Ligands induce conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody.

Weigel, Nancy L.; Beck, Candace A.; Estes, Patricia A.; Prendergast, Paul; Altmann, Magda; Christensen, Kurt D.; Edwards, Dean P.

doi:10.1210/mend.6.10.1448113

Cited by 41 publications

(43 citation statements)

References 34 publications

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“…In an experiment with a larger amount of unlabeled androgen receptors, obtained from LNCaP cells, it was shown that the interaction of HSP90 with an immunopurified AR is disturbed upon a salt wash, whereas ligand binding remained (dissociation of HSP90 shown in Fig. 3, lane [15][16][17][18]. Strikingly, when the in vitro produced 35 Slabeled AR on the affinity matrix was liganded with an antiandrogen, trypsinization now resulted in the formation of a 29-kDa fragment (Fig.…”

Section: Resultsmentioning

confidence: 96%

“…The model argues that agonists and antagonists recognize distinct regions of the ligand binding domain. Antagonists induce an incomplete conformational change that results in dimerization and DNA binding, but leave the C terminus of the ligand binding domain in a form still available for protease (11) and antibody recognition (14,15). As a result, a surface repressor function is not removed and the receptor is not able to induce transcription.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the monoclonal antibody C-262, raised against the last 14 amino acids of the progesterone receptor, could discriminate agonistand antagonist-bound progesterone receptors. The antibody bound to the full-length receptor only in the presence of antagonist, whereas progesterone prevented the recognition of the receptor by C-262 (14,15). Deletion of 42 amino acids abolished the binding of progesterone, but had no effect on binding of the antagonist RU486.…”

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confidence: 96%

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Ligand-induced Conformational Alterations of the Androgen Receptor Analyzed by Limited Trypsinization

Kuil

Berrevoets

Mulder

1995

Journal of Biological Chemistry

131

103

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Limited proteolysis of in vitro produced human androgen receptor was used to probe the different conformations of the receptor after binding of androgens and several antiandrogens. The results provide evidence for five different conformations of the receptor, as detected by the formation of proteolysis resisting fragments: 1) an initial conformation of the unoccupied receptor not resisting proteolytic attack; and receptor conformations characterized by 2) a 35-kDa proteolysis resisting fragment spanning the ligand binding domain and part of the hinge region, obtained with most antagonists, and in an initial step after agonist binding; 3) a 29-kDa proteolysis resisting fragment spanning the ligand binding domain, obtained in the presence of agonists after an activation process; 4 and 5) 30-and 25-kDa fragments, derived from 2 and 3, but missing part of the C terminus, obtained with RU486 (RU486 has antiandrogenic properties, besides its effects as an antiprogestagen/antiglucocorticoid). Concomitantly with the change from 2 to 3 (and of 4 to 5 for RU486), dissociation of the 8 S complex of receptor with associated proteins occurred. With a mutant receptor (LNCaP cell mutation in C-terminal region), some antagonists activated transcription analogous to agonists, and induced the activated receptor conformation 3. A mutant lacking the C-terminal 12 amino acids bound RU486 but not androgens, and formed with RU486 conformation 5. These data imply that, after the initial rapid binding of ligand, androgens induce a conformational change of the receptor, a process that also involves release of associated proteins. RU486 induces an inappropriate conformation of the C-terminal end, similar as found for its effect on the progesterone receptor. In contrast, the other antiandrogens act at a different step in the mechanism of action: they do not induce an abnormal conformation, but act earlier and prevent a conformation change by stabilizing a complex with associated proteins.The biological effects of androgens and other steroid hormones are mediated through intracellular receptors, belonging to the steroid and thyroid hormone receptor superfamily (1). Upon activation by the hormone, steroid receptors interact with specific DNA sequences, located in the flanking regions of target genes, resulting in modulation of the expression of these genes (2-4). Steroid hormone receptor antagonists inhibit the biological effects of agonists, and are frequently used in the treatment of hormone-based dysfunctions in human. Furthermore, the synthetic antagonists are important tools to define the molecular mechanism of transactivation by steroid hormones (5, 6).Agonists and antagonists may change the spatial structure of the receptor in distinct ways, as was first indicated by gel retardation experiments: antagonist-and agonist-receptor-DNA complexes showed slightly different mobilities (7-10). Recently, limited proteolysis of progesterone, estrogen, and glucocorticoid receptors pinpointed the distinction in conformation between hormone-and ant...

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Section: Resultsmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 96%

See 1 more Smart Citation

Ligand-induced Conformational Alterations of the Androgen Receptor Analyzed by Limited Trypsinization

Kuil

Berrevoets

Mulder

1995

Journal of Biological Chemistry

131

103

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“…Depending on the cell and the promoter context, RU486 can function as either an agonist or an antagonist. Accumulating evidence reveals that mixed antagonists induce a unique PR conformation that is different from those conformations induced by either pure agonists or pure antagonists (22), and that ligand-induced conformational changes in a receptor may regulate the interactions of steroid receptors with coregulators (25,28). To explain the capacity of mixed antagonists to manifest different activities in different cellular contexts, we hypothesized that a differential ratio of coactivator to corepressor can directly modulate the ability of RU486 to activate transcription in different cellular contexts.…”

