Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 May 15;88(10):4463–4467. doi: 10.1073/pnas.88.10.4463

Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

A M Molinari 1, C Abbondanza 1, I Armetta 1, N Medici 1, S Minucci 1, B Moncharmont 1, V Nigro 1, G A Puca 1
PMCID: PMC51680  PMID: 1709742

Abstract

The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4-hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity.

Full text

PDF
4463

Images in this article

4464Image
on p.4464
4466Image
on p.4466
4466Image
on p.4466
4466Image
on p.4466

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arriza J. L., Weinberger C., Cerelli G., Glaser T. M., Handelin B. L., Housman D. E., Evans R. M. Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science. 1987 Jul 17;237(4812):268–275. doi: 10.1126/science.3037703. [DOI] [PubMed] [Google Scholar]
  2. Arànyi P., Radanyi C., Renoir M., Devin J., Baulieu E. E. Covalent stabilization of the nontransformed chick oviduct cytosol progesterone receptor by chemical cross-linking. Biochemistry. 1988 Feb 23;27(4):1330–1336. doi: 10.1021/bi00404a036. [DOI] [PubMed] [Google Scholar]
  3. Atger M., Milgrom E. Chromatographic separation on phosphocellulose of activated and nonactivated forms of steroid-receptor complex. Purification of the activated complex. Biochemistry. 1976 Sep 21;15(19):4298–4304. doi: 10.1021/bi00664a025. [DOI] [PubMed] [Google Scholar]
  4. Beato M. Gene regulation by steroid hormones. Cell. 1989 Feb 10;56(3):335–344. doi: 10.1016/0092-8674(89)90237-7. [DOI] [PubMed] [Google Scholar]
  5. Berry M., Metzger D., Chambon P. Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J. 1990 Sep;9(9):2811–2818. doi: 10.1002/j.1460-2075.1990.tb07469.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chin D. T., Goff S. A., Webster T., Smith T., Goldberg A. L. Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La. J Biol Chem. 1988 Aug 25;263(24):11718–11728. [PubMed] [Google Scholar]
  7. Chung C. H., Goldberg A. L. The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4931–4935. doi: 10.1073/pnas.78.8.4931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Denis M., Poellinger L., Wikstöm A. C., Gustafsson J. A. Requirement of hormone for thermal conversion of the glucocorticoid receptor to a DNA-binding state. Nature. 1988 Jun 16;333(6174):686–688. doi: 10.1038/333686a0. [DOI] [PubMed] [Google Scholar]
  9. Desautels M., Goldberg A. L. Demonstration of an ATP-dependent, vanadate-sensitive endoprotease in the matrix of rat liver mitochondria. J Biol Chem. 1982 Oct 10;257(19):11673–11679. [PubMed] [Google Scholar]
  10. Evans R. M. The steroid and thyroid hormone receptor superfamily. Science. 1988 May 13;240(4854):889–895. doi: 10.1126/science.3283939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Green S., Walter P., Kumar V., Krust A., Bornert J. M., Argos P., Chambon P. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature. 1986 Mar 13;320(6058):134–139. doi: 10.1038/320134a0. [DOI] [PubMed] [Google Scholar]
  12. Howard K. J., Distelhorst C. W. Evidence for intracellular association of the glucocorticoid receptor with the 90-kDa heat shock protein. J Biol Chem. 1988 Mar 5;263(7):3474–3481. [PubMed] [Google Scholar]
  13. Hubbard J. R., Barrett A. J., Kalimi M. Tosyl-lysyl chloromethane alters glucocorticoid-receptor complex nuclear binding and physical properties. Endocrinology. 1984 Jul;115(1):65–72. doi: 10.1210/endo-115-1-65. [DOI] [PubMed] [Google Scholar]
  14. Katzenellenbogen B. S., Elliston J. F., Monsma F. J., Jr, Springer P. A., Ziegler Y. S., Greene G. L. Structural analysis of covalently labeled estrogen receptors by limited proteolysis and monoclonal antibody reactivity. Biochemistry. 1987 Apr 21;26(8):2364–2373. doi: 10.1021/bi00382a043. [DOI] [PubMed] [Google Scholar]
  15. King R. J. Structure and function of steroid receptors. J Endocrinol. 1987 Sep;114(3):341–349. doi: 10.1677/joe.0.1140341. [DOI] [PubMed] [Google Scholar]
