Heterogeneity of Nuclear Glucocorticoid Receptor Interactions

  • John A. Cidlowski
  • Allan Munck
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 117)

Abstract

When thymocytes are incubated with glucocorticoids at 37°, 60–70% of the receptor bound steroid is associated with the nucleus. Under conditions where the rate of steroid-receptor formation is not limiting the transfer of steroid-receptors from the cytoplasm to the nucleus occurs rapidly with a T1/2 of 30 seconds. These observations have led us to investigate whether or not all glucocorticoid receptor complexes are associated with the nucleus in the same manner. To this end, nuclear glucocorticoid-receptor complexes have been extracted by differential salt extraction and DNase I and DNase II digestion. Of the nuclear dexamethasone receptor complex initially bound, 70–75% is resistant to 0.2 M KC1 extraction (designated N2) and 25–39% is resistant to 0.4 extraction (designated N4). N2 be further extracted with 0.4 M KC1 whereas N4 is resistant to reextraction with either 0.2 M KC1, suggesting that N2–N4 (N2–4) and N4 represent distinct physical forms of nuclear dexamethasone receptor. In intact cells, N2 and N differ under the following physiological condition. (1) N4 binding occurs prior to N2–4 (2) a cold chase of unlabeled dexamethasone decreases N2–4 by 70% but N4 binding by only 10%; (3) N4 binding decreases mor apidly than N2–4 following a decrease in hormone concentration by dilution; (4) a cold chase of either cortexolone or progesterone preferentially decreases N2–4 and has little effect on N4. In addition, the nuclear N2–4 and N4 distribution differ for cortisol, dexamethasone and triamcinolone acetonide, three steroids having different in vitro biological potencies. DNase I treatment of nuclei solubilizes approximately 60% of nuclear DNA yet releases only 20–30% of nuclear receptor, whereas DNase II solubilizes only 10% of nuclear DNA and releases 75–80% of nuclear receptor. As seen with salt extraction, the resistance of nuclear glucocorticoid-receptor complexes to a DNase I and II is dependent on the steroid molecule which is associated with the receptor. Of the steroids we have tested, nuclear triamcinolone acetonide and dexamethasone receptor complexes are most resistant to nuclease attack. Nuclear cortisol receptor complexes are readily solubilized by either DNase I or II under conditions where little dissociation of steroid from receptor occurs. These data represent evidence for physiologically distinct forms of nuclear glucocorticoid receptor interaction. In addition, they demonstrate the importance of the steroid portion of the steroid receptor in directing the nature and/or location of steroid receptors within or on the nucleus.

