Unspecific and Sequence-Specific Deoxyribonucleic Acid Binding of the Partially Purified Human Progesterone Receptor

Abstract

The progesterone receptor (PR) was partially purified from T47D human breast cancer cells by sequential chromatography on phosphocellulose, heparin-Sepharose, and DNA-cellulose. Heparin-Sepharose chromatography resulted in an efficient conversion of the receptor to a DNA-binding form (activation) since more than 85% of the3H-R5020 labeled eluate from heparin-Sepharose was retained on DNA-cellulose and since the cytosolic 8S receptor was converted to a 4S moiety after chromatography on heparin-Sepharose. The3H-R5020 labeled human PR eluted from DNA-cellulose as a single symmetrical peak at 0.2M NaCI; after photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this species was shown to consist of about equal amounts of two proteins of Mr (symbol missing) 96, 000 and 120, 000 (the so called A- and B-subunits, respectively). This partially purified receptor preparation (SA 490 pmol/mg protein) did not contain any glucocorticoid receptor (GR) as shown by immunoblotting with a monoclonal antirat GR antibody that cross-reacts with the human GR. Therefore, this preparation was used to compare the specific DNA-binding properties of the human PR with those of the purified rat GR. The human PR bound specifically to the promoter region of mouse mammary tumor virus (MMTV) at a molar ratio between receptor and DNA similar to the molar ratio between GR and DNA needed for binding of rat GR to MMTV, indicating that the PR was purified in a biologically active form. The human PR generated a DNase I protection pattern (footprint) between nucleotides -189 and -166, identical in length to that caused by the rat GR. However, two other rat GR-binding sites in the 5′-long terminal repeat of MMTV did not interact with the human PR, indicating similar but not identical binding properties of PR and GR, respectively. © 1988 by The Endocrine Society.