A. O. (1994) Mol. Cell. Endocrinol. 100, 3. In-situ Hybridisation 103-107), and several phosphorylation sites identified in the 65 In- Situ hybridisation was carried out as previously N-terminal domain and LBD of ERC. (Arnold, S. F., Obourn, described (Dagerlind A., Friber, K., Bean, A. J., & Hokfelt, J. D., Jaffe, H., & Notides. A. C. (1995) Mol. Endocrinol 9, T (1992) Histochemistry 98, 39–49). Briefly, two oligo 5,958,710 7 8 nucleotide probes directed against nucleotides 994-1041 containing 5% serum substitute (SRC 3000, Tissue Culture and 1981–2031 were each labelled at the 3'-end with P Services Ltd., Botolph Claydon, Buckinghnam, UK) 2 mM dATP using terminal deoxynucleotidyltransferase Lglutamine and 50 lug/ml gentamicin plus hormones as (Amersham, UK). Adult male and female Sprague-Dawley indicated. After 48 h the medium was assayed for alkaline rats (age 2 to 3 months n=10) were used for this study. The phosphatase (ALP) activity by a chemiluminescence assay. rats were decapitated and the tissues were rapidly excised A 10 ul aliquot of the cell culture medium Was mixed with and frozen on dry ice. The tissues were Sectioned in a 200 ul assay buffer (10 mM diethanolamine pH 10.01mM Microm HM500 cryostat at 14 um and thawed onto Probe MgCl, and 0.5 mM CSPD (Tropix Inc. Boston, USA)) and On glass slides (Fisher Scientific, PA, USA). The slides were incubated for 20 min at 37 C. before measurement in a stored at -20° C. until used. The slides were incubated in microplate luminometer (Luminoskan; Labsystems, humidifed boxes at 42° C. for 18 h with 1x10 cpm of the Finland) with integral measurement for 1 second. The ALP probe in a hybridization solution containing 50% tonnamide. activity of ERE-reporter alone was set at 1. 4xSSC (1xSSC=0.15 M NaCl, 0.015 M sodium citrate), 5.Ligand Binding Characteristics and Transactivation Func 1xDenhardt (0.02% BSA, 0.02% Ficoll, 0.02% PVP), 1% tion of Clone 29 Protein Sarkosyl, 0.02 M sodium phosphate (pH7.), 10% 15 On the basis of the described high homology between dextransulphate, 500 tug/ml salmon sperm DNA and 200 clone 29 protein and rat ERC. in the DBD and LBD it was mnMDTT. Slides were subsequently rinsed in 1xSSC at 55° hypothesized that clone 29 protein might encode a novel ER. C. for 60 min with four changes of SSC and finally in 1xSSC Furthermore, biological effects of estrogens on rat prostate Starting at 55 C. and slowly cooled to room temperature, and ovary, which show high expression of clone 29 RNA, transferred through distilled water and briefly dehydrated in are well known (Griffiths, K., Davies, P., Eaton, C. I., 50% and 95% ethanol for 30 sec each, air-dried, and covered Harper, M. E., Turkes, A., & Peeling W. B. (1991) in with Amersham B-man autoradiography film for 15 to 30 Endocrine Dependent Tumours, eds Voigt, K-D. & Knabbe, days. Alternatively the slides were dipped in Kodak NTB2 C. (Raven Press), pp 83-125, Richards, J. S (1994) Endo nuclear track emulsion (diluted 1:1 with distilled water) and crine Rev. 15, 725-745; and Habenicht, U-F., Tunn, U.W., exposed for 30 to 60 days at 4 C. Finally, the sections were 25 Senge, Th., Schroder, R. H., Schweikert, H. U., Bartsch, G., stained with cresyl violet. & E1 Etreby. M. F. (1993).J. Steroid Biochem. Molec. Biol. Clear expression of clone 29 was observed in the repro 44, 557-563). In order to analyze the steroid binding prop ductive tract of both male and female rats, while in all other erties of clone 29 protein synthesized in vitro, the reticulo rat tissues the expression was very low or below the level of cyte lysate was incubated at 8 C. for 16 h with increasing detection with in-situ hybridisation (not shown). In male concentrations (0.3–6.0 nM) of HE2 in the presence or reproductive organs high expression was Seen in the prostate absence of a 200 fold molar excess of unlabelled E2. Linear gland (FIG. 3), while very low expression was observed in transformation of Saturation data revealed a Single popula testis, epididymis and Vesicula Seminalis (not shown). In tion of binding sites for E2 with a K (dissociation constant) dipped Sections, expression was clearly visible in prostate of 0.6 nM (FIG. 5A and C). Steroid binding specificity was epithelial cells (Secreting alveoli) while the expression in 35 measured by incubating reticulocvte lysate with 5 nMH) Smooth muscle cells and fibroblasts in the stroma was low E2 in the presence of 0.5, 50, 500 and 5,000 nM unlabelled (FIG. 3). In female reproductive organs expression was seen competitors. Competition curves generated are indicative of in the ovary (FIG. 4), while uterus and vagina were negative an estrogen receptor in that only estrogens competed effi (not shown). In dipped sections high expression was seen in ciently with HE2 for binding (FIG. 5B). Fifty percent the granulosa cell layer of primary, Secondary and mature 40 inhibition of specific binding occured by 0.6 fold excess of follicles (FIG. 4), whereas primordial follicles, oocytes and unlabelled E2; diethylstilbestrol, estriol, estrone and corpora lutea appeared completely negative. Low expression 5C.-androstane-3?,17(3-diol were 5, 15, 50 and 150 times, was seen in the interStitial cells of the ovary. Both anti-Sense respectively, less effective as competitors. Neither oligonucleotide probes used produced similar results. Addi testosterone, progesterone, corticosterone nor 5C.- tion of a 100 fold excess of the respective unlabelled 45 androstane-3C, 173-diol were efficient competitors, even at oligonucleotide probes during the hybridisation reactions the highest concentrations used (1000 fold excess). The abolished all signals. dissociation constant and the Steroid binding Specificities 4. Transactivation Analysis in CHO-Cells measured are in good agreement with data previously The expression vector pCMV29 was constructed by reported for ERS in rat and human prostate, rat granulosa inserting the 2.6 kb clone 29 fragment in the EcoRI site of 50 cells, rat antral follicles and whole rat ovarian tissue the expression vector pCMV5 (Andersson, S., Davis, D. L., (Ekman, P., Barrack, E. R., Greene, G. L., Jensen, E. V., & Dahlback, H., Jornwall, H., & Russell, D. W. (1989) J. Biol. Walsh. P. C (1983).J. Clin. Endocrinol. Metab. 57, 166–176; Chem. 264, 8222-8229). The pERE-ALP reporter construct van Beurden-Lamers, W. M. O., Brinkmann, A. O., Mulder, contains a Secreted form of the plancental alkaline phos E., & van der Molen, H. (1974) Biochem. J 140, 495-502; phatase gene (Berger, J., Hauber, J., Hauber, R., Geiger, R., 55 Kudolo, G. B., Elder, M. G., & Myatt, L. (1984) J. Endo & Cullen, B. R. (1988) Gene 66, 1-10) and the MMTV-LTR crinol. 102, 83-91, and Kawashima, M., & GreenWald, G. in which the glucocorticoid response elements were replaced S. (1993) Biology of Reprod. 48 172–179). by the Vitellogenin promoter estrogen response element When clone 29 protein was labelled with a saturating dose (ERE). of HE2 and analyzed on Sucrose density gradients, a CHO-K1 cells were seeded in 12-well plates at approxi 60 Single peak of Specifically bound radioactivity was mately 1.7x10 cells per well in phenol-red free Ham F12 observed. The sedimentation coefficient of this complex was medium with 5% FCS (dextran-coated charcoal treated) and about 7S, and it shifted to 4S in the presence of 0.4M NaCl 2 mM Lglutamine. After 24 h the cells were transfected with (not shown). To investigate the transcriptional regulatory 250 ng peRL-ALP vector and 50 ng pCMV29 using lipo properties of clone 29 protein, we performed co-transfection fectamine (Gibco) according to the manufacturers instruc 65 experiments in which CHO cells were transfected with a tions. After five hours of incubation the cells were washed clone 29 protein expression vector and/or an estrogen and refed with 0.5 ml phenol-red free Coon's F-12 medium responsive reporter gene construct. Cells were incubated in 5,958,710 9 10 the absence of E2 (clone 29) or in the presence of 100 nM Same cells. In addition, it will be seen that in human E2 (Clone 29--F2) or in the presence of 100 nM E2 and 12 osteosarcoma cell line (HOS-D4), hERB is expressed in uM Tamoxifen (Clone 29+E2/Tam). In the absence of exog greater quantities compared to hERC. enously added E2 clone 29 protein showed considerable The partial DNA sequence of hERB is shown in FIG. 13B transcriptional activity which could be further increased by (SEQID NO:4) and a derived amino acid sequence is shown the addition of 100 nM E2 (FIG. 6). Simultaneous addition in FIG. 13A (SEQ ID NO:3). of a 10 fold excess of the antiestrogen Tamoxifen partially Cloning of Human ERB from testis A commercially available cDNA from human testis suppressed the E2 stimulated activity (FIG. 6). The consti (Clontech, article no. HL1161X) was Screened, using a tutive transcriptional activity of clone 29 protein could be fragment containing the ligand-binding domain of the rat suppressed by the anti-estrogen ICI-1624384 (not shown). It ERB cDNA as probe. Approximately 10 recombinants were has been shown previously that the wild-type mouse and Screened, resulting