Keywords

Triamcinolone Acetonide Thymus Cell Nuclear Binding Biological Potency Hormone Receptor Complex 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Munck, A. and Brinck-Johnsen, T. (1968) J. Biol. Chem. 243, 5556–5565.Google Scholar
  2. 2.
    Wira, C.R. and Munck, A. (1974) J. Biol. Chem.249, 5328–5336.Google Scholar
  3. 3.
    Munck, A. and Leung, K. (1976) In: Receptors and Mechanism of Action of Steroid Hormones (Pasqualini, J.R. ed) Part III. pp. 311–397. Marcel Dekker, New York.Google Scholar
  4. 4.
    Munck, A. and Foley, R. (1976) J. Steroid Biochem.7, 1117–1122.Google Scholar
  5. 5.
    Yamamoto, K.R. and Alberts, B.M. (1976) An. Rev. Biochem. 45, 721–744.Google Scholar
  6. 6.
    Spelsberg, T.C., Pikler, G.M. and Webster, R.A. (1976) Science 194, 197–198.Google Scholar
  7. 7.
    Spelsberg T.C., Steggles, A.W. and O’Malley, B.W. (1974) J. Biol. Chem. 246, 4188–4197.Google Scholar
  8. 8.
    Clark, J.H. and Peck, E.J., Jr. (1976) Nature 260, 635–637.Google Scholar
  9. 9.
    Klyzsehko-Stefanowicz, L., Chiu, J., Tsai, Y. and Hnilica, L. (1976) Pro. Nat. Acad. Sciences 73, 1954–1958.Google Scholar
  10. 10.
    Simons, S.S., Jr., Martinez, H.M., Garcea, R.L., Baxter, J.D. and Tomkins, G.M. (1976) J. Biol. Chem. 251, 334 343.Google Scholar
  11. 11.
    Defer, N., Dastugue, B. and Kruh, J. (1974) Biochemie 56, 1549–1557.Google Scholar
  12. 12.
    Puca, G.A., Nola, E., Hibner, U., Cicala, G. and Sica, V. (1975) J. Biol. Chem. 250, 6452–6459.Google Scholar
  13. 13.
    Baudenistel, L.J. and Ruh, T.S. (1976) Steroids 28, 223–237.Google Scholar
  14. 14.
    Jackson, V. and Chalkley, R. (1974) 249, 1615–1626.Google Scholar
  15. 15.
    Clark, J.H., Anderson, J.N. and Peck, E.J., Jr. (1973) In: Receptors for Reproductive Hormones (O’Malley, B.W. and Means, A.R) Vol. 36, pp. 15–59 Plenum Press, NY.CrossRefGoogle Scholar
  16. 16.
    Baxter, J.D., Rousseau, G.G., Benson, M.C., Garcea, R.C., Ito, J. and Tomkins, G M. (1972) Proc. Nat. Acad. Sci. 69, 18921896.Google Scholar
  17. 17.
    Rousseau, G.G., Higgins, S.J., Baxter, J.D., Gelfand, D. and Tomkins, G.M. (1975) J. Biol. Chem. 250, 6015–6021.Google Scholar
  18. 18.
    Middlebrook, J.L., Wong, M.D., Ishii, D.N. and Aronow, L. (1975) Biochemistry 14, 180–186.Google Scholar
  19. 19.
    Clark, R.J. and Felsenfeld, G. (1971) Nature New Biology 229, 101–106.Google Scholar
  20. 20.
    Gottesfeld, J.M., Garmad, W.T., Bagi, G., Wilson, R.F., and Bonner, J. (1974) Proc. Natl. Acad. Sci. 71, 2193–2197.Google Scholar
  21. 21.
    Weintraub, H. and Groudine, M. (1976) Science 193, 848–856.PubMedCrossRefGoogle Scholar
  22. 22.
    Simpson, R T. (1978) Cell 13, 691–699.Google Scholar
  23. 23.
    Munck, A. and Wira, C In: Methods in Enzymology (O’Malley B, and Hardman, J.) Academic Press, NY vol. 36, (1975) pp. 255–266.Google Scholar
  24. 24.
    Schneider, W.C. Determination of Nucleic Acids in Tissues by Pentose Analysis In: Methods in Enzymology. vol. 3 p. 680–684, 1957 (S.P. Colowick and N.O. Kaplan ed ) 1957.Google Scholar
  25. 25.
    Dixon, G.H. Non-histone chromatin proteins and chromatin structure. presented at American Society of Biological Chemists, June 4, 1978, Atlanta, GA.Google Scholar
  26. 26.
    Mueller, R.E., Traish, A.M. and Wotiz, H.A. (1977) J. Biol. Chem. 252, 8206, 8211.Google Scholar
  27. 27.
    Mulvihill, E.R. and Palmiter, R.D. (1977) J. Biol. Chem. 252, 2060–2068.Google Scholar

Copyright information

© Springer Science+Business Media New York 1979

Authors and Affiliations

  • John A. Cidlowski
    • 1
  • Allan Munck
    • 1
  1. 1.Burlington, VT Dartmouth Medical SchoolUniversity of Vermont Medical SchoolHanoverUSA

Personalised recommendations