Application of a Protein-Blotting Procedure to the Study of Human Glucocorticoid Receptor Interactions with DNA

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 84; no. 7; pp. 1744 - 1748
• Main Authors: Silva, Corinne M., Tully, Douglas B., Petch, Leslie A., Jewell, Christine M., Cidlowski, John A.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.04.1987; National Acad Sciences
• Subjects: Biochemistry; Biological and medical sciences; Cell nucleus; Cell physiology; Cellulose nitrate; Cytosol; Cytosol - metabolism; Dexamethasone - analogs & derivatives; Dexamethasone - metabolism; DNA; DNA, Viral - metabolism; Fundamental and applied biological sciences. Psychology; Gels; Genes, Regulator; Glucocorticoid receptors; HeLa cells; HeLa Cells - metabolism; Hormonal regulation; Humans; Mammary Tumor Virus, Mouse - genetics; Mesylates; Molecular and cellular biology; Neoplasm Proteins - metabolism; Receptors; Receptors, Glucocorticoid - genetics; Receptors, Glucocorticoid - isolation & purification; Receptors, Glucocorticoid - metabolism; Human; Proteins; Steroid hormone; DNA; Hormonal regulation; Molecular interaction; Method; Glucocorticoid; Hormonal receptor
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.84.7.1744

To exert their effects, glucocorticoid receptor complexes interact selectively with DNA sequences known as glucocorticoid regulatory elements. We have studied the interaction between human glucocorticoid receptors and mouse mammary tumor virus (MMTV) DNA by means of a procedure that permits analysis after immobilization of the receptor on nitrocellulose. Proteins from crude cytosolic or nuclear extracts were electrophoresed on NaDodSO4/PAGE gels, soaked in a urea buffer to remove NaDodSO4, transferred to nitrocellulose, and probed with nick-translated MMTV [32P]DNA in a 5% nonfat dry milk buffer, which minimizes nonselective DNA-protein interactions. We present evidence that MMTV [32P]DNA interacts selectively with the glucocorticoid receptor. These data include (i) comigration of [3H]dexamethasone mesylate-labeled band and bound MMTV [32P]DNA on gel electrophoresis systems; (ii) localization of DNA-binding activity in the cytosol of cells incubated with steroid at 0 degrees C and in the nucleus and cytosol of cells incubated at 37 degrees C; (iii) binding of the MMTV DNA to highly purified receptor; and (iv) absence of MMTV DNA binding activity in extracts from cells whose receptor has been down-regulated. Furthermore, glucocorticoid receptors analyzed under these conditions exhibit selective binding to DNA fragments that contain glucocorticoid regulatory elements.