Characterization and Sequence-Specific Binding to Mouse Mammary Tumor Virus DNA of Purified Activated Human Glucocorticoid Receptor

Mikael Brönnegård, Lorenz Poellinger, Sam Okret, Ann Charlotte Wikström, Oddmund Bakke, Jan-Ake Gustafsson

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Activated glucocorticoid receptor (GR) from the human cell line HeLa S3 was purified by differential chromatography on DNA-cellulose followed by DEAE-Sepharose chromatography to 50-60% homogeneity according to sodium dodecyl sulfate gel electrophoresis and densitometric scanning of silver-stained gels. These gels routinely demonstrated a main band of Mr 94 000 (94K band) and two minor bands of Mr 79 000 (79K band) and 39 000 (39K band), respectively. Photoaffinity labeling indicated that the hormone was bound to the 94K and 79K components. In some preparations, a 72K band was observed. Further characterization of the purified receptor by gel permeation chromatography on Sephadex G-200 revealed a receptor complex with a Stokes radius of 5.8 nm. The sedimentation coefficient of the purified receptor was 4.4 Sw. In analogy to the rat hepatic GR, limited proteolysis of the purified GR with trypsin or α-chymotrypsin led to degradation of the 94K and 79K components and appearance of 28K and 39K fragments, respectively. In addition, no difference in the protease digestion pattern using Staphylococcus aureus V8 protease was observed. Immunoblotting using a monoclonal antibody raised against the 94K GR from rat liver demonstrated cross-reactivity with the human 94K and 79K proteins from HeLa S3 cells, indicating similar antigenic characteristics between rat and human GR. In our study, five out of nine tested monoclonal antibodies against the rat liver GR cross-reacted with human GR. DNase I and exonuclease III protection experiments demonstrated binding of the purified human GR to specific GR binding regions in mouse mammary tumor virus DNA. Both the purified rat and human receptors recognized the same nucleotide sequences in mouse mammary tumor virus DNA exhibited a similar degree of DNase I and exonuclease III protection at similar concentrations of receptor. The similarity in structure and specific DNA binding suggests highly conserved properties of GR from different species.

Original languageEnglish (US)
Pages (from-to)1697-1704
Number of pages8
JournalBiochemistry
Volume26
Issue number6
DOIs
StatePublished - Mar 1 1987

ASJC Scopus subject areas

  • Biochemistry

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