in one positive clone. Upon Sequencing human ERS are constitutive activators of transcription, and of this clone, it was seen that the insert was 1156 bp (FIGS. that the transcriptional activity can be stimulated further by 13A and 13B). This corresponds to most of the translated the addition of E2 (Txukerman, M., Xiao-Kun Zhang., region of a receptor with an overall homology of 90.0% to Hermann, T., Wills, K. N., Graupner, G., & Phal. M. (1990) 15 rat ERB, therefore deduced to represent the human form of New Biologist 2, 613-620 and Lees, J. A., Fawell, S. E., & ERB. Parker, M. G. (1989) Nucl. Acids Res. 17, 5477–5488). To The cloned hERB, however, lacks approximately 47 obtain more insight into what concentrations of E2 effect amino acids at the N-teninal end and 61 amino acids at the clone 29 protein transcriptional activity, transient transfec C-terminal end (as compared to the rat Sequence). Further tion experiments were carried out in the presence of increas Screening of the same library was unsuccessful. PCR tech ing concentrations of E2. CHO-cells were transiently trans nology was therefore used to obtain the remaining parts. For fected with the ERE-reporter plasmid and the clone 29 oligonucleotides were Synthesised, two degenerate oligo protein expression plasmid. Cells were incubated with nucleotides containing all possible codons for the amino increasing concentrations of E2 (0.1-1000 nM), estrone (E1, acids adjacent to the initiation methlionine and the Stop 1000 nM).5o-androstane-3,3,17|3-diol (3.B-AD, 1000 nM) or 25 codon, respectively, of the rat ERB, and two Specific oligo no ligand added. Alkaline phosphatase activity (ALP) was nucleotides containing the Sequence of the clone isolated from the human testis library and Situated approximately measured as described and the activity in the absence of 100 bp from respective end of this clone. PCR with the ligand (control) was set at 1. The figure shows relative N-terminal and C-terminal pair of oligos yielded specific ALP-activities (t-SD) from three independent experiments. bands, that were Subcloned and Sequenced. The parts of Clone 29 protein began to respond at 0.1 nM E2 and these new clones that overlap the original cDNA clone are maximal stimulation was observed between 1 nm and 10 nM identical to this. It was thus possible to construct peptide and E2 (FIG. 7). The maximal stimulation factor was 2.6+0.5 DNA sequences corresponding to the whole open reading fold (meantSD. n=9) as compared to incubation in the frame (FIGS. 13A and 13B). absence of E2. Apart from E2 also estrone and When comparing the human ERB to rat ERB, this receptor 5C.-androstane-3,3,173-diol could stimulate transcriptional 35 is 79.6% identical in the N-terminal domain, 98.5% in the activity, albeit at higher concentrations (FIG. 7). DNA-binding domain, 85.6% in the hinge and 91.6% in the Dexamethasone, testosterone, progesterone, 5C-androstane ligand-binding and F-domains. These numbers match very 3C,17(3-diol, thyroid hormone and all-trans-retinoic acid well those found when comparing the rat and human forms could not stimulate transcriptional activity of clone 29 of ERC. protein, even at the highest concentration (1000 nM) tested 40 Studies of the expression of human ERB using Northern (not shown). The results of the co-transfection experiments blot show expression in testis and in ovaries. The expression are in agreement with the ligand binding and Specificity data in prostate, however, appears lower than found in the rat. of clone 29 protein presented in FIG. 5. In control The human ERB gene has been mapped to chromosome experiments, wild-type human ERC. also showed transcrip 14 using PCR and to region 14q22-23 using the FISH tional activity in the absence of E2, which could be 45 technique, whereas the human ERB gene has been mapped increased by the addition of E2 (not shown). to chromosome 6q25. 6. Detection of Rat ER Expression by RT-PCR 2. Comparison of Ligand Binding Affinity of hERC. and The tissue specificity of expression of rat ERB and ERC. rERB was determined using reverse transcriptase polymerase The ligand affinity of the two estrogen receptors, human chain reaction (RT-PCR). The results of the experiment are 50 ERC. (ovary) (hERC) and rat ERB (rBRf8) was tested in shown in FIG. 8. binding Saturation experiments and in binding competition B. Isolation of Human ERB experiments. 1 A human version of ERB (hERf8) has also been cloned cDNA of the receptor subtypes hERC. and rBRB were in from human ovary. The tissue specificity of hERB expres Vitro translated in rabbit reticulocyte lysate in presence of Sion in a variety of cells was also determined using the 55 non-radioactive amino acids according to the instructions RT-PCR technique. The results are shown in FIG. 9. It will Supplied by the manufacturer (Promega). be noticed that there is a very high level of mRNA of hERB The radioactive ligand used in all experiments was 16C.- in human umbilical vein endothelial cells (HUVEC) but no I-17B-estradiol (II-E2) (NEX-144, New England detection of hERC. in the same cells. In addition, it will be Nuclear). The method for the binding experiments was seen that in human osteosarcoma cell line (HOS-D4), hERB 60 previously described in: Salomonsson M, Carlsson B, Hag is expressed in greater quantities compared to hERC. gblad J. J. Steroid Biochem. Molec. Biol. Vol. 50, No. 5/6 pp. I. A human version of ERB (hERB) has also been cloned. 313-18, 1994. In brief, estrogen receptors are incubated The tissue specificity of hERB expression in a variety of with I-E2 to equilibrium (16–18 h at +4° C.). The cells was also determined using the RT-PCR technique. The incubation was stopped by Separation of protein-bound results are shown in FIG. 9. It will be noticed that there is 65 'Il-E2 from free 'I-E2 on Sephadex G25 columns. a very high level of mRNA of hERB in human umbilical vein The radioactivity of the eluate is measured in a gamma endothelial cells (HUVEC) but no detection of hERC. in the COunter. 5,958,710 11 12 In the competition experiments, non-radioactive ligands I is the added concentration of binding inhibitor, ICso is the were diluted in DMSO, mixed with I-E2 concentration of inhibitor at half maximal binding and S is (approximately 100-200 pM), aliquoted in parallel, and a slope factor. The free concentration of ''Il-E2 was finally hERC. or rBRB was added. The final concentration of determined by Sampling an aliquot from the Wells at the end DMSO in the binding buffer was 2%. 5 of the incubation and then substract bound radioactivity The s used in the experiments was of the following from Sampled total radioactivity. composiuon: Since the equilibrium binding experiments (above Hepes (pH=7.5) 20 mM, KC1 150 mM, EDTA 1 mM, C gig, exp ( ) glycerol (8.7%), monothioglycerol 6 mM, Na-MO 10 mM. showed that the K-values for "f I-E2 differed between the- - - 3.. EquilibriEquilibrium Binding Saturation EExperiments tS (K-(K. two ER:s, K-values (from the Cheng-Prusoff1251 equation: Determinations) 10 K=ICso/(1+L/K) where L is free ('''Il-E2) were calcu A range of concentrations of I-E2 were mixed with lated for the compounds investigated. Two approaches for the ER:s and incubated as described above, free 'I-E2 calculating RBA (Relative Binding Affinitv) were used. The was determined by subtracting bound 'I-E2 from added RBA values were derived using either the ICso values or the 'I-E2. Binding data was analysed by Hill-plots and by K values. In both approaches, the value for the compound Scatchard plots (FIG. 11). The equilibrium binding results 15 16C.-bromo-cStradiol was selected as the reference value are shown in Table 1. The apparent K-values for 'I-E2 (100%). Both approaches gave Similar results. The results differed between the two ER:s with approximately a factor are summarized in FIGS. 11A and 11B. In these Figures of four; K(hERC):K(rERB)=1:4. 4-OH Tam"=4-hydroxy-tamoxifen; “DES diethylstilbestrol; “Hexestr'=hexestrol: “ICI-164384'-ICI TABLE 1. 20 plc compound no. 164382; “17B-E2'=17 B-estradiol; “16a B- E2 = 16C.-bromo-estradiol; “Ralox'=Raloxifen; and Equilibrium dissociation constants for 'I-E2 “17a-E2'-17C, diol. - to the two subtypes The results show that ERC. and ERB have significant Receptor subtype K (Hill-plot) K (Scatchard-plot) different ligand binding affinities—the apparent K-values 25 for 'I-E2 differed between the two ELR's by a factor of hERCi. O.O6 nM O.O9 nM about 4 (K(hERC): K (rBRB) s1:4). Some compounds rERB O.24 nM O.42 nM investigated showed significant differences in the competi tion for binding of I-E2 to the ER's. Certain compounds 4. Competition Experiments (ICso Determinations) were found to be more potent inhibitors of ''Il-E2 binding The experiments were performed as described above. 30 to hERC. as compared to rBRB whereas others were found to ICso values were obtained by applying a four parameter be more potent inhibitors of I-E2 binding to reR? than logistic analysis, b=(b-bit)/(1+(I/ICso)))+b, where to hERO.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 19

(2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2568 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus rattus (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

GGAATTCCGG GGGAGCTGGC. CCAGGGGGAG CGGCTGGTGC TGCCACTGGC ATCCCTAGGC 60

ACCCAGGTCT, GCAATAAAGT CTGGCAGCCA CTGCATGGCT GAGCGACAAC CAGTGGCTGG 120

GAGTCCGGCT. CTGTGGCTGA. GGAAAGCACC TGTCTGCATT TAGAGAATGC AAAATAGAGA 18O

ATGTTTACCT GCCAGTCATT ACATCTGAGT CCCATGAGTC. TCTGAGAACA. TAATGTCCAT 240

CTGTACCTCT, TCTCACAAGG AGTTTTCTCA GCTGCGACCC. TCTGAAGACA TGGAGATCAA 3OO

AAACT CACCG TCGAGCCTTA GTTCCCTGCT, TCCTATAACT GTAGCCAGTC CATCCTACCC 360

CTGGAGCACG GCCCCATCTA CATCCCTTCC. TCCTACGTAG ACAACCGCCA TGAGTATTCA 420

GCTATGACAT. TCTACAGTCC TGCTGTGATG AACTACAGTG TTCCCGGCAG CACCAGTAAC 480

CTGGACGGTG GGCCTGTCCG ACTGAGCACA AGCCCAAATG TGCTATGGCC AACTTCTGGG 540

5,958,710 15 16 -continued

Met Thr Phe Ser Pro Ala Wall Met Asn Ser Val Pro Gly Ser 10 15

Thr Ser Asn Telu Asp Gly Gly Pro Wall Arg Teu Ser Thr Ser Pro Asn 25 30

Wall Telu Trp Pro Thr Ser Gly His Telu Ser Pro Teu Ala Thr His Cys 35 40 45

Glin Ser Ser Telu Teu Ala Glu Pro Glin Ser Pro Trp Cys Glu 50 55 60

Ala Arg Ser Telu Glu His Thr Telu Pro Wall Asn Arg Glu Thr Telu Lys 65 70 75

Arg Telu Ser Gly Ser Ser Ala Ser Pro Wall Thr Ser Pro Asn 85 90 95

Ala Arg Asp Ala His Phe Pro Wall Ser Asp Tyr Ala Ser 100 105 110

Gly Tyr His Gly Wall Trp Ser Glu Gly Lys Ala Phe Phe 115 120 125

Arg Ser Ile Glin Gly His Asn Asp Ile Cys Pro Ala Thr Asn 130 135 1 4 0

Glin Thr Ile Asp Lys Asn Arg Arg Ser Glin Ala Cys Arg 145 15 O 155 160

Teu Arg Tyr Glu Wall Gly Met Wall Gly Ser Arg Arg 1.65 170 175

Glu Arg Gly Tyr Arg Ile Wall Arg Arg Glin Arg Ser Ser Ser Glu 18O 185 190

Glin Wall His Teu Ser Ala Arg Asn Gly Gly His Ala Pro 195 200

Arg Wall Glu Teu Teu Teu Ser Thr Telu Ser Pro Glu Glin Telu Wall 210 215 220

Teu Thr Telu Telu Glu Ala Glu Pro Pro Asn Wall Teu Wall Ser Pro 225 230 235 240

Ser Met Pro Phe Thr Glu Ala Ser Met Met Met Ser Teu Thr Lys Telu 245 250 255

Ala Asp Glu Teu Wall His Met Ile Gly Trp Ala Tys Ile Pro 260 265 27 O

Gly Phe Wall Glu Teu Ser Teu Telu Asp Glin Wall Arg Teu Teu Glu Ser 275 280 285

Trp Met Glu Wall Teu Met Wall Gly Telu Met Trp Arg Ser Ile Asp 29 O 295

His Pro Gly Lys Teu Ile Phe Ala Pro Asp Teu Wall Teu Asp Asp 305 310 315 320

Glu Gly Lys Wall Glu Gly Ile Telu Glu Ile Phe Asp Met Telu Telu 325 330 335

Ala Thr Thr Ser Arg Phe Arg Glu Telu Teu Glin His Tys Glu Tyr 340 345 350

Teu Wall Ala Met Ile Telu Telu Asn Ser Ser Met Pro Telu 355 360 365

Ala Ser Ala Asn Glin Glu Ala Glu Ser Ser Arg Lys Teu His Telu 370 375

Teu Asn Ala Wall Thr Asp Ala Telu Wall Trp Wall Ile Ala Ser Gly 385 390 395 400

Ile Ser Ser Glin Glin Glin Ser Wall Arg Telu Ala Asn Teu Teu Met Telu 405 410 415

Teu Ser His Wall Arg His Ile Ser Asn Gly Met Glu His Telu Telu 5,958,710 17 -continued

420 425 430 Ser Met Lys Cys Lys Asn Val Val Pro Val Tyr Asp Leu Lleu Lieu Glu 435 4 40 4 45 Met Lieu. Asn Ala His Thr Lieu Arg Gly Tyr Lys Ser Ser Tle Ser Gly 450 455 460 Ser Glu Cys Ser Ser Thr Glu Asp Ser Lys Asn Lys Glu Ser Ser Glin 465 470 475 480

Asn Lieu Glin Ser Glin 485

(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 485 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi). SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Thr Phe Tyr Ser Pro Ala Val Met Asn Tyr Ser Ile Pro Ser Asn 1 5 10 15 Val Thr Asn Leu Glu Gly Gly Pro Gly Arg Gln Thr Thr Ser Pro Asn 2O 25 30 Val Leu Trp Pro Thr Pro Gly. His Leu Ser Pro Leu Val Val His Arg 35 40 45 Glin Leu Ser His Leu Tyr Ala Glu Pro Gln Lys Ser Pro Trp Cys Glu 50 55 60 Ala Arg Ser Lieu Glu His Thr Lieu Pro Val Asn Arg Glu Thir Lieu Lys 65 70 75 8O Arg Llys Val Ser Gly Asn Arg Cys Ala Ser Pro Val Thr Gly Pro Gly 85 90 95 Ser Lys Arg Asp Ala His Phe Cys Ala Val Cys Ser Asp Tyr Ala Ser 100 105 110 Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe 115 120 125 Lys Arg Ser Ile Glin Gly. His Asn Asp Tyr Ile Cys Pro Ala Thr Asn 130 135 1 4 0 Glin Cys Thir Ile Asp Lys Asn Arg Arg Lys Ser Cys Glin Ala Cys Arg 145 15 O 155 160 Leu Arg Lys Cys Tyr Glu Val Gly Met Wall Lys Cys Gly Ser Arg Arg 1.65 170 175 Glu Arg Cys Gly Tyr Arg Lieu Val Arg Arg Glin Arg Ser Ala Asp Glu 18O 185 190 Glin Lieu. His Cys Ala Gly Lys Ala Lys Arg Ser Gly Gly His Ala Pro 195 200 2O5 Arg Val Arg Glu Lieu Lleu Lleu. Asp Ala Leu Ser Pro Glu Gln Leu Val 210 215 220 Leu Thr Leu Leu Glu Ala Glu Pro Pro His Val Leu Ile Ser Arg Pro 225 230 235 240 Ser Ala Pro Phe Thr Glu Ala Ser Met Met Met Ser Leu Thr Lys Leu 245 250 255 Ala Asp Lys Glu Lieu Val His Met Ile Ser Trp Ala Lys Lys Ile Pro 260 265 27 O Gly Phe Val Glu Lieu Ser Leu Phe Asp Glin Val Arg Lieu Lieu Glu Ser 275 280 285 5,958,710 19 20 -continued

Cys Trp Met Glu Val Leu Met Met Gly Leu Met Trp Arg Ser Ile Asp 29 O 295

His Pro Gly Lys Lieu. Ile Phe Ala Pro Asp Lieu Val Lieu. Asp Arg Asp 305 310 315 320

Glu Gly Lys Cys Val Glu Gly Ile Leu Glu Ile Phe Asp Met Telu Telu 325 330 335 Ala Thr Thr Ser Arg Phe Arg Glu Lieu Lys Lieu Gln His Lys Glu Tyr 340 345 350

Lieu. Cys Wall Lys Ala Met Ile Telu Lieu. Asn. Ser Ser Met Tyr Pro Leu 355 360 365

Val Thr Ala Thr Glin Asp Ala Asp Ser Ser Arg Lys Lieu Ala His Lieu 370 375

Lieu. Asn Ala Val Thr Asp Ala Telu Val Trp Val Ile Ala Lys Ser Gly 385 390 395 400

Ile Ser Ser Glin Glin Glin Ser Met Arg Lieu Ala Asn Lieu. Telu Met Leu 405 410 415

Leu Ser His Val Arg His Ala Ser Asn Lys Gly Met Glu. His Telu Telu 420 425 430

Asn Met Lys Cys Lys Asn Wall Wall Pro Val Tyr Asp Leu Lieu Leu Glu 435 4 40 4 45

Met Leu Asn Ala His Wall Teu Arg Gly Cys Lys Ser Ser Ile Thr Gly 450 455 460 Ser Glu Cys Ser Pro Ala Glu Ser Lys Ser Lys Glu Gly Ser Glin 465 470 475 480

Asn Lieu Glin Ser Glin 485

(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1460 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 4:

CTATGACATT CTACAGTCCT. GCTGTGATGA ATTACAGCAT TCCCAGCAAT GTCACTAACT 60

TGGAAGGTGG GCCTGGTCGG CAGACCACAA. GCCCAAATGT GTTGTGGCCA ACACCTGGGC 120

ACCTTTCTCC TTTAGTGGTC CATCGCCAGT TATCACATCT GTATGCGGAA CCT CAAAAGA 18O

GTCCCTGGTG TGAAGCAAGA TCGCTAGAAC ACACCTTACC TGTAAACAGA GAGACACTGA 240

AAAGGAAGGT TAGTGGGAAC CGTTGCGCCA GCCCTGTTAC TGGTCCAGGT TCAAAGAGGG

ATGCTCACTT CTGCGCTGTC TGCAGCGATT ACGCATCGGG ATATCACTAT GGAGTCTGGT 360

CGTGTGAAGG ATGTAAGGCC TTTTTTAAAA GAAGCATTCA AGGACATAAT GATTATATTT 420

GTCCAGCTAC AAATCAGTGT ACAATCGATA AAAACCGGCG CAAGAGCTGC CAGGCCTGCC 480

GACTTCGGAA GTGTTACGAA. GTGGGAATGG TGAAGTGTGG CTCCCGGAGA GAGAGATGTG 540

GGTACCGCCT TGTGCGGAGA CAGAGAAGTG CCGACGAGCA GCTGCACTGT GCCGGCAAGG 600

CCAAGAGAAG TGGCGGCCAC. GCGCCCCGAG TGCGGGAGCT GCTGCTGGAC GCCCTGAGCC 660

CCGAGCAGCT AGTGCTCACC CTCCTGGAGG CTGAGCCGCC CCATGTGCTG ATCAGCCGCC 720

CCAGTGCGCC CTTCACCGAG GCCTCCATGA TGATGTCCCT GACCAAGTTG GCCGACAAGG 5,958,710 21 22 -continued

AGTTGGTACA CATGATCAGC TGGGCCAAGA AGATTCCCGG CTTTG GGAG CTCAGCCTGT 840

TCGACCAAGT GCGGCTCTTG GAGAGCTGTT GGATGGAGGT GTTAA GATG GGGCTGATGT 9 OO

GGCGCTCAAT TGACCACCCC GGCAAGCTCA TCTTTGCTCC AGATC TGTT CTGGACAGGG 96.O

ATGAGGGGAA ATGCGTAGAA GGAATTCTGG AAATCTTTGA CATGC CCTG GCAACTACTT O20

CAAGGTTTCG AGAGTTAAAA CTCCAACACA. AAGAATATCT CTGTG CAAG GCCATGATCC

TGCTCAATTC CAGTATGTAC CCTCTGGTCA CAGCGACCCA GGATGCTGAC AGCAGCCGGA 14 O

AGCTGGCTCA CTTGCTGAAC GCCGTGACCG ATGCTTTGGT TTGGG GATT GCCAAGAGCG 200

GCATCTCCTC CCAGCAGCAA TCCATGCGCC TGGCTAACCT CCTGA GCTC CTGTCCCACG 260

TCAGGCATGC GAGTAACAAG GGCATGGAAC ATCTGCTCAA CATGAAGTGC AAAAATGTGG 320

TCCCAGTGTA TGACCTGCTG CTGGAGATGC TGAATGCCCA TCGC GGGTGCAAGT

CCTCCATCAC GGGGTCCGAG TGCAGCCCGG CAGAGGACAG TAAAAGCAAA. GAGGGCTCCC 4 40

AGAACCTACA GTCTCAGTGA 460

(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: A) LENGTH: 485 amino acids B) TYPE: amino acid D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (xi). SEQUENCE DESCRIPTION: SEQ ID NO:5: Met Ala Phe Tyr Ser Pro Ala Val Met Asn Tyr Ser Wall Pro Ser Ser 5 10 15

Thr Gly Asn Leu Glu Gly Gly Pro Val Arg Glin Thir Ala Ser Pro Asn 2O 25 30

Val Leu Trp Pro Thir Ser Gly His Leu Ser Pro Leu Ala Thr His Cys 35 40 45

Glin Ser Ser Lieu Lleu Tyr Ala Glu Pro Gln Lys Ser Pro Trp Cys Glu 50 55 60

Ala Arg Ser Leu Glu His Thr Telu Pro Wall Asn Arg Glu Thr Lieu Lys 65 70 75

Arg Lys Lieu Gly Gly Ser Gly Cys Ala Ser Pro Wall Thir Ser Pro Ser 85 90 95

Thr Lys Arg Asp Ala His Phe Cys Ala Val Cys Ser Asp Tyr Ala Ser 100 105 110

Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe 115 120 125

Lys Arg Ser Ile Glin Gly His Asn Asp Tyr Ile Cys Pro Ala Thr Asn 130 135 1 4 0

Gln Cys Thr Ile Asp Lys Asn Arg Lys Asn Cys Glin Ala 145 15 O 155 160

Leu Arg Lys Cys Tyr Glu Wall Gly Met Val Lys Cys Gly Ser Arg Arg 1.65 170 175

Glu Arg Cys Gly Tyr Arg Ile Wall Arg Arg Glin Arg Ser Ala Ser Glu 18O 185 190

Glin Wal His Cys Lieu. Asn Ala Lys Arg Thr Ser Gly. His Thr Pro 195 200 2O5

Arg Val Lys Glu Lieu Lieu Teu Asn Ser Lieu. Ser Pro Glu Glin Leu Wall 210 215 220

Lieu. Thir Lieu Leu Glu Ala Glu Pro Pro Asn. Wall Leu Wal Ser Arg Pro 5,958, 710 23 24 -contin ued

225 230 235 240

Ser Met Pro Phe Thr Glu Ala Ser Met Met Met Ser Lieu. Thr Lys Lieu 245 250 255

Ala Asp Lys Glu Lieu Val His Met Ile Gly Trp Ala Lys Lys Ile Pro 260 265 27 O

Gly Phe Val Glu Leu Ser Teu Telu Asp Glin Val Arg Lieu Lieu Glu Ser 275 280 285

Cys Trp Met Glu Val Leu Met Wall Gly Leu Met Trp Arg Ser Ile Asp 29 O 295 3OO

His Pro Gly Lys Lieu. Ile Phe Ala Pro Asp Lieu Val Lieu. Asp Arg Asp 305 310 315 320

Glu Gly Lys Cys Val Glu Gly Ile Leu Glu Ile Phe Asp Met Telu Telu 325 330 335

Ala Thr Thr Ala Arg Phe Glu Lieu Lys Lieu Gln His Lys Glu Tyr 340 345 350

Lieu. Cys Wall Lys Ala Met Ile Telu Lieu. Asn. Ser Ser Met Tyr His Lieu 355 360 365

Ala Thr Ala Ser Glin Glu Ala Glu Ser Ser Arg Lys Lieu. Thr His Lieu 370 375 38O

Lieu. Asn Ala Val Thr Asp Ala Telu Val Trp Val Ile Ser Lys Ser Arg 385 390 395 400

Ile Ser Ser Glin Glin Glin Ser Wall Arg Lieu Ala Asn Lieu. Telu Met Leu 405 410 415

Leu Ser His Val Arg His Ile Ser Asn Lys Gly Met Glu. His Telu Telu 420 425 430 Ser Met Lys Cys Lys Asn Wall Wall Pro Val Tyr Asp Lieu Lieu Leu Glu 435 4 40 4 45

Met Leu Asn Ala His Thr Teu Arg Gly Tyr Lys Ser Ser Ile Ser Gly 450 455 460

Ser Gly Cys Cys Ser Thr Glu Ser Lys Ser Lys Glu Gly Ser Glin 465 470 475 480

Asn Lieu Glin Ser Glin 485

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1458 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 6:

ATGGCATTCT ACAGTCCTGC TGTGATGAAC TACAGTGTTC CCAGCAGCAC CGGTAACCTG 60

GAAGGTGGGC CTGTTCGCCA GACTGCAAGC CCAAATGTGC TATGGCCAAC TTCTGGACAC 120

CTCTCTCCTT TAGCCACCCA CTGCCAATCA TCGCTTCTCT ATGCAGAACC TCAAAAGAGT 18O

CCTTGGTGTG AAGCAAGATC ACTAGAACAC ACCTTGCCTG TAAACAGAGA GACCCTGAAG 240

AGGAAGCTTG GCGGGAGCGG TTGTGCCAGC CCTGTTACTA GTCCAAGCAC CAAGAGGGAT

GCTCACTTCT GTGCCGTCTG CAGTGATTAT GCATCTGGGT ATCATTACGG TGTCTGGTCC 360

TGTGAAGGAT GTAAGGCCTT TTTTAAAAGA AGCATTCAAG GACATAATGA CTATATCTGT 420

CCAGCCACGA ATCAGTGTAC GATAGACAAG AACCGGCGTA AAAACTGCCA GGCCTGCCGA 480 5,958,710 25 26 -continued

CTTCGCAAGT GTTACGAAGT AGGAATGGTC AAGTGTGGAT CCAGGAGAGA AAGGTGTGGG 540

TACCGAATAG TACGAAGACA GAGAAGTGCC AGCGAGCAGG TGCATTGCCT GAACAAAGCC 600

AAGAGAACCA. GTGGGCACAC ACCCCGGGTG AAGGAGCTAC TGCTGAACTC TCTGAGTCCC 660

GAGCAGCTGG TGCTCACCCT GCTGGAAGCT GAGCCACCCA ATGTGCTAGT GAGTCGTCCC 720

AGCATGCCCT TCACCGAGGC CTCCATGATG ATGTCCCTTA CGAAGCTGGC TGACAAGGAA

CTGGTGCACA TGATTGGCTG GGCCAAGAAA ATCCCTGGCT TTGTGGAGCT CAGCCTGTTG 840

GACCAAGTCC GCC CTTGGA AAGCTGCTGG ATGGAGGTGC TGA GGTGGG GCTGATGTGG 9 OO

CGCTCCATCG ACCACCCCGG CAAGCTCATC TTTGCTCCAG ACC CGTTCT GGACAGGGAT 96.O

GAGGGGAAGT GGAAGG GATTCTGGAA. ATCTTTGACA CCTGGC GACGACGGCA O20

CGGTTCCGTG AGT AAAACT, GCAGCACAAA GAATATCTGT GAAGGC CATGATTCTC

CTCAACTCCA GTA GTACCA CTTGGCTACC GCAAGCCAGG AAGCAGAGAG TAGCCGGAAG 14 O

CTGACACACC TAT GAACGC AGTGACAGAT GCCCTGGTCT GGG GATTTC GAAGAGTAGA 200

ATCTCTTCCC AGCAGCAGTC AGTCCGTCTG GCCAACCTCC TGA GCTTCT TTCTCATGTC 260

AGGCACATCA. GTAACAAGGG CATGGAACAT CTGCTCAGCA TGAAGTGCAA AAATGTGGTC 320

CCGGTGTACG ACCTGCTGCT GGAGATGCTG AATGCTCACA CGC TCGAGG GTACAAGTCC

TCAATCTCGG GGTCTGGGTG CTGCTCGACA GAGGACAGTA AGAGCAA AGA GGGCTCCCAG 4 40

AACCTCCAGT CTCAGTGA 458

(2) INFORMATION FOR SEQ ID NO: 7 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 226 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus rattus (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 7 : Glu Lieu Val His Met Ile Gly Trp Ala Lys Lys Ile Pro Gly Phe Val 5 10 15