  16. Klein-Hitpass L., Tsai S. Y., Greene G. L., Clark J. H., Tsai M. J., O'Malley B. W. Specific binding of estrogen receptor to the estrogen response element. Mol Cell Biol. 1989 Jan;9(1):43–49. doi: 10.1128/mcb.9.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kost S. L., Smith D. F., Sullivan W. P., Welch W. J., Toft D. O. Binding of heat shock proteins to the avian progesterone receptor. Mol Cell Biol. 1989 Sep;9(9):3829–3838. doi: 10.1128/mcb.9.9.3829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krust A., Green S., Argos P., Kumar V., Walter P., Bornert J. M., Chambon P. The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors. EMBO J. 1986 May;5(5):891–897. doi: 10.1002/j.1460-2075.1986.tb04300.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kumar V., Chambon P. The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell. 1988 Oct 7;55(1):145–156. doi: 10.1016/0092-8674(88)90017-7. [DOI] [PubMed] [Google Scholar]
  20. Kumar V., Green S., Stack G., Berry M., Jin J. R., Chambon P. Functional domains of the human estrogen receptor. Cell. 1987 Dec 24;51(6):941–951. doi: 10.1016/0092-8674(87)90581-2. [DOI] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Metzger D., White J. H., Chambon P. The human oestrogen receptor functions in yeast. Nature. 1988 Jul 7;334(6177):31–36. doi: 10.1038/334031a0. [DOI] [PubMed] [Google Scholar]
  23. Molinari A. M., Medici N., Armetta I., Nigro V., Moncharmont B., Puca G. A. Particulate nature of the unoccupied uterine estrogen receptor. Biochem Biophys Res Commun. 1985 Apr 30;128(2):634–642. doi: 10.1016/0006-291x(85)90093-2. [DOI] [PubMed] [Google Scholar]
  24. Moncharmont B., Su J. L., Parikh I. Monoclonal antibodies against estrogen receptor: interaction with different molecular forms and functions of the receptor. Biochemistry. 1982 Dec 21;21(26):6916–6921. doi: 10.1021/bi00269a046. [DOI] [PubMed] [Google Scholar]
  25. Nigro V., Medici N., Abbondanza C., Minucci S., Molinari A. M., Puca G. A. Aprotinin inhibits the hormone binding of the estrogen receptor from calf uterus. Biochem Biophys Res Commun. 1989 Nov 15;164(3):1206–1211. doi: 10.1016/0006-291x(89)91797-x. [DOI] [PubMed] [Google Scholar]
  26. Nigro V., Medici N., Abbondanza C., Minucci S., Moncharmont B., Molinari A. M., Puca G. A. An aprotinin binding site localized in the hormone binding domain of the estrogen receptor from calf uterus. Biochem Biophys Res Commun. 1990 Jul 31;170(2):930–936. doi: 10.1016/0006-291x(90)92180-8. [DOI] [PubMed] [Google Scholar]
  27. Puca G. A., Abbondanza C., Nigro V., Armetta I., Medici N., Molinari A. M. Estradiol receptor has proteolytic activity that is responsible for its own transformation. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5367–5371. doi: 10.1073/pnas.83.15.5367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Puca G. A., Medici N., Armetta I., Nigro V., Moncharmont B., Molinari A. M. Interaction between estrogen receptor and subcellular structures of target cells: nuclear localization of unoccupied receptor and its modification induced by estradiol. Ann N Y Acad Sci. 1986;464:168–189. doi: 10.1111/j.1749-6632.1986.tb16003.x. [DOI] [PubMed] [Google Scholar]
  29. Sakai D., Gorski J. Reversible denaturation of the estrogen receptor and estimation of polypeptide chain molecular weight. Endocrinology. 1984 Dec;115(6):2379–2383. doi: 10.1210/endo-115-6-2379. [DOI] [PubMed] [Google Scholar]
  30. Titani K., Torff H. J., Hormel S., Kumar S., Walsh K. A., Rödl J., Neurath H., Zwilling R. Amino acid sequence of a unique protease from the crayfish Astacus fluviatilis. Biochemistry. 1987 Jan 13;26(1):222–226. doi: 10.1021/bi00375a029. [DOI] [PubMed] [Google Scholar]
  31. Tora L., White J., Brou C., Tasset D., Webster N., Scheer E., Chambon P. The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell. 1989 Nov 3;59(3):477–487. doi: 10.1016/0092-8674(89)90031-7. [DOI] [PubMed] [Google Scholar]
  32. Webster N. J., Green S., Jin J. R., Chambon P. The hormone-binding domains of the estrogen and glucocorticoid receptors contain an inducible transcription activation function. Cell. 1988 Jul 15;54(2):199–207. doi: 10.1016/0092-8674(88)90552-1. [DOI] [PubMed] [Google Scholar]
  33. Webster N. J., Green S., Tasset D., Ponglikitmongkol M., Chambon P. The transcriptional activation function located in the hormone-binding domain of the human oestrogen receptor is not encoded in a single exon. EMBO J. 1989 May;8(5):1441–1446. doi: 10.1002/j.1460-2075.1989.tb03526.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES