Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1987 May;6(5):1309–1315. doi: 10.1002/j.1460-2075.1987.tb02369.x

Functional dissection of the hormone and DNA binding activities of the glucocorticoid receptor.

S Rusconi, K R Yamamoto
PMCID: PMC553934  PMID: 2440676

Abstract

We have identified two separate regions of the 795 amino acid rat glucocorticoid receptor that interact with hormonal ligands and DNA respectively. The functional regions were defined by direct assays of segments of the receptor coding sequence translated in vitro. Hormone affinity measurements suggested that residues near the receptor C-terminus are the primary determinants of ligand binding, whereas sequence-specific DNA binding activity resides between amino acids 440 and 546. DNA binding efficiency was stimulated only modestly by prior hormone binding. The receptor regions identified in these in vitro studies correspond to those that mediate ligand-dependent transcriptional enhancement in vivo.

Full text

PDF
1309

Images in this article

Selected References

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

  1. Becker P. B., Gloss B., Schmid W., Strähle U., Schütz G. In vivo protein-DNA interactions in a glucocorticoid response element require the presence of the hormone. Nature. 1986 Dec 18;324(6098):686–688. doi: 10.1038/324686a0. [DOI] [PubMed] [Google Scholar]
  2. Buetti E., Diggelmann H. Glucocorticoid regulation of mouse mammary tumor virus: identification of a short essential DNA region. EMBO J. 1983;2(8):1423–1429. doi: 10.1002/j.1460-2075.1983.tb01601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carlstedt-Duke J., Okret S., Wrange O., Gustafsson J. A. Immunochemical analysis of the glucocorticoid receptor: identification of a third domain separate from the steroid-binding and DNA-binding domains. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4260–4264. doi: 10.1073/pnas.79.14.4260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chandler V. L., Maler B. A., Yamamoto K. R. DNA sequences bound specifically by glucocorticoid receptor in vitro render a heterologous promoter hormone responsive in vivo. Cell. 1983 Jun;33(2):489–499. doi: 10.1016/0092-8674(83)90430-0. [DOI] [PubMed] [Google Scholar]
  5. Dahmer M. K., Tienrungroj W., Pratt W. B. Purification and preliminary characterization of a macromolecular inhibitor of glucocorticoid receptor binding to DNA. J Biol Chem. 1985 Jun 25;260(12):7705–7715. [PubMed] [Google Scholar]
  6. Danielsen M., Northrop J. P., Ringold G. M. The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins. EMBO J. 1986 Oct;5(10):2513–2522. doi: 10.1002/j.1460-2075.1986.tb04529.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eisen H. J., Schleenbaker R. E., Simons S. S., Jr Affinity labeling of the rat liver glucocorticoid receptor with dexamethasone 21-mesylate. Identification of covalently labeled receptor by immunochemical methods. J Biol Chem. 1981 Dec 25;256(24):12920–12925. [PubMed] [Google Scholar]
  8. Eisen L. P., Reichman M. E., Thompson E. B., Gametchu B., Harrison R. W., Eisen H. J. Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain. J Biol Chem. 1985 Sep 25;260(21):11805–11810. [PubMed] [Google Scholar]
  9. Fasel N., Pearson K., Buetti E., Diggelmann H. The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription. EMBO J. 1982;1(1):3–7. doi: 10.1002/j.1460-2075.1982.tb01115.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Finer-Moore J., Stroud R. M. Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor. Proc Natl Acad Sci U S A. 1984 Jan;81(1):155–159. doi: 10.1073/pnas.81.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Firzlaff J. M., Diggelmann H. Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences. Mol Cell Biol. 1984 Jun;4(6):1057–1062. doi: 10.1128/mcb.4.6.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Giguère V., Hollenberg S. M., Rosenfeld M. G., Evans R. M. Functional domains of the human glucocorticoid receptor. Cell. 1986 Aug 29;46(5):645–652. doi: 10.1016/0092-8674(86)90339-9. [DOI] [PubMed] [Google Scholar]
  13. Godowski P. J., Rusconi S., Miesfeld R., Yamamoto K. R. Glucocorticoid receptor mutants that are constitutive activators of transcriptional enhancement. Nature. 1987 Jan 22;325(6102):365–368. doi: 10.1038/325365a0. [DOI] [PubMed] [Google Scholar]
