We have developed a series of in vitro models with which to evaluate the biological activity of estrogen receptor (ER) agonists and antagonists. Using a protease digestion assay we show that the conformational changes induced within ER are distinct for agonists and antagonists. However, this assay is unable to discriminate between pure antagonists like ICI164,384 and partial agonists such as 4-OH tamoxifen or keoxifene. Using a chimeric ER-VP16 construct, we demonstrate that both pure antagonists and partial agonists deliver ER to its DNA target within cells. However, the ability of the DNA-bound receptor to activate transcription in the presence of a given antagonist is dependent on cell and promoter context. These data, suggesting functional differences among ER antagonists, were confirmed by additional experiments demonstrating that their ability to modulate the transcriptional activity of a series of ER mutants is dramatically different. Depending on the cell and promoter context and the particular ER form expressed, 4-OH tamoxifen and the related compound, keoxifene, functioned as partial agonists. Importantly, the transcriptional profiles of these two compounds were dissimilar, suggesting that they are functionally different from each other and from ICI164,384, which does not display agonist activity under any context examined. Our results reveal functional differences between these clinically important antiestrogens and suggest that the distinct biologies manifest by these compounds in vivo relate to their ability to differentially regulate ER function.

Human progesterone receptor (PR) is phosphorylated on multiple serine residues (at least seven sites) in a manner that involves distinct groups of sites coordinately regulated by hormone and different kinases. Progress on defining a functional role for PR phosphorylation has been hampered both by the complexity of phosphorylation and the lack of simple, nonradioactive methods to detect the influence of ligands and other signaling pathways on specific PR phosphorylation sites in vivo. Toward this end, we have produced monoclonal antibodies (MAbs) that recognize specific phosphorylation sites within human PR including a basal site at Ser 190 (MAb P190) and a hormone-induced site at Ser 294 (MAb P294). Biochemical experiments showed the differential reactivity of the P190 and P294 MAbs for phosphorylated and unphosphorylated forms of PR. Both MAbs recognize specific phosphorylated forms of PR under different experimental conditions including denatured PR protein by Western blots and PR in its native conformation in solution or complexed to specific target DNA. As detected by Western blot of T47D cells treated with hormone for different times, hormone-dependent down-regulation of total PR and the Ser 190 phosphorylation site occurred in parallel, whereas the Ser 294 phosphorylation site was down-regulated more rapidly. This difference in kinetics suggests that the Ser 294 site is more labile than basal sites and is acted upon by distinct phosphatases. A strong preferential hormone-dependent phosphorylation of Ser 294 was observed on PR-B as compared with the amino-terminal truncated A form of PR. This was unexpected because Ser 294 and flanking sequences are identical on both proteins, suggesting that a distinct conformation of the N-terminal domain of PR-A inhibits phosphorylation of this site. That Ser 294 lies within an inhibitory domain that mediates the unique repressive functions of PR-A raises the possibility that differential phosphorylation of Ser 294 is involved in the distinct functional properties of PR-A and PR-B.

No abstract

Glucocorticoid receptor (GR) is expressed at essentially equal levels in almost all tissues and cell types. Remarkably, glucocorticoids themselves regulate transcription in vivo in both a promoter- and tissue-specific manner. Thus, specific systems must be in place to regulate receptor action within certain cells and at certain promoters. To address two specific aspects of these systems, we have analyzed promoter-specific activity of GR using two different, well studied promoters (termed simple and composite promoters) from which GR activates transcription. The simple promoter depends only on the receptor for glucocorticoid-responsive transcriptional activation, while GR activity at the composite promoter depends on additional transcription factors. We have compared the action of several GR ligands at these promoters and demonstrate fundamental differences in the activities of these ligands on receptor activity. Furthermore, these compounds induce unique conformational changes in receptor, resulting in promoter-specific receptor function. We have identified critical amino acid residues within GR which, when mutated, genetically distinguish the action of GR at these promoters. Taken together, the data indicate that the presence of only the receptor and the ligand is not sufficient to allow activation of transcription. An additional system of regulation influences receptor action in both a tissue- and promoter-selective fashion, suggesting that multiple, regulated surfaces of the receptor respond to the cellular environment and determine the spectrum of GR activities. These functional surfaces may be induced or regulated by ligand binding, by the DNA sequence to which receptor is bound, or by the nonreceptor factors resident at the promoter or in the tissue.

No abstract