Glu Lieu Ser Lieu Lleu. Asp Glin Wall Arg Lieu Lieu Glu Ser Cys Trp Met 25 30

Glu Wall Leu Met Val Gly Teu Met Trp Arg Ser Ile Asp His Pro Gly 35 40 45

Lys Lieu. Ile Phe Ala Pro Asp Telu Val Lieu. Asp Asp Glu Gly Lys 50 55 60

Cys Val Glu Gly Ile Lieu Glu Ile Phe Asp Met Teu Leu Ala Th Thr 65 70 75 8O

Ser Arg Phe Arg Glu Lieu Telu Gln His Lys Glu Tyr Lieu Cys Val 85 90 95

Lys Ala Met Ile Leu Lieu Asn Ser Ser Met Tyr Pro Leu Ala Ser Ala 100 105 110

Asn Glin Glu Ala Glu Ser Ser Arg Lys Lieu. Thr His Teu Telu Asn Ala 115 120 125

Val Thr Asp Ala Leu Wall Trp Wall Ile Ala Lys Ser Gly Ile Ser Ser 130 135 1 4 0

Glin Glin Glin Ser Val Arg Teu Ala Asn Lieu. Lieu Met Teu Telu Ser His 145 15 O 155 160

Val Arg His Ile Ser Asn Gly Met Glu. His Teu Teu Ser Met Lys 1.65 170 175 5,958,710 27 28 -continued Lys Asn Val Val Pro Val Tyr Asp Leu Lleu Lleu Glu Met Lieu. Asn 18O 185 190

Ala His Thr Lieu Arg Gly Tyr Lys Ser Ser Ile Ser Gly Ser Glu Cys 195 200 2O5

Ser Ser Thr Glu Asp Ser Lys Asn Lys Glu Ser Ser Glin Asn Lieu Glin 210 215 220

Ser Glin 225

(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 243 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus rattus (xi). SEQUENCE DESCRIPTION: SEQ ID NO:8: Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Gly 5 10 15 Asp Lieu. Asn Lieu. His Asp Glin Val His Lieu Lieu Glu Cys Ala Trp Lieu 2O 25 30

Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly 35 40 45 Lieu Lleu Phe Ala Pro Asn Lieu Lleu Lieu. Asp Arg Asn Glin Gly Lys 50 55 60 Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 65 70 75 8O

Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 85 90 95 Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 100 105 110 Lieu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Lieu. Asp 115 120 125 Ile Asin Asp Thr Lieu. Ile His Leu Met Ala Lys Ala Gly Lieu. Thr 130 135 1 4 0

Teu Glin Glin Gln His Arg Arg Lieu Ala Glin Leu Lleu Lleu. Ile Leu Ser 145 15 O 155 160 His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Asn Met 1.65 170 175 Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Lleu Lleu Glu Met Lieu 18O 185 190 Asp Ala His Arg Leu. His Ala Pro Ala Ser Arg Met Gly Val Pro Pro 195 200 2O5

Glu Glu Pro Ser Glin Ser Glin Leu Thir Thr Thr Ser Ser Thr Ser Ala 210 215 220 His Ser Leu Gln Thr Tyr Tyr Ile Pro Pro Glu Ala Glu Gly Phe Pro 225 230 235 240

Asn Thir Ile

(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 243 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 5,958,710 29 30 -continued (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 9: Glu Lieu Val His Met Ile Asn Trp Ala Lys Arg Wall Pro Phe Gly 5 10 15

Asp Lieu. Asn Lieu. His Asp Glin Val His Lieu Lieu Glu Ala Trp Telu 2O 25 30

Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly 35 40 45

Lieu Lleu Phe Ala Pro Asn Lieu Lleu Lieu. Asp Arg Asn Glin Gly Lys 50 55 60

Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Teu Teu Ala Thr Ser 65 70 75

Ser Arg Phe Arg Met Met Asn Lieu Glin Gly Glu Glu Phe Wall Cys Telu 85 90 95

Ser Ile Ile Lieu Lleu. Asn. Ser Gly Val Tyr Thr Phe Teu Ser Ser 100 105 110

Lieu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Wall Telu Asp 115 120 125

Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Telu Thr 130 135 1 4 0

Teu Glin Glin Gln His Arg Arg Lieu Ala Glin Lieu Teu Teu Telu Ser 145 15 O 155 160

His Ile Arg His Met Ser Asn Lys Gly Met Glu His Teu Asn Met 1.65 170 175

Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Lieu Lieu Glu Met Lieu 18O 185 190

Asp Ala His Arg Lieu. His Ala Pro Ala Ser Arg Met Gly Wall Pro Pro 195 200

Glu Glu Pro Ser Glin Thr Glin Leu Ala Thir Thr Ser Ser Thr Ser Ala 210 215 220

His Ser Leu Gln Thr Tyr Tyr Ile Pro Pro Glu Ala Glu Gly Phe Pro 225 230 235 240

Asn Thir Ile

(2) INFORMATION FOR SEQ ID NO : 10 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 243 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 10 Glu Lieu Val His Met Ile Asn Trp Ala Lys Arg Wall Pro Phe Wall 5 10 15

Asp Lieu. Thir Lieu. His Asp Glin Val His Lieu Lieu Glu Ala Trp Telu 2O 25 30

Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Wall 35 40 45

Lieu Lleu Phe Ala Pro Asn Lieu Lleu Lieu. Asp Arg Asn Glin Gly Lys 50 55 60

Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Teu Teu Ala Thr Ser 65 70 75 8O 5,958,710 31 32 -continued Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 85 90 95 Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 100 105 110 Thr Lieu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Lieu. Asp 115 120 125 Lys Ile Thr Asp Thr Lieu. Ile His Leu Met Ala Lys Ala Gly Lieu. Thr 130 135 1 4 0 Leu Glin Glin Gln His Glin Arg Lieu Ala Glin Leu Lleu Lieu. Ile Leu Ser 145 15 O 155 160 His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met 1.65 170 175 Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Lleu Lleu Glu Met Lieu 18O 185 190 Asp Ala His Arg Lieu. His Ala Pro Thir Ser Arg Gly Gly Ala Ser Val 195 200 2O5 Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser 210 215 220 His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 225 230 235 240

Ala Thr Wall

(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus rattus (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 11: Cys Pro Val Cys Ser Asp Tyr Ala Ser Gly Tyr His Tyr Gly Val Trp 1 5 10 15 Ser Cys Glu Gly Cys Lys Ala Phe Phe Lys Arg Ser Ile Glin Gly His 2O 25 30 Asn Asp Tyr Ile Cys Pro Ala Thr Asn Glin Cys Thr Ile Asp Lys Asn 35 40 45 Arg Arg Lys Ser Cys Glin Ala Cys Arg Lieu Arg Lys Cys Tyr Glu Val 50 55 60 Gly Met 65

(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi). SEQUENCE DESCRIPTION: SEQ ID NO:12: Cys Ala Val Cys Asn Asp Tyr Ala Ser Gly Tyr His Tyr Gly Val Trp 1 5 10 15 Ser Cys Glu Gly Cys Lys Ala Phe Phe Lys Arg Ser Ile Glin Gly His 2O 25 30 Asn Asp Tyr Met Cys Pro Ala Thr Asn Glin Cys Thr Ile Asp Lys Asn 35 40 45 Arg Arg Lys Ser Cys Glin Ala Cys Arg Lieu Arg Lys Cys Tyr Glu Val 5,958,710 33 34 -continued