  14. Hollenberg S. M., Weinberger C., Ong E. S., Cerelli G., Oro A., Lebo R., Thompson E. B., Rosenfeld M. G., Evans R. M. Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature. 1985 Dec 19;318(6047):635–641. doi: 10.1038/318635a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kumar V., Green S., Staub A., Chambon P. Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J. 1986 Sep;5(9):2231–2236. doi: 10.1002/j.1460-2075.1986.tb04489.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Loose D. S., Stover E. P., Feldman D. Ketoconazole binds to glucocorticoid receptors and exhibits glucocorticoid antagonist activity in cultured cells. J Clin Invest. 1983 Jul;72(1):404–408. doi: 10.1172/JCI110982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mendel D. B., Bodwell J. E., Gametchu B., Harrison R. W., Munck A. Molybdate-stabilized nonactivated glucocorticoid-receptor complexes contain a 90-kDa non-steroid-binding phosphoprotein that is lost on activation. J Biol Chem. 1986 Mar 15;261(8):3758–3763. [PubMed] [Google Scholar]
  19. Miesfeld R., Okret S., Wikström A. C., Wrange O., Gustafsson J. A., Yamamoto K. R. Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature. 1984 Dec 20;312(5996):779–781. doi: 10.1038/312779a0. [DOI] [PubMed] [Google Scholar]
  20. Miesfeld R., Rusconi S., Godowski P. J., Maler B. A., Okret S., Wikström A. C., Gustafsson J. A., Yamamoto K. R. Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA. Cell. 1986 Aug 1;46(3):389–399. doi: 10.1016/0092-8674(86)90659-8. [DOI] [PubMed] [Google Scholar]
  21. Miller J., McLachlan A. D., Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun;4(6):1609–1614. doi: 10.1002/j.1460-2075.1985.tb03825.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Okret S., Wikström A. C., Wrange O., Andersson B., Gustafsson J. A. Monoclonal antibodies against the rat liver glucocorticoid receptor. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1609–1613. doi: 10.1073/pnas.81.6.1609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Payvar F., Wrange O., Carlstedt-Duke J., Okret S., Gustafsson J. A., Yamamoto K. R. Purified glucocorticoid receptors bind selectively in vitro to a cloned DNA fragment whose transcription is regulated by glucocorticoids in vivo. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6628–6632. doi: 10.1073/pnas.78.11.6628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ponta H., Kennedy N., Skroch P., Hynes N. E., Groner B. Hormonal response region in the mouse mammary tumor virus long terminal repeat can be dissociated from the proviral promoter and has enhancer properties. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1020–1024. doi: 10.1073/pnas.82.4.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rüther U., Müller-Hill B. Easy identification of cDNA clones. EMBO J. 1983;2(10):1791–1794. doi: 10.1002/j.1460-2075.1983.tb01659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Scheidereit C., Geisse S., Westphal H. M., Beato M. The glucocorticoid receptor binds to defined nucleotide sequences near the promoter of mouse mammary tumour virus. Nature. 1983 Aug 25;304(5928):749–752. doi: 10.1038/304749a0. [DOI] [PubMed] [Google Scholar]
  27. Ucker D. S., Yamamoto K. R. Early events in the stimulation of mammary tumor virus RNA synthesis by glucocorticoids. Novel assays of transcription rates. J Biol Chem. 1984 Jun 25;259(12):7416–7420. [PubMed] [Google Scholar]
  28. Weinberger C., Hollenberg S. M., Ong E. S., Harmon J. M., Brower S. T., Cidlowski J., Thompson E. B., Rosenfeld M. G., Evans R. M. Identification of human glucocorticoid receptor complementary DNA clones by epitope selection. Science. 1985 May 10;228(4700):740–742. doi: 10.1126/science.2581314. [DOI] [PubMed] [Google Scholar]
  29. Willmann T., Beato M. Steroid-free glucocorticoid receptor binds specifically to mouse mammary tumour virus DNA. Nature. 1986 Dec 18;324(6098):688–691. doi: 10.1038/324688a0. [DOI] [PubMed] [Google Scholar]
  30. Wrange O., Okret S., Radojćić M., Carlstedt-Duke J., Gustafsson J. A. Characterization of the purified activated glucocorticoid receptor from rat liver cytosol. J Biol Chem. 1984 Apr 10;259(7):4534–4541. [PubMed] [Google Scholar]
  31. Yamamoto K. R., Gehring U., Stampfer M. R., Sibley C. H. Genetic approaches to steroid hormone action. Recent Prog Horm Res. 1976;32:3–32. doi: 10.1016/b978-0-12-571132-6.50008-7. [DOI] [PubMed] [Google Scholar]
  32. Yamamoto K. R., Stampfer M. R., Tomkins G. M. Receptors from glucocorticoid-sensitive lymphoma cells and two clases of insensitive clones: physical and DNA-binding properties. Proc Natl Acad Sci U S A. 1974 Oct;71(10):3901–3905. doi: 10.1073/pnas.71.10.3901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yamamoto K. R. Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet. 1985;19:209–252. doi: 10.1146/annurev.ge.19.120185.001233. [DOI] [PubMed] [Google Scholar]
  34. Zaret K. S., Yamamoto K. R. Reversible and persistent changes in chromatin structure accompany activation of a glucocorticoid-dependent enhancer element. Cell. 1984 Aug;38(1):29–38. doi: 10.1016/0092-8674(84)90523-3. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES