Photoaffinity labelling of the human mineralocorticoid receptor with steroids having a reactive group at position 3, 18 or 21.

The ability of a glucocorticoid (triamcinolone acetonide: TA) and three progesterone derivatives with photoreactive groups at different positions (promegestone: R5020; 18-oxo-18-vinylprogesterone: 18OVP; 21-diazoprogesterone: 21DP) to bind covalently to the human mineralocorticoid receptor (hMR) expressed in Sf9 insect cells was assessed. Sedimentation gradient analysis and exchange assays with aldosterone showed that [3H]TA, a partial mineralocorticoid agonist, and [3H]R5020, a pure antimineralocorticoid, were covalently bound to hMR after UV irradiation, with a labelling efficiency of approx. 3-5%. UV irradiation did not alter the heterooligomeric structure of the hMR, since the irradiated [3H]TA- and [3H]R5020-hMR complexes sedimented at approx. 9-10 S, as did the non-irradiated complexes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a band labelled by [3H]TA or [3H]R5020, having a molecular mass of 120 kDa. This band was not detected in the presence of an excess of the corresponding unlabelled steroid or when the cytosol was recovered from non-infected Sf9 cells. Electrophoresis of a truncated hMR (hMRDelta(1-351)) photolabelled with [3H]TA revealed a 80 kDa band, compatible with the molecular mass of the truncated hMR. Limited chymotrypsin proteolysis of the [3H]TA photolabelled hMR generated a 30 kDa fragment covalently associated with [3H]TA. As the 30 kDa fragment generated by chymotrypsin has been shown to encompass the entire ligand-binding domain of the hMR (B. Couette, J. Fagart, S. Jalaguier, M. Lombès, A. Souque, M.E. Rafestin-Oblin, Biochem. J. 315 (1996) 421-427), the present experiments provide evidence that [3H]TA is covalently bound to the ligand binding domain of the hMR. Exchange assays with [3H]A also revealed that unlabelled 18OVP and 21DP, two mineralocorticoid agonists bearing photoreactive groups at skeleton positions crucial for the ligand-MR interaction, are covalently bound to hMR with an approx. 30-35% labelling efficiency.

Effect of photoaffinity labeling on rabbit uterine progesterone receptor.

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.

In situ photolinked nuclear progesterone receptors of human breast cancer cells: subunit molecular weights after transformation and translocation.

The subunit structure of mammalian cytoplasmic progesterone receptors (PR) has been difficult to study because these proteins are subject to in vitro proteolysis; the structure of nuclear PR is unknown. We have now developed an in situ photoaffinity labeling method for PR that permits study of their subunits with minimal in vitro incubations. The strategy is to use [3H]R5020, a synthetic photoactive progestin, and suitable incubation temperatures to place receptors into their precise intracellular sites in intact cells. The cells, still intact, are then irradiated with UV at 300 nm for 2 min. This irradiation efficiently (approximately 15%) yields covalently linked hormone-receptor complexes at any intracellular location. Cells are than rapidly ruptured, nuclei are separated, and receptors are extracted with salt and/or directly solubilized with detergents before the subunits are displayed on denaturing polyacrylamide gels. With this as well as with modified in vitro methods, we show here that untransformed human breast cancer PR have two dissimilar subunits (mol wt, 115,000 and 81,000) present in equimolar amounts. The same subunits, with apparently unmodified mass, can be demonstrated in nuclei after they have been translocated by progestin treatment. Therefore, PR transformation and acquisition of nuclear binding capacity does not require prior proteolytic processing of subunits or other major structural modifications that are detectable on single dimension gels.

Photoaffinity labeling of glucocorticoid receptors.

The cross-reactivity of progestins for glucocorticoid receptors was exploited to photoaffinity label glucocorticoid receptors from cultured rat hepatoma (HTC) and mouse lymphoma (S49) cell cytosol. The synthetic progestin, 17 alpha, 21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020), rapidly forms covalent bonds with protein upon irradiation of either cytosol with 350 nm light. Polyacrylamide gel electrophoresis under denaturing conditions reveals a single band photolabeled by R5020 that is not observed when excess nonradioactive dexamethasone is included in the incubation. This protein band corresponds to a molecular weight of about 87,000 in both HTC and S49 cell cytosol; it is entirely absent in cytosol from glucocorticoid-resistant S49(r-) cells which lack receptor-binding activity. Another steroid-resistant mutant, S49 (nti), which exhibits normal levels of steroid-binding activity but increased binding of receptor-steroid complexes by the nucleus, yields a receptor which, when photolabeled, has an apparent molecular weight of only 39,000. These results demonstrate that glucocorticoid receptors can be photoaffinity-labeled; the data are consistent with the notion that the binding form of the receptor consists of a single polypeptide chain, Mr = 87,000, in two different species, rat and mouse, and in cells of either hepatic or lymphoid origin. The data also suggest that the lesion in the steroid-resistant S49 (nti) lymphoma cell line is a mutation of the structural gene for the glucocorticoid receptor which results in the synthesis of a truncated protein.