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  • Title: Differences in the association of the progesterone receptor ligated by antiprogestin RU38486 or progestin ORG 2058 to chromatin components.
    Author: Geier A, Bella R, Beery R, Haimsohn M, Lunenfeld B.
    Journal: Biochim Biophys Acta; 1987 Oct 22; 931(1):78-86. PubMed ID: 3651513.
    Abstract:
    We assessed the hypothesis that due to variations in the conformation of the progesterone receptor induced by the antiprogestin RU38486 compared to the progestin ORG 2058, differences may result in the association of the receptor with some of the chromatin components. The physical properties of the receptor-bound chromatin fragments released by micrococcal nuclease digestion were characterized by sucrose gradient sedimentation and by gel filtration on Agarose A-1.5m or Agarose A-5m columns. The nuclear fraction was isolated from T47D cells previously exposed to 0.1 microM [3H]RU38486 or 0.1 microM [3H]ORG 2058. Micrococcal nuclease digestion solubilized two receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only one receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking with the cross-linker 2-iminothiolane of the micrococcal nuclease solubilized receptor forms resulted in 6.7-S and 4.4-S forms sedimenting on 0.4 M KCl gradients for the antiprogestin and progestin ligated receptors, respectively. Stokes radii of 7.3 nm and 6.4 nm were determined by gel filtration in 0.4 M KCl for the 6.7-S and the 4.4-S receptor forms, respectively. Using the sedimentation coefficient and the Stokes radius, molecular weights of 202,000 and 116,000 were calculated for the antiprogestin and progestin ligated receptors. We conclude that the micrococcal nuclease solubilized antiprogestin ligated receptor is associated with additional or different chromatin components compared to the progestin bound receptor. The hypothesis that variations in the conformation of the progesterone receptor induced by the antiprogesterone RU38486 compared to the progestin ORG2058 may cause differences in the association of the receptor with some of the chromatin components was investigated. The approach to analyzing the functional organization of the receptor in the chromatin was to study the receptor-bound fragments released by nuclear digestion. The physical properties of these receptor-bound chromatin fragments were characterized by sucrose gradient sedimentation and gel filtration. Micrococcal nuclease digestion solubilized 2 receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only 1 receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking resulted in 6.7 S forms for the antiprogestin receptors and 4.4 S forms for the progestin ligated receptors. Use of the sedimentation coefficient and the Stokes radius yielded molecular weights of 202,000 and 116,000 for the antiprogestin and progestin ligated receptors, respectively. Overall, these kinetic studies were unable to explain the variation in responses of the target cell to agonist or antagonist ligands. It is postulated that the antagonist exerts a subtle, but important, conformational change in the receptor, which alters the receptor's ability to associate with some chromatin components and thus affects the normal events in gene expression.
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