50 55 60 Gly Met 65

(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 484 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus rattus (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 13: Met Thr Phe Tyr Ser Pro Ala Val Met Asn Tyr Ser Val Pro Gly Ser 1 5 10 15 Thr Ser Asn Leu Asp Gly Gly Pro Val Arg Leu Ser Thr Ser Pro Asn 2O 25 30 Val Leu Trp Pro Thr Ser Gly. His Leu Ser Pro Leu Ala Thr His Cys 35 40 45 Glin Ser Ser Lieu Lleu Tyr Ala Glu Pro Gln Lys Ser Pro Trp Cys Glu 50 55 60 Ala Arg Ser Lieu Glu His Thr Lieu Pro Val Asn Arg Glu Thir Lieu Lys 65 70 75 8O Arg Lys Leu Ser Gly Ser Ser Cys Ala Ser Pro Val Thr Ser Pro Asn 85 90 95 Ala Lys Arg Asp Ala His Phe Cys Pro Val Cys Ser Asp Tyr Ala Ser 100 105 110 Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe 115 120 125 Lys Arg Ser Ile Glin Gly. His Asn Asp Tyr Ile Cys Pro Ala Thr Asn 130 135 1 4 0 Glin Cys Thir Ile Asp Lys Asn Arg Arg Lys Ser Cys Glin Ala Cys Arg 145 15 O 155 160 Leu Arg Lys Cys Tyr Glu Val Gly Met Wall Lys Cys Gly Ser Arg Arg 1.65 170 175 Glu Arg Cys Gly Tyr Arg Ile Val Arg Arg Glin Arg Ser Ser Ser Glu 18O 185 190 Glin Val His Cys Lieu Ser Lys Ala Lys Arg Asn Gly Gly His Ala Pro 195 200 2O5 Arg Val Lys Glu Lieu Lleu Lleu Ser Thr Lieu Ser Pro Glu Gln Leu Val 210 215 220 Leu Thr Leu Leu Glu Ala Glu Pro Pro Asn Val Leu Val Ser Arg Pro 225 230 235 240 Ser Met Pro Phe Thr Glu Ala Ser Met Met Met Ser Leu Thr Lys Leu 245 250 255 Ala Asp Lys Glu Lieu Val His Met Ile Gly Trp Ala Lys Lys Ile Pro 260 265 27 O Gly Phe Val Glu Lieu Ser Lieu Lieu. Asp Glin Val Arg Lieu Lieu Glu Ser 275 280 285 Cys Trp Met Glu Val Leu Met Val Gly Leu Met Trp Arg Ser Ile Asp 29 O 295 3OO His Pro Gly Lys Lieu. Ile Phe Ala Pro Asp Leu Val Lieu. Asp Arg Asp 305 310 315 320 Glu Gly Lys Cys Val Glu Gly Ile Leu Glu Ile Phe Asp Met Leu Lieu 325 330 335 5,958,710 35 36 -continued

Ala Thir Thr Ser Arg Phe Arg Glu Lieu Lys Lieu Glin His Tys Glu Tyr 340 345 350

Teu Cys Wall Lys Ala Met Ile Leu Lieu. Asn. Ser Ser Met Tyr Pro Telu 355 360 365

Ala Ser Ala Asn Glin Glu Ala Glu Ser Ser Arg Lys Teu His Telu 370 375

Teu Asn Ala Val Thr Asp Ala Leu Val Trp Val Ile Ala Ser Gly 385 390 395 400

Ile Ser Ser Glin Glin Glin Ser Val Arg Lieu Ala Asn Teu Teu Met Telu 405 410 415

Teu Ser His Val Arg His Ile Ser Asn Lys Gly Met Glu His Telu Telu 420 425 430

Ser Met Lys Cys Lys Asn Val Val Pro Val Tyr Asp Teu Teu Telu Glu 435 4 40 4 45

Met Lieu. Asn Ala His Thr Lieu Arg Gly Tyr Lys Ser Ser Ile Ser Gly 450 455 460

Ser Glu Cys Ser Ser Thr Glu Asp Ser Lys Asn Glu Ser Ser Glin 465 470 475 480

Asn Leu Glin Ser

(2) INFORMATION FOR SEQ ID NO: 14 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 484 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (xi). SEQUENCE DESCRIPTION: SEQ ID NO: 14 Met Ala Phe Tyr Ser Pro Ala Val Met Asn Tyr Ser Wall Pro Ser Ser 5 10 15

Thr Gly Asn Lieu Glu Gly Gly Pro Val Arg Glin Thr Ala Ser Pro Asn 2O 25 30

Wall Leu Trp Pro Thr Ser Gly. His Leu Ser Pro Teu Ala Thr His Cys 35 40 45

Glin Ser Ser Lieu Lleu Tyr Ala Glu Pro Glin Lys Ser Pro Trp Cys Glu 50 55 60

Ala Arg Ser Leu Glu His Thr Leu Pro Val Asn Arg Glu Thr Telu Lys 65 70 75

Arg Lys Lieu Gly Gly Ser Gly Cys Ala Ser Pro Wall Thr Ser Pro Ser 85 90 95

Thr Lys Arg Asp Ala His Phe Cys Ala Val Cys Ser Asp Tyr Ala Ser 100 105 110

Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Lys Ala Phe Phe 115 120 125

Arg Ser Ile Glin Gly. His Asn Asp Tyr Ile Cys Pro Ala Thr Asn 130 135 1 4 0

Glin Cys. Thir Ile Asp Lys Asn Arg Arg Lys Asn Glin Ala Arg 145 15 O 155 160

Teu Arg Lys Cys Tyr Glu Val Gly Met Wall Lys Gly Ser Arg Arg 1.65 170 175

Glu Arg Cys Gly Tyr Arg Ile Val Arg Arg Glin Arg Ser Ala Ser Glu 18O 185 190

Glin Val His Cys Lieu. Asn Lys Ala Lys Arg Thr Ser Gly His Thr Pro 5,958,710 37 38 -continued

195 200 2O5 Arg Val Lys Glu Lieu Lleu Lleu. Asn. Ser Lieu Ser Pro Glu Gln Leu Val 210 215 220 Leu Thr Leu Leu Glu Ala Glu Pro Pro Asn Val Leu Val Ser Arg Pro 225 230 235 240 Ser Met Pro Phe Thr Glu Ala Ser Met Met Met Ser Leu Thr Lys Leu 245 250 255 Ala Asp Lys Glu Lieu Val His Met Ile Gly Trp Ala Lys Lys Ile Pro 260 265 27 O Gly Phe Val Glu Lieu Ser Lieu Lieu. Asp Glin Val Arg Lieu Lieu Glu Ser 275 280 285 Cys Trp Met Glu Val Leu Met Val Gly Leu Met Trp Arg Ser Ile Asp 29 O 295 3OO His Pro Gly Lys Lieu. Ile Phe Ala Pro Asp Leu Val Lieu. Asp Arg Asp 305 310 315 320 Glu Gly Lys Cys Val Glu Gly Ile Leu Glu Ile Phe Asp Met Leu Lieu 325 330 335 Ala Thir Thr Ala Arg Phe Arg Glu Lieu Lys Lieu Gln His Lys Glu Tyr 340 345 350 Lieu. Cys Wall Lys Ala Met Ile Leu Lieu. Asn. Ser Ser Met Tyr His Lieu 355 360 365 Ala Thr Ala Ser Glin Glu Ala Glu Ser Ser Arg Lys Lieu. Thir His Lieu 370 375 38O Lieu. Asn Ala Val Thr Asp Ala Lieu Val Trp Val Ile Ser Lys Ser Arg 385 390 395 400 Ile Ser Ser Glin Gln Gln Ser Val Arg Leu Ala Asn Leu Leu Met Leu 405 410 415 Leu Ser His Val Arg His Ile Ser Asn Lys Gly Met Glu His Lieu Lieu 420 425 430 Ser Met Lys Cys Lys Asn Val Val Pro Val Tyr Asp Leu Lleu Lieu Glu 435 4 40 4 45 Met Lieu. Asn Ala His Thr Lieu Arg Gly Tyr Lys Ser Ser Tle Ser Gly 450 455 460 Ser Gly Cys Cys Ser Thr Glu Asp Ser Lys Ser Lys Glu Gly Ser Glin 465 470 475 480

Asn Lieu Glin Ser

(2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 384 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi). SEQUENCE DESCRIPTION: SEQ ID NO:15: Ala Leu Ser Pro Leu Val Val His Arg Glin Leu Ser His Leu Tyr Ala 1 5 10 15 Glu Pro Gln Lys Ser Pro Trp Cys Glu Ala Arg Ser Lieu Glu His Thr 2O 25 30 Leu Pro Val Asn Arg Glu Thir Lieu Lys Arg Lys Val Ser Gly Asn Arg 35 40 45 Cys Ala Ser Pro Val Thr Gly Pro Gly Ser Lys Arg Asp Ala His Phe 50 55 60 5,958,710 39 40 -continued Cys Ala Val Cys Ser Asp Tyr Ala Ser Gly Tyr His Tyr Gly Val Trp 65 70 75 8O Ser Cys Glu Gly Cys Lys Ala Phe Phe Lys Arg Ser Ile Glin Gly His 85 90 95 Asn Asp Tyr Ile Cys Pro Ala Thr Asn Glin Cys Thr Ile Asp Lys Asn 100 105 110 Arg Arg Lys Ser Cys Glin Ala Cys Arg Lieu Arg Lys Cys Tyr Glu Val 115 120 125 Gly Met Wall Lys Cys Gly Ser Arg Arg Glu Arg Cys Gly Tyr Arg Lieu 130 135 1 4 0 Val Arg Arg Glin Arg Ser Ala Asp Glu Glin Lieu. His Cys Ala Gly Lys 145 15 O 155 160 Ala Lys Arg Ser Gly Gly His Ala Pro Arg Val Arg Glu Lieu Lleu Lieu 1.65 170 175 Asp Ala Leu Ser Pro Glu Glin Leu Val Lieu. Thir Lieu Lleu Glu Ala Glu 18O 185 190 Pro Pro His Val Leu Ile Ser Arg Pro Ser Ala Pro Phe Thr Glu Ala 195 200 2O5 Ser Met Met Met Ser Lieu. Thir Lys Lieu Ala Asp Lys Glu Lieu Val His 210 215 220 Met Ile Ser Trp Ala Lys Lys Ile Pro Gly Phe Val Glu Leu Ser Leu 225 230 235 240 Phe Asp Glin Val Arg Leu Leu Glu Ser Cys Trp Met Glu Val Leu Met 245 250 255 Met Gly Lieu Met Trp Arg Ser Ile Asp His Pro Gly Lys Lieu. Ile Phe 260 265 27 O Ala Pro Asp Leu Val Lieu. Asp Arg Asp Glu Gly Lys Cys Val Glu Gly 275 280 285 Ile Leu Glu Ile Phe Asp Met Leu Leu Ala Thr Thr Ser Arg Phe Arg 29 O 295 3OO Glu Lieu Lys Lieu Gln His Lys Glu Tyr Lieu. Cys Wall Lys Ala Met Ile 305 310 315 320 Leu Leu Asn Ser Ser Met Tyr Pro Leu Val Thr Ala Thr Glin Asp Ala 325 330 335 Asp Ser Ser Arg Lys Lieu Ala His Lieu Lieu. Asn Ala Val Thr Asp Ala 340 345 350 Leu Val Trp Val Ile Ala Lys Ser Gly Ile Ser Ser Glin Glin Glin Ser 355 360 365 Met Arg Lieu Ala Asn Lieu Lleu Met Lieu Lleu Ser His Val Arg His Ala 370 375 38O

