Agonistic and antagonistic activities of RU486 on the functions of the human progesterone receptor.

RU486 induced the binding to a palindromic progestin responsive element (PRE) in vitro of homo- and heterodimers of the human progesterone receptor (hPR) isoforms A and B present in T47D breast cancer cells or in HeLa cells transiently expressing the recombinant proteins. The resulting complexes were indistinguishable from those induced with the agonist R5020 with regard to specificity affinity and stability. Ligand exposure was a necessary prerequisite to observe PR/PRE complexes. Antagonist-induced complexes migrated more rapidly during electrophoresis than agonist-induced ones and no "mixed" PR/RU486- PR/R5020 complexes were observed suggesting that the dimerization interfaces of agonist- and antagonist-bound molecules are noncompatible. The analysis of a series of deletion mutants and chimeric receptors revealed the presence of 2 transcription activation functions (TAFs) located in the N-terminal region A/B (TAF-1) and the hormone binding domain (TAF-2). In the presence of agonist both TAFs were active in the HeLa cells. In the presence of RU486 TAF-2 was inactive while TAF-1 within the hPR form B/RU486 complex activated transcription from a reporter gene containing a single palindromic PRe. The authors consider this to be the most convincing evidence that the receptor/RU486-complex does bind to PREs in vivo. No transcriptional activation was observed in the presence of RU486 from a reporter gene containing the complex MMTV-LTR PRE. In contrast to hPR form B form A was not able to activate transcription from PRE/GRE-tk-CAT in the presence of RU486. In vivo competition between hPR/RU486 and either cPR/R5020 or the human glucocorticoid receptor/dexamethasone (hGR/Dex) complex further supported that of hPR/RU486 bound in vivo to its cognate responsive element. Indeed the observed inhibition of transcription was show to be due to competition for the MMTV PRE since no transcriptional interference by the hPR/RU486 was observed and since no heterodimers were formed between hPR/RU486 and cPR/R5020 or hGR/Dex. That the ligand-free hPR however was unable to complete demonstrated that ligand binding is the prerequisite for DNA binding of hPR in vivo. (authors)