Europaisches Patentamt (19) European Patent Office Office des brevets europeenpeen (11) EP 0 287 653 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) intci.6: C12N 15/00, C12Q 1/68, of the grant of the patent: C07K 14/705 14.07.1999 Bulletin 1999/28 (86) International application number: (21) Application number: 87907643.8 PCT/US87/02782 Date of 23.10.1987 (22) filing: (87) International publication number: WO 88/03168 (05.05.1988 Gazette 1988/10) (54) HORMONE RECEPTOR COMPOSITIONS AND METHODS HORMON-REZEPTOR-VERBINDUNGEN UND METHODEN COMPOSITIONS RECEPTRICES D'HORMONES ET PROCEDES (84) Designated Contracting States: (74) Representative: AT BE CH DE FR GB IT LI LU NL SE Kolb, Helga, Dr. Dipl.-Chem. et al Hoffmann Eitle, (30) Priority: 24.10.1986 US 922585 Patent- und Rechtsanwalte, 20.10.1987 US 108471 Postfach 81 04 20 81904 Miinchen (DE) (43) Date of publication of application: 26.10.1988 Bulletin 1988/43 (56) References cited: WO-A-88/00975 (60) Divisional application: 95120305.8/0 733 705 • CELL, vol. 46, 29 August 1986, Cell Press; V. (73) Proprietor: THE SALK INSTITUTE FOR GIGUERE et al., pp. 645-652 BIOLOGICAL STUDIES • NATURE, vol. 324, 18-25 December 1986; C. La Jolla California 92037 (US) WEINBERGER et al., pp. 641-646 • SCIENCE, vol. 237, 25 September 1987; C.C. (72) Inventors: THOMPSON et al., pp. 1610-1614 • EVANS, Ronald, Mark • NATURE, vol. 329, 22 October 1987; C.K. GLASS San Diego, CA 92110 (US) et al., pp. 738-741 • WEINBERGER, Cary, A. • SCIENCE, vol. 231, 07 March 1986; G.L. GREENE Silver Spring, MD 20906 (US) et al., pp. 1150-1154 • HOLLENBERG, Stanley, Mark • SCIENCE, vol. 237, 17 July 1987; J.L. ARRIZAet San Diego, CA 92122 (US) al., pp. 268-275 • GIGUERE, Vincent • NATURE, vol. 331, 07 January 1988; V. GIGUERE San Diego, CA 92122 (US) et al., pp. 91-94 • ARRIZA, Jeffrey, Louis • SCIENCE, vol. 240, 13 May 1988; R.M. EVANS, Carlsbad, CA 92008 (US) pp. 889-891 • THOMPSON, Catherine, Caroline • SCIENCE, vol. 237, 17 July 1987, Washington, LaJolla, CA 92037 (US) DC (US); ARRIZA et al., p. 268 • ONG, Estelita, Sebastian • JOURNAL OF BIOLOGICAL CHEMISTRY, vol. San Diego, CA 921 17 (US) 261, 25 August 1986, Washington, DC (US); NORTHROP et al., p. 11064 • PROCEEDINGS OF THE NATL. ACADEMY OF DO SCIENCES USA, vol. 81, August 1984, CO Washington, DC (US); NARAYAN et al., p. 4687 io • THE EMBO JOURNAL, vol. 5, May 1986, Oxford CO (GB); KRUST et al., p. 891 Is- 00 CM Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice the Patent Office of the Notice of shall be filed in o to European opposition to European patent granted. opposition a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. a. 99(1) European Patent Convention). LU Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 0 287 653 B1 • THE EMBO JOURNAL, vol. 5, October 1986, • BIOCHEMISTRY, vol. 23, 18 December 1984, Oxford (GB); DANIELSEN et al., p. 2513 Washington, DC (US); MILLER et al., p. 6883 • THE EMBO JOURNAL, vol. 5, September 1986, • NATURE, vol. 320, 13 March 1986, London (GB); Oxford (GB); CATO et al., p. 2237 GREEN etal., p. 134 • MOLECULAR & CELLULAR BIOLOGY, vol. 3, • MOLECULAR & CELLULAR BIOLOGY, vol. 6, July 1983, Washington, DC (US); DANIELSEN et April 1986, Washington, DC (US); DEFRANCOet al., p. 1310 al., p. 993 • CELL, vol. 33, June 1983, Cambridge, MA (US); • NATURE, vol. 31 2, 20/27 December 1 984, London CHANDLER et al., p. 489 (GB); MIESFILD et al., p. 779 • CELL, vol. 31, December 1982, Cambridge, MA • MOLECULAR & CELLULAR BIOLOGY, vol. 4, (US); PFAHL, p. 475 June 1984, Washington, DC (US); FIRZLAFF et • PROCEEDINGS OF THE NATL. ACADEMY OF al., p. 1057 SCIENCES USA, vol. 79, September 1982, • THE EMBO JOURNAL, vol. 1, December 1984, Washington, DC (US); GOVINDAN et al., p. 5157 London (GB); GEISSE et al., p. 1613 • PROCEEDINGS OF THE NATL. ACADEMY OF SCIENCES USA, vol. 82, February 1985, Remarks: Washington, DC (US); MOORE et al., p. 699 Divisional application 951 20305.8 filed on 21/1 2/95. 2 EP 0 287 653 B1 Description FIELD OF THE INVENTION 5 [0001] The present invention relates to hormone receptor proteins and genes encoding them, modification of such receptors and genes by recombinant DNA and other genetic engineering techniques, plus uses of such receptors and genes, both unmodified and modified. More particularly, the invention concerns mineralocorticoid receptors and genes for them. In addition the invention relates to a novel bioassay system for determining the functionality of hormone receptor proteins coded for by receptor DNA clones, plus novel methods for inducing and controlling expression of 10 genes whose transcription is activated by hormones complexed with receptor proteins. BACKGROUND OF THE INVENTION [0002] Transcriptional regulation of development and homeostasis in complex eukaryotes, including humans and is other mammals, birds, and fish, is controlled by a wide variety of regulatory substances, including steroid and thyroid hormones. These hormones exert potent effects on development and differentiation in phylogenetically diverse organ- isms and their actions are mediated as a consequence of their interactions with specific, high affinity binding proteins referred to as receptors. See generally, Jensen, etal., (1972); Gorski, etal., (1976); Yamamoto, etal., (1976); O'Malley, et al., (1969); Hayward, et al., (1982); andAsburner, et al., (1978). 20 [0003] Receptor proteins, each especially specific for one of the several classes of cognate steroid hormones (i.e., estrogens (estrogen receptor), progestogens (progesterone receptor), glucocorticoids (glucocorticoid receptor), an- drogens (androgen receptor), aldosterones (mineralocorticoid receptor) or for cognate thyroid hormones (thyroid hor- mone receptor), are known and distributed in a tissue specific fashion. See Horwitz, et al., (1978) and Pamiter, et al., (1976). 25 [0004] Turning now to the interaction of hormones and receptors, it is known that a steroid or thyroid hormone enters cells by facilitated diffusion and binds to its specific receptor protein, initiating an allosteric alteration of the protein. As a result of this alteration, the hormone/receptor complex is capable of binding to certain specific sites on chromatin with high affinity. See Yamamoto, et al., (1972) and Jensen, et al., (1968). [0005] It is also known that many of the primary effects of steroid and thyroid hormones involve increased transcription 30 of a subset of genes in specific cell types. See Peterkofsky, et al., (1968) and McKnight, et al., (1968). Moreover, there is evidence that activation of transcription (and, consequently, increased expression) of genes which are responsive to steroid and thyroid hormones (through interaction of chromatin with hormone receptor/hormone complexj is effected through binding of the complex to enhancers associated with the genes. (See Khoury, et al., 1983.) [0006] In any case, a number of steroid hormone and thyroid hormone responsive transcriptional control units, some 35 of which have been shown to include enhancers, have been identified. These include the mouse mammary tumor virus 5'-long terminal repeat (MTV LTR), responsive to glucocorticoid, aldosterone and androgen hormones; the transcrip- tional control units for mammalian growth hormone genes, responsive to glucocorticoids, estrogens, and thyroid hor- mones; the transcriptional control units for mammalian prolactin genes and progesterone receptor genes, responsive to estrogens; the transcriptional control units for avian ovalbumin genes, responsive to progesterones; mammalian 40 metallothionein gene transcriptional control units, responsive to glucocorticoids; and mammalian hepatic alpha2u-glob- ulin gene transcriptional control units, responsive to androgens, estrogens, thyroid hormones and glucocorticoids. (See the Introduction portion of Experimental Section I of this Specification for references.) [0007] A major obstacle to further understanding and more practical use of the steroid and thyroid hormone receptors has been the lack of availability of the receptor proteins, in sufficient quantity and sufficiently pure form, to allow them 45 to be adequately characterized. The same is true for the DNA gene segments which encode them. Lack of availability of these DNA segments has prevented in vitro manipulation and in vivo expression of the receptor-coding genes, and consequently the knowledge such manipulation and expression will yield. [0008] The present invention is directed to overcoming these problems of short supply of adequately pure receptor material and lack of DNA segments which encode the receptors. 50 REFERENCE LIST [0009] The Background section of the specification refers to the following publications. 55 3 EP 0 287 653 B1 PUBLICATIONS [0010] 5 1. Asburner, M., and Berendes, H.D. in The Genetics and Biology of Drosophila, Eds. Ashburner, M., and Wright, T.R.F., Vol. 2, pp. 315-395, Academic, London (1978). 2. Gorski, J., and Gannon, E, A. Rev. Physiol., 38:425-450 (1976). 3. Hayward, M.A., Brock, M.L. and Shapiro, D.J., Nucleic Acids Res., 10:8273-8284 (1982). 4. Horwitz, K.B., and McGuire, W.L., J. Biol. Chem., 253:2223-2228 (1978). 10 5. Jensen, E.V, and DeSombre, E.R., A. Rev. Biochem., 41:203-230 (1972). 6. Jensen, E.V, et al., Proc. Natl. Acad. Sci. U.S.A., 59:632-638 (1968). 7. Khoury, G., and Gruss, P., Cell, 33:313-314 (1983). 8. McKnight, G.S., and Palmiter, R.D., J. Biol. Chem., 254:9050-9058 (1968). 9. O'Malley, B.W., McGuire, W.L., Kohler, P.O., and Kornman, S.G., Recent Prog. Horm. Res., 25:105-160 (1969). is 10. Pamiter, R.D., Moore, P.B., Mulvihill, E.R. and Emtage, S., CeN, 8:557-572 (1976). 11. Peterkofsky, B., and Tomkins, G., Proc. Natl. Acad. Sci. U.S.A., 60:222-228 (1968).