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 the 3H-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. The 3H-R5020 labeled human PR eluted from DNA-cellulose as a single symmetrical peak at 0.2 M NaCl; after photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this species was shown to consist of about equal amounts of two proteins of M(r) ≃ 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 retion 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.
ASJC Scopus subject areas
- Molecular Biology