Rat Uterine Progesterone Receptor Analyzed by [3H]R5020 Photoaffinity Labeling: Evidence that the A and B Subunits Are Not Equimolar*

Abstract

The hormone-binding components of the rat uterine progesterone receptor were investigated by the methods of [3H]R5020 photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Two specifically labeled peaks were observed at mol wt of 85,600 .+-. 1,200 and 109,600 .+-. 1,200 (n = 31), resembling the A and B progesterone receptor components previously described in other systems. However, in contrast to the equimolar ratio reported in other systems, the level of subunit A observed was consistently greater than that of B (A/B ratio = 3.2 .+-. 0.3; n = 31). The unusual A/B ratio prompted a complete validation of the photolabeling procedure in this system. Although the levels of specific binding increased, there was no change in the A/B ratio with varying [3H]R5020 concentrations (5-80nM) or with time of UV exposure (0.5 min to 3 h). Although adsorption to hydroxylapatite indicated that specific [3H]R5020 binding was reduced by 72.0 .+-. 6.4% within 5 min of UV exposure, relabeling the irradiated preparations with [3H]R5020 resulted in little change in specific [3H]R5020 binding. TLC analysis of [3H] R5020 (Rf = 0.48 .+-. 0.01; n = 4) after irradiation demonstrated rapid photolysis resulting in a 94.3 .+-. 2.5% (n = 3) loss of authentic [3H]R5020 within 5 min. After photolysis, at least two new tritiated products were recovered with Rf values of 0.20 .+-. 0.03 and 0.72 .+-. 0.02. Analysis by adsorption to hydroxylapatite indicated that the photolysis products competed for specific [3H]R5020-binding sites in cytosol with only 10-fold lower relative binding activity than authentic R5020. Thus these compounds probably account for the increase in specific photolabeling of the A and B peaks achieved when UV exposure is prolonged from 5 to 30 min. Further study indicated that the A/B subunit ratio in this system was not changed under a variety of in vitro conditions, including the absence or presence of molybdate, sulfhydryl protective reagents (dithiothreitol and thioglycerol), protease inhibitors (phenylmethylsulfonylfluoride and leupeptin), glycerol (0%, 10%, and 30%, vol/vol), or 1.5 mM EGTA, or after precipitation with 40% ammonium sulfate. This consistency of the A/B ration under a wide variety of adverse in vitro conditions suggests that in vitro artefacts may not account for the ratio''s deviation from unity. Estrogen withdrawal (48 h) enhanced by progesterone treatment (0.5 mg for 24 h) resulted in only a modest reduction in the A/B ratio to 1.9 .+-. 0.1. Additionally, seasonal variation did not account for the observed A/B subunit ratio, which remained high throughout the year. Resolving whether the high A/B ratio represents a physiological adaptation of the rat uterine progesterone receptor system can be important in elucidating the functional significance of the A component common to progesterone receptors.