“Defective” Receptors in Steroid-Resistant Conditions may be Proteolytic Artifacts
The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to stuctural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio.
In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue.
The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7-amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by “lysine-specific” endopeptidases, detected with t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for its ability to digest the glucocorticoid receptors from rat liver cytosol. Under the conditions used, about 40% of the receptors were converted to the mero-receptor, the smallest fragment containing the steroid-binding site.
Since the cleavage of steroid receptors in vitro has been amply documented, we conclude that the observation of altered receptors in extracts of tissues or tumors that are resistant to steroid treatment does not prove that this resistance is due to intrinsic structural defects in the receptors.
KeywordsAcute Lymphoblastic Leukemia Glucocorticoid Receptor Axial Ratio Triamcinolone Acetonide Limited Proteolysis
Unable to display preview. Download preview PDF.
- 1.E. V. Jensen, G. E. Block, S. Smith, E. R. DeSombre, Hormonal dependency of breast cancer. In: M. L. Griem, E. V. Jensen, J. E. Ultmann, R. W. Wissler (eds) Recent Results in Cancer Research. Springer-Verlag, Berlin, 42:55 (1973).Google Scholar
- 3.J. L. Wittliff, B. W. Beatty, E. D. Savlov, W. B. Patterson, R. A. Cooper, Jr., Estrogen receptors and hormone dependency in human breast cancer. In: G. St. Arneault, P. Band, L. Israël (eds) Recent Results in Cancer Research. Springer-Verlag, Berlin, 57:59 (1976).Google Scholar
- 4.E. B. Thompson, M. E. Lippman (eds), Steroid Receptors and the Management of Cancer. CRC Press, Boca Raton, FL, Vol. 1 and 2 (1979).Google Scholar
- 5.J. Stevens, Y.-W. Stevens, Glucocorticoid receptors in human leukemia and lymphoma: Quantitation and clinical significance. In: V. P. Hollander (ed) Hormonally Responsive Tumors. Academic Press, Orlando, FL, p. 155 (1985).Google Scholar
- 10.M. R. Sherman, F. B. Tuazon, Y.-W. Stevens, E.-M. Niu, Oligomeric steroid receptor forms and the products of their dissociation and proteolysis. In: H. Eriksson, J.-X. Gustafsson (eds) Steroid Hormone Receptors: Structure and Function (Nobel Symposium No. 57). Elsevier, Amsterdam, p. 3 (1983).Google Scholar
- 11.E.-M. Niu, J. L. Lewis, Jr., M. R. Sherman, Steroid receptors in human breast cancer and uterine specimens: Quantitation and structural analysis. Reprod. Contracep. 2(4):10 (1982) (In Chinese).Google Scholar
- 12.M. R. Sherman, M. C. Moran, R. M. Neal, E.-M. Niu, F. B. Tuazon, Characterization of molybdate-stabilized glucocorticoid receptors in healthy and malignant tissues. In: H. J. Lee, T. J. Fitzgerald (eds) Progress in Research and Clinical Applications of Corticosteroids. Heyden, Philadelphia, p. 45 (1982).Google Scholar
- 14.M. R. Sherman, D. Barzilai, P. R. Pine, F. B. Tuazon, Glucocorticoid receptor cleavage by leupeptin-sensitive enzymes in rat kidney cytosol. In: W. W. Leavitt, J. H. Clark (eds) Steroid Hormone Receptor Systems. Plenum Press, New York, p. 357 (1979).Google Scholar
- 16.M. R. Sherman, F. B. Tuazon, G. J. Sömjen, Stabilization and cleavage of steroid receptors: Effects of leupeptin and molybdate on rat kidney glucocorticoid receptors. In: R. J. Soto, A. De Nicola, J. Blaquier (eds) Physiopathology of Endocrine Diseases and Mechanisms of Hormone Action. Alan R. Liss, New York, p. 321 (1981).Google Scholar
- 18.A. Bfóyum, Isolation of lymphocytes, granulocytes and macrophages. Scand. J. Immunol. 5 (Suppl 5): 9 (1976).Google Scholar
- 22.H. K. Schachman, Ultracentrifugation in Biochemistry. Academic Press, New York, p. 239 (1959).Google Scholar
- 23.H. Kato, N. Adachi, Y. Ohno, S. Iwanaga, K. Takada, S. Sakakibara, New fluorogenic peptide substrates for plasmin. J. Biochem. (Tokyo) 88: 183 (1980).Google Scholar
- 31.W. V. Vedeckis, Steroid hormone receptor structure in normal and neoplastic cells. In: V. P. Hollander (ed) Hormonally Responsive Tumors. Academic Press, Orlando, FL, p. 3 (1985).Google Scholar
- 39.A. C. Notides, S. Sasson, The positive cooperativity of the estrogen receptor and its relationship to receptor activation. In: H. Eriksson, J.-Â. Gustafsson (eds) Steroid Hormone Receptors: Structure and Function (Nobel Symposium No. 57). Elsevier, Amsterdam, p. 103 (1983).Google Scholar
- 40.T. Aoyagi, H. Umezawa, Structures and activities of protease inhibitors of microbial origin. In: E. Reich, D. B. Rifkin, E. Shaw (eds) Proteases and Biological Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 429 (1975).Google Scholar
- 41.J.-X. Gustafsson, N. Einhorn, G. Elfstróm, B. Nordenskjöld, O. Wrange, Progestin receptor in endometrial carcinoma. In: W. L. McGuire, J.-P. Raynaud, E.-E. Baulieu (eds) Progesterone Receptors in Normal and Neoplastic Tissues (Progress in Cancer Research and Therapy, Vol. 4). Raven Press, New York, p. 299 (1977).Google Scholar
- 42.M. A. Costello, M. R. Sherman, Modification of mouse mammary tumor glucocorticoid receptor forms by ribonuclease treatment. Program of the 62nd Annual Meeting of The Endocrine Society, Washington, D. C., p. 174 (1980).Google Scholar
- 48.M. Mead, Blackberry Winter: My Earlier Years. Pocket Books, New York, p. 234 (1972).Google Scholar
- 51.C. A. Barnett, G. Litwack, Additional evidence that corticosteroid binder IB is not derived from binder II by limited proteolysis. Biochem. Biophys. Res. Commun. 108: 1670 (1982).Google Scholar