(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 596 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Rattus rattus (xi). SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Thr Met Thr Leu. His Thr Lys Ala Ser Gly Met Ala Leu Leu. His 1 5 10 15 Glin Ile Glin Gly Asn. Glu Lieu Glu Pro Leu Asn Arg Pro Glin Leu Lys 2O 25 30 Met Pro Met Glu Arg Ala Leu Gly Glu Val Tyr Val Asp Asn. Ser Lys 5,958,710 41 42 -continued

35 40 45

Pro Ala Wall Phe Asn Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60

Ala Ala Ala Ala Ala Ala Ala Gly Ala Ser Ala Pro Wall Tyr Gly Glin 65 70 75

Ser Ser Ile Thr Tyr Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ala 85 90 95

Asn Ser Telu Gly Ala Phe Pro Glin Telu Asn Ser Wall Ser Pro Ser Pro 100 105 110

Ile Met Ile Telu His Pro Pro Pro His Wall Ser Pro Phe Teu His Pro 115 120 125

His Gly His Glin Wall Pro Tyr Telu Glu Asn Glu Pro Ser Ala Tyr 130 135 1 4 0

Ala Wall Arg Asp Thr Gly Pro Pro Ala Phe Tyr Arg Ser Asn Ser Asp 145 15 O 155 160

Asn Arg Arg Glin Asn Gly Arg Glu Arg Telu Ser Ser Ser Ser Glu Lys 1.65 170 175

Gly Asn Met Ile Met Glu Ser Ala Lys Glu Thr Arg Cys Ala Wall 18O 185 190

Asn Asp Ala Ser Gly Tyr His Gly Wall Trp Ser Cys Glu 195 200 2O5

Gly Cys Ala Phe Phe Lys Arg Ser Ile Glin Gly His Asn Asp Tyr 210 215 220

Met Pro Ala Thr Asn Glin Thr Ile Asp Asn Arg Lys 225 230 235 240

Ser Glin Ala Cys Arg Lieu Arg Cys Glu Wall Gly Met Met 245 250 255

Gly Gly Ile Arg Asp Arg Arg Gly Gly Arg Met Teu Lys His 260 265 27 O

Arg Glin Arg Asp Asp Teu Glu Gly Arg Asn Glu Met Thr Ser 275 280 285

Gly Asp Met Arg Ala Ala Asn Telu Trp Pro Ser Pro Teu Wall Ile Lys 29 O 295

His Thr Asn Ser Pro Ala Telu Ser Teu Thr Ala Asp Glin Met 305 310 315 320

Wall Ser Ala Telu Teu Asp Ala Glu Pro Pro Teu Ile Ser Glu Tyr 325 330 335

Asp Pro Ser Arg Pro Phe Ser Glu Ala Ser Met Met Gly Teu Telu Thr 340 345 350

Asn Telu Ala Asp Arg Glu Teu Wall His Met Ile Asn Trp Ala Lys 355 360 365

Wall Pro Gly Phe Gly Asp Teu Asn Telu His Asp Glin Wall His Telu Telu 370 375

Glu Ala Trp Teu Glu Ile Telu Met Ile Gly Teu Wall Trp Ser 385 390 395 400

Met Glu His Pro Gly Teu Telu Phe Ala Pro Asn Teu Teu Telu Asp 405 410 415

Arg Asn Glin Gly Wall Glu Gly Met Wall Glu Ile Phe Asp Met 420 425 430

Teu Telu Ala Thr Ser Ser Arg Phe Arg Met Met Asn Teu Glin Gly Glu 435 4 40 4 45

Glu Phe Wall Teu Ser Ile Ile Telu Teu Asn Ser Wall Tyr 450 455 460 5,958,710 43 44 -continued Thr Phe Leu Ser Ser Thr Lieu Lys Ser Lieu Glu Glu Lys Asp His Ile 465 470 475 480 His Arg Val Lieu. Asp Lys Ile Asn Asp Thir Lieu. Ile His Leu Met Ala 485 490 495 Lys Ala Gly Lieu. Thir Lieu Glin Glin Gln His Arg Arg Lieu Ala Glin Lieu 5 OO 505 510 Leu Lieu. Ile Leu Ser His Ile Arg His Met Ser Asn Lys Gly Met Glu 515 52O 525 His Leu Tyr Asn Met Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Lieu 530 535 540 Leu Lieu Glu Met Leu Asp Ala His Arg Lieu. His Ala Pro Ala Ser Arg 545 550 555 560 Met Gly Val Pro Pro Glu Glu Pro Ser Glin Ser Gln Leu Thir Thr Thr 565 570 575 Ser Ser Thr Ser Ala His Ser Leu Gln Thr Tyr Tyr Ile Pro Pro Glu 58O 585 59 O Ala Glu Gly Phe 595

(2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 591 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi). SEQUENCE DESCRIPTION: SEQ ID NO:17: Met Thr Met Thr Leu. His Thr Lys Ala Ser Gly Met Ala Leu Leu. His 1 5 10 15 Glin Ile Glin Gly Asn. Glu Lieu Glu Pro Leu Asn Arg Pro Glin Leu Lys 2O 25 30 Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Lieu. Asp Ser Ser Lys 35 40 45 Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60 Ala Ala Ala Ala Asn Ala Glin Val Tyr Gly Glin Thr Gly Lieu Pro Tyr 65 70 75 8O Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Lieu Gly Gly 85 90 95

Phe Pro Pro Leu Asn. Ser Wal Ser Pro Ser Pro Ile Met Ile Leu. His 100 105 110 Pro Pro Pro Gln Leu Ser Pro Phe Leu Glin Pro His Gly Glin Glin Val 115 120 125 Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 1 4 0 Gly Pro Pro Ala Phe Tyr Arg Pro Asn. Ser Asp Asn Arg Arg Glin Gly 145 15 O 155 160 Gly Arg Glu Arg Lieu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met 1.65 170 175 Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 18O 185 190 Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 2O5 Phe Lys Arg Ser Ile Glin Gly His Asn Asp Tyr Met Cys Pro Ala Thr 5,958,710 45 46 -continued

210 215 220

Asn Glin Thr Ile Asp Asn Arg Arg Lys Ser Glin Ala Cys 225 230 235 240

Arg Telu Arg Cys Glu Wall Gly Met Met Gly Gly Ile Arg 245 250 255

Asp Arg Arg Gly Gly Arg Met Telu His Arg Glin Arg Asp 260 265 27 O

Asp Gly Glu Gly Arg Gly Glu Wall Gly Ser Ala Gly Asp Met Arg Ala 275 280 285

Ala Asn Telu Trp Pro Ser Pro Telu Met Ile Arg Ser Lys Asn 29 O 295

Ser Telu Ala Telu Ser Teu Thr Ala Asp Glin Met Wall Ser Ala Telu Telu 305 310 315 320

Asp Ala Glu Pro Pro Ile Teu Ser Glu Asp Pro Thr Arg Pro 325 330 335

Phe Ser Glu Ala Ser Met Met Gly Telu Telu Thr Asn Teu Ala Asp 340 345 350

Glu Telu Wall His Met Ile Asn Trp Ala Arg Wall Pro Phe Wall 355 360 365

Asp Telu Thr Telu His Asp Glin Wall His Telu Teu Glu Ala Trp Telu 370 375

Glu Ile Telu Met Ile Gly Teu Wall Trp Arg Ser Met Glu His Pro Wall 385 390 395 400

Telu Telu Phe Ala Pro Asn Telu Telu Telu Asp Arg Asn Glin Gly Lys 405 410 415

Wall Glu Gly Met Wall Glu Ile Phe Asp Met Lieu Lieu Ala Thr Ser 420 425 430

Ser Arg Phe Arg Met Met Asn Telu Glin Gly Glu Glu Phe Wall Cys Telu 435 4 40 4 45

Ser Ile Ile Teu Teu Asn Ser Gly Wall Thr Phe Teu Ser Ser 450 455 460

Thr Telu Ser Teu Glu Glu Asp His Ile His Arg Wall Telu Asp 465 470 475 480

Ile Thr Asp Thr Teu Ile His Telu Met Ala Ala Telu Thr 485 490 495

Teu Glin Glin Glin His Glin Arg Lel Ala Glin Teu Teu Teu Ile Telu Ser 5 OO 505 510

His Ile Arg His Met Ser Asn Lys Gly Met Glu His Teu Ser Met 515 52O 525

Cys Asn Wall Wall Pro Lel Asp Teu Teu Teu Glu Met Telu 530 535 540

Asp Ala His Arg Teu His Ala Thr Ser Arg Gly Gly Ala Ser Wall 545 550 555 560

Glu Glu Thr Asp Glin Ser His Lel Ala Thr Ala Gly Ser Thr Ser Ser 565 570 575

His Ser Telu Glin Ile Thr Gly Glu Ala Glu Gly Phe 58O 585 59 O

(2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 518 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: 5,958,710 47 48 -continued (A) ORGANISM: Homo sapiens (xi). SEQUENCE DESCRIPTION: SEQ ID NO:18: Met Gly Lieu Glu Met Ser Ser Lys Asp Ser Pro Gly Ser Lieu. Asp Gly 1 5 10 15 Arg Ala Trp Glu Asp Ala Glin Lys Pro Glin Ser Ala Trp Cys Gly Gly 2O 25 30 Arg Lys Thr Arg Val Tyr Ala Thr Ser Ser Arg Arg Ala Pro Pro Ser 35 40 45 Glu Gly. Thir Arg Arg Gly Gly Ala Ala Arg Pro Glu Glu Ala Ala Glu 50 55 60 Glu Gly Pro Pro Ala Ala Pro Gly Ser Lieu Arg His Ser Gly Pro Leu 65 70 75 8O Gly Pro His Ala Cys Pro Thr Ala Leu Pro Glu Pro Glin Val Thr Ser 85 90 95 Ala Met Ser Ser Glin Val Val Gly Ile Glu Pro Leu Tyr Ile Lys Ala 100 105 110 Glu Pro Ala Ser Pro Asp Ser Pro Lys Gly Ser Ser Glu Thr Glu Thr 115 120 125 Glu Pro Pro Val Ala Leu Ala Pro Gly Pro Ala Pro Thr Arg Cys Leu 130 135 1 4 0 Pro Gly. His Lys Glu Glu Glu Asp Gly Glu Gly Ala Gly Pro Gly Glu 145 15 O 155 160 Glin Gly Gly Gly Lys Lieu Val Lieu Ser Ser Lieu Pro Lys Arg Lieu. Cys 1.65 170 175 Leu Val Cys Gly Asp Val Ala Ser Gly Tyr His Tyr Gly Val Ala Ser 18O 185 19 O Cys Glu Ala Cys Lys Ala Phe Phe Lys Arg Thr Ile Glin Gly Ser Ile 195 200 2O5 Glu Tyr Ser Cys Pro Ala Ser Asn. Glu Cys Glu Ile Thr Lys Arg Arg 210 215 220 Arg Lys Ala Cys Glin Ala Cys Arg Phe Thr Lys Cys Ile Arg Val Gly 225 230 235 240 Met Leu Lys Glu Gly Val Arg Lieu. Asp Arg Val Arg Gly Gly Arg Glin 245 250 255 Lys Tyr Lys Arg Arg Pro Glu Val Asp Pro Leu Pro Phe Pro Gly Pro 260 265 27 O Phe Pro Ala Gly Pro Leu Ala Val Ala Gly Gly Pro Arg Lys Thr Ala 275 280 285

Ala Pro Wall Asn Ala Leu Val Ser His Leu Leu Val Val Glu Pro Glu 29 O 295 3OO Lys Lieu. Tyr Ala Met Pro Asp Pro Ala Gly Pro Asp Gly His Leu Pro 305 310 315 320 Ala Val Ala Thr Lieu. Cys Asp Leu Phe Asp Arg Glu Ile Val Val Thr 325 330 335 Ile Ser Trp Ala Lys Ser Ile Pro Gly Phe Ser Ser Leu Ser Leu Ser 340 345 350 Asp Gln Met Ser Val Leu Gln Ser Val Trp Met Glu Val Leu Val Leu 355 360 365 Gly Val Ala Glin Arg Ser Leu Pro Leu Glin Asp Glu Lieu Ala Phe Ala 370 375 38O Glu Asp Lieu Val Lieu. Ile Glu Glu Gly Ala Arg Ala Ala Gly Lieu Gly 385 390 395 400

Glu Lieu Gly Ala Ala Lieu Lleu Glin Leu Val Arg Arg Lieu Glin Ala Lieu 5,958,710 49 SO -continued

405 410 415

Arg Leu Glu Arg Glu Glu Tyr Val Lieu Lleu Lys Ala Teu Ala Telu Ala 420 425 430

Asn Ser Asp Ser Val His Ile Glu Asp Glu Pro Arg Teu Trp Ser Ser 435 4 40 4 45

Glu Lys Lieu Lleu. His Glu Ala Lieu Lieu Glu Tyr Glu Ala Gly Arg 450 455 460

Ala Gly Pro Gly Gly Gly Ala Glu Arg Arg Arg Ala Gly Telu Telu 465 470 475 480

Teu Thir Lieu Pro Leu Lleu Arg Glin Thr Ala Gly Wall Teu Ala His 485 490 495

Phe Tyr Gly Wall Lys Lieu Glu Gly Lys Val Pro Met His Tys Telu Phe 5 OO 505 510

Teu Glu Met Leu Glu Ala 515

(2) INFORMATION FOR SEQ ID NO : 19 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 431 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi). SEQUENCE DESCRIPTION: SEQ ID NO : 19 Ser Ser Glu Asp Arg His Leu Gly Ser Ser Gly Ser Phe Ile 5 10 15

Thr Glu Pro Ser Ser Pro Ser Ser Gly Ile Asp Ala Teu Ser His 2O 25 30

His Ser Pro Ser Gly Ser Ser Asp Ala Ser Gly Gly Phe Met Ala 35 40 45

Teu Gly Thr His Ala Asn Gly Lieu. Asp Ser Pro Pro Met Phe Ala Gly 50 55 60

Ala Gly Lieu Gly Gly Asn Pro Cys Arg Lys Ser Glu Asp Cys Thr 65 70 75

Ser Gly Ile Met Glu Asp Ser Ala Ile Lys Cys Glu Met Telu Asn 85 90 95

Ala Ile Pro Lys Arg Lieu. Cys Lieu Val Cys Gly Asp Ile Ala Ser Gly 100 105 110

His Tyr Gly Val Ala Ser Cys Glu Ala Cys Ala Phe Phe Lys 115 120 125

Arg Thr Ile Glin Gly Asn Ile Glu Tyr Ser Cys Pro Ala Thr Asn Glu 130 135 1 4 0

Cys Glu Ile Thr Lys Arg Arg Arg Lys Ser Cys Glin Ala Phe 145 15 O 155 160

Met Lys Cys Ile Lys Val Gly Met Leu Lys Glu Gly Wall Arg Telu Asp 1.65 170 175

Arg Val Arg Gly Gly Arg Glin Lys Tyr Lys Arg Arg Teu Asp Ser Glu 18O 185 190

Asn Ser Pro Tyr Leu Ser Leu Glin Ile Ser Pro Pro Ala Lys Pro 195 200

Teu Thr Lys Ile Val Ser Tyr Leu Leu Val Ala Glu Pro Lys Telu 210 215 220

Tyr Ala Met Pro Pro Asp Asp Val Pro Glu Gly Asp Ile Ala Telu 225 230 235 240 5,958,710 S1 52 -continued

Thir Thr Lieu. Cys Asp Leu Ala Asp Arg Glu Leu Wall Phe Teu Ile Ser 245 250 255

Trp Ala Lys His Ile Pro Gly Phe Ser Asn Lieu. Thir Lieu Gly Asp Glin 260 265 27 O

Met Ser Telu Telu Glin Ser Ala Trp Met Glu Ile Teu Ile Teu Gly Ile 275 280 285

Wall Tyr Arg Ser Leu Pro Tyr Asp Asp Lys Teu Ala Ala Glu Asp 29 O 295 3OO

Tyr Ile Met Asp Glu Glu. His Ser Arg Telu Wall Gly Teu Teu Glu Lieu 305 310 315 320

Arg Ala Ile Leu Gln Leu Wall Arg Arg Teu Lys Wall 325 330 335

Glu Glu Glu Phe Wal Met Leu Lys Ala Ile Ala Lieu Ala Asn. Ser 340 345 350

Asp Ser Met Tyr Ile Glu Asn Leu Glu Ala Wall Glin Lys Teu Glin Asp 355 360 365

Teu Lieu. His Glu Ala Teu Glin Asp Glu Teu Ser Glin Arg His Glu 370 375

Glu Pro Ala Gly Lys Telu Telu Telu Thr Leu Pro Teu Leu Arg 385 390 395 400

Glin Thr Ala Ala Lys Ala Val Glin His Phe Ser Wall Leu Glin 405 410 415

Gly Wall Pro Met His Telu Phe Leu Glu Met Leu Glu Ala 420 425 430

We claim: or SEQ ID NO:5, with a molecule in vitro, under 1. An isolated polypeptide displaying the biological activ 35 conditions which permit Said polypeptide to bind to ity of ERB, Said polypeptide having the amino acid Sequence Said molecule; and of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:5. (b) detecting the binding of said polypeptide with said 2. An isolated nucleic acid which codes for a polypeptide molecule. displaying the biological activity of ERB, Said polypeptide 5. An isolated polypeptide displaying the biological activ having the amino acid sequence of SEQ ID NO:2, SEQ ID 40 ity of ERB, Said polypeptide having the amino acid Sequence NO:3, or SEQ ID NO:5. of SEO ID NO:2. 3. The isolated nucleic acid according to claim 2, Said 6. An isolated nucleic acid which codes for a polypeptide nucleic acid having the nucleic acid Sequence of SEQ ID displaying the biological activity of ERB, Said polypeptide NO:1, SEQ ID NO:4, or SEQ ID NO:6. having the amino acid sequence of SEQ ID NO:2. 4. A method for identifying a molecule which binds with 45 7. The isolated nucleic acid according to claim 6, Said ERB, comprising the Steps of: nucleic acid having the nucleic acid Sequence of SEQ ID (a) contacting an isolated polypeptide displaying the NO:1. biological activity of ERB, Said polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3