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Photoaffinity labeling with [17 alpha-methyl-3H]promegestone ([ 3H]R5020) is an effective technique for the covalent labeling of the progesterone receptor (PR), which allows monitoring of the steroid receptor complex under denaturing conditions. The present study was initiated to evaluate whether photolabeled PR could be used also as a marker for PR under nondenaturing conditions. Accordingly, the effect of irradiation on each component of the reaction was evaluated separately. When [3H]R5020 alone was irradiated, there was a rapid (less than 5 min), light dependent destruction of [3H]R5020, as evident from increased formation of a more polar tritiated product on TLC and a concomitant decrease in the ability of the irradiated preparation to bind to PR. When rabbit uterine PR was irradiated in the absence of steroid, a gradual decrease in the binding capacity was observed, reaching 70% of the nonirradiated control in 10 min. The optimal irradiation time for covalent [3H]R5020-PR complex formation was determined by irradiation for up to 5 min, and separation of the products by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Specific labeling of proteins of Mr 116,000 and 85,000 was observed, with the rate of labeling of the two being similar, and reaching a plateau by 4 min of irradiation. The photolabeling efficiency ranged from 2 to 12%. Sucrose gradient ultracentrifugation of photolabeled PR revealed that both the irradiated sample and the nonirradiated control sedimented to the same position. Subsequent SDS-polyacrylamide gel electrophoresis of the sucrose gradient peak from the photolabeled sample showed the presence of both labeled proteins of Mr 116,000 and 85,000. In addition, photolabeled rabbit uterine PR (Mr 116,000 and 85,000) could be immunoprecipitated with a guinea pig antiserum raised against rabbit uterine PR. Analysis of the photoaffinity labeling procedure in our system revealed that the photodestruction of [3H]R5020 was very rapid. However, maximal labeling with [3H]R5020 was obtainable with minimal photodestruction of PR which suggests that photolabeled receptor can be used as a marker for PR under nondenaturing conditions.

Chick progesterone receptor subunits A and B have been photoaffinity-labeled using [3H]R5020 ([17 alpha-methyl-3H]17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione) by a modification of the procedures previously reported by our laboratory (Dure, L. S., IV, Schrader, W. T., and O'Malley, B. W. (1980) Nature (Lond.) 283, 784-786). [3H]R5020 binds to the same receptor sites as authentic progesterone, and has an apparent Kdiss of 2.0 nM. Use of a CuSO4 filter raised the coupling efficiency to 5% and labeled exclusively the receptor proteins. Smaller labeled macromolecules were found to be proteolytic fragments of receptors. The protease(s) could not be inhibited by any of the commonly used protease inhibitors. However, the proteolytic activity was completely removed by passage of crude receptor preparations through phosphocellulose columns. Receptor preparations, photoaffinity-labeled after this procedure, showed exclusively one radioactive band at Mr = 79,000 (subunit A) or Mr = 108,000 (subunit B) with no detectable side-reaction products. Labeled receptors A and B were digested with Staphylococcus aureus V8 protease to yield smaller [3H]R5020-protein fragments derived from both. Molecular weight estimates (Mr = 9,500) and apparent isoelectric points indicate similarities of these regions of both A and B. The photoaffinity protocol described here thus provides a method for study of the hormone-binding domain of progesterone receptors and of receptor proteolysis in crude extracts.

The hormone-binding components of the rat uterine progesterone receptor were investigated by the methods of [3H]R5020 photoaffinity labeling and sodium dodecyl sulfatepolyacrylamide 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 (5-80 nM) 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 ratio 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.(ABSTRACT TRUNCATED AT 400 WORDS)