Section: Discussionmentioning

confidence: 99%

“…In fact, progesterone and RU486 appear to contact noncoincident, but overlapping, amino acids in the ligand-binding domain (LBD) of PR (22)(23)(24). The precise manner in which multiple conformations of PR induced by SPRMs affects its agonist or antagonist activity is open to question.…”

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confidence: 99%

Coactivator/corepressor ratios modulate PR-mediated transcription by the selective receptor modulator RU486

Liu

Auboeuf

Wong

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

145

102

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Selective receptor modulators, such as the antiprogestin RU486, are known to exhibit partial agonist activities in a cell-typedependent manner. Employing an in vitro chromatin transcription system that recapitulates progesterone receptor (PR)-mediated transcription in vivo, we have investigated the molecular basis by which the antiprogestin RU486 regulates transcription in a celltype-specific manner. We have compared the effects of RU486 on PR-dependent transcription in vitro using T47D and HeLa cell nuclear extracts. RU486 exhibits a differential ability to activate transcription within these two cell types. The differential effect on transcription correlates with different ratios of endogenous coactivators͞corepressors in these cells. Unlike agonist-bound PR that interacts only with coactivators such as steroid receptor coactivator-1 (SRC-1), RU486-bound PR binds to both coactivator SRC-1 and corepressor silencing mediator for retinoid and thyroid hormone receptor (SMRT) in vitro. Both SRC-1 and SMRT have the capacity to modulate RU486-dependent activity. Moreover, a change in the relative levels of SRC-1 and SMRT contained in our chromatin transcription system modulates agonist͞antagonist effects of RU486 on transcription by PR. Our data indicate that the ability of RU486 to activate transcription is modulated by the ratio of coactivators to corepressors and substantiate the important roles of coregulators in the regulation of steroid receptor mediated transactivation in response to selective receptor modulators. Biological actions of progesterone are mediated primarily by the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors (1-3). In the absence of progesterone, PR exists in a transcriptionally inactive form associated with heat shock proteins (4). On hormone binding, the receptor undergoes a conformational change, resulting in dissociation from heat shock proteins, translocation to the nucleus, and dimerization and binding to progesteroneresponsive elements (PREs) within the promoter regions of target genes (1-3). When bound to the PRE, the receptor can modulate target gene transcription by directly contacting components of the transcriptional machinery (5) or indirectly by means of coactivators, such as steroid receptor coactivator-1 (SRC-1) (6) and p300͞CBP (CREB-binding protein) (7).Progesterone antagonists are synthetic pharmaceutical agents that suppress the transcriptional activity of the natural steroid agonist, progesterone (8, 9). They are used in the treatment of a variety of endocrine disorders as well as certain hormonedependent tumors (8, 9), and exhibit a spectrum of activity ranging from pure antagonists to mixed antagonists. The pure antagonists, such as onapristone (ZK98299), are generally incapable of activating transcription and fully antagonize PR functions. The molecular basis of the effects of ZK98299 remains unclear (10-14). In contrast, mixed antagonists, known as selective progesterone receptor modulators (SPRMs) (15), may stimulate P...

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Induction of the acrosome reaction in dog sperm cells is dependent on epididymal maturation: The generation of a functional progesterone receptor is involved

et al. 2001

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In the current study we investigated the progesterone receptor exposure on the sperm from the testis and different parts of the epididymis, the relation to the sperm maturation stage, the functionality of the progesterone receptor and the capacity of sperm to undergo acrosome reaction. Exposed progesterone receptors on spermatozoa were detected using Progesterone–BSA conjugate labeled with fluorescein isothiocyanate (P‐BSA‐FITC) or a monoclonal antibody against progesterone receptor, C‐262. Either progesterone or calcium ionophore was used to induce acrosome reaction. A high percentage (69 ± 8%; mean ± SD) of spermatozoa from the cauda epididymis showed P‐BSA‐FITC labeling at the onset of incubation, whereas only 0.1 ± 1 and 4 ± 2%, of spermatozoa from the testes, caput, and corpus epididymis, respectively, were labeled. There was no significant increase in P‐BSA‐FITC binding during the course of a 6 hr incubation. Treatment with either 10 μM progesterone or 5 μM calcium ionophore induced acrosome reaction in cauda epididymal sperm but not in testicular sperm, caput or corpus epipidymal sperm. It is concluded that the matured sperm of the dog from cauda epididymis and freshly ejaculated sperm demonstrate a functional membrane‐bound progesterone receptor while less matured spermatozoa from the testicle, caput, and corpus epididymis fail to demonstrate such a receptor. Acrosome reaction of dog sperm can be induced using either progesterone or calcium ionophore; however, the maturation stages of spermatozoa influence this occurrence. Mol. Reprod. Dev. 58:451–459, 2001. © 2001 Wiley‐Liss, Inc.

show abstract

Ligands induce conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody.

Cited by 41 publications

References 34 publications

Ligand-induced Conformational Alterations of the Androgen Receptor Analyzed by Limited Trypsinization

Ligand-induced Conformational Alterations of the Androgen Receptor Analyzed by Limited Trypsinization

Coactivator/corepressor ratios modulate PR-mediated transcription by the selective receptor modulator RU486

Induction of the acrosome reaction in dog sperm cells is dependent on epididymal maturation: The generation of a functional progesterone receptor is involved

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