Heterogeneous deoxyribonucleic acid-binding forms of rabbit uterine progesterone receptor

Abstract

This study investigated the subunit structure of the rabbit uterine progesterone receptor (PR) using ion-exchange and DNA-cellulose chromatography. The mol wts of receptor were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling the receptors with the progestin [3H]R5020. Cytosols were labeled with [3H]progesterone or 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione [( 3H]R5020) and chromatographed on DE-52 cellulose. Greater than 80% of the PR bound to DE-52 and elution with a KCl gradient gave two peaks of activity at 50-75 mM (peak I) and at 125-200 mM KCl (peak II). Chromatography on QAE-Sephadex also separated two peaks. Peak I was abolished by addition of molybdate, or by passage over phosphocellulose. All (greater than 80%) of the PR from peaks I and II bound to DNA-cellulose and eluted as a single, symmetrical peak at 0.29 M KCl in both cases. Peak I could not be directly generated from peak II by exposure to salt or by ammonium sulfate precipitation. After peak II receptor had been bound and eluted from a DNA-cellulose column, however, it eluted subsequently from QAE as peak I. Photoaffinity labeling of peak I and peak II with [3H] R5020 in the presence of 10 microM cortisol revealed two proteins in each peak with mol wt of 102,000 and 78,000 (n = 13). Both mol wt forms were present in the DNA-eluates both of peak I and peak II. The [3H]R5020 binding to these mol wt forms could be completely displaced under exchange conditions with 10 nM progesterone or R5020. Deoxycorticosterone, dihydrotestosterone, and estradiol were nearly ineffective competitors at 10 nM. Without phosphocellulose chromatography before chromatography on DE-52 and DNA-cellulose, numerous receptor fragments were found. Fragments containing both steroid- and DNA-binding domains were found at 102,000, 78,000, 54-60,000, 43,000, 33-34,000, and 21,000, suggesting proteolysis. This proteolytic activity was removed by passage over phosphocellulose. The rabbit uterine PR contains at least two major proteins of 78,000 and 102,000 mol wt which cannot be distinguished on the basis of their ionic or DNA-binding characteristics or steroid-binding specificity.