Meyer M E, Pornon A, Ji J W, Bocquel M T, Chambon P, Gronemeyer H
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique de l'INSERM, Faculté de Médecine, Strasbourg, France.
EMBO J. 1990 Dec;9(12):3923-32. doi: 10.1002/j.1460-2075.1990.tb07613.x.
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 respect 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 non-compatible. The analysis of a series of deletion mutants and chimeric receptors revealed the presence of two 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 agonists, both TAFs were active in 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. We consider this to be the most convincing evidence that the receptor/RU486-complex does in fact 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 hPR/RU486 bound in vivo to its cognate responsive element. Indeed, the observed inhibition of transcription was shown 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 compete, demonstrated that ligand binding is the prerequisite for DNA binding of hPR in vivo.
RU486可诱导人孕酮受体（hPR）A和B亚型的同二聚体和异二聚体在体外与人T47D乳腺癌细胞或瞬时表达重组蛋白的HeLa细胞中的回文孕酮反应元件（PRE）结合。就特异性、亲和力和稳定性而言，所形成的复合物与激动剂R5020诱导形成的复合物无法区分。配体暴露是观察PR/PRE复合物的必要前提条件。拮抗剂诱导的复合物在电泳过程中比激动剂诱导的复合物迁移得更快，并且未观察到“混合”的PR/RU486 - PR/R5020复合物，这表明激动剂结合分子和拮抗剂结合分子的二聚化界面不兼容。对一系列缺失突变体和嵌合受体的分析揭示了位于N端A/B区域（TAF - 1）和激素结合域（TAF - 2）的两种转录激活功能（TAF）。在激动剂存在的情况下，两种TAF在HeLa细胞中均具有活性。在RU486存在的情况下，TAF - 2无活性，而hPR B型/RU486复合物中的TAF - 1可激活来自含有单个回文PRE的报告基因的转录。我们认为这是受体/RU486复合物在体内确实与PRE结合的最有说服力的证据。在含有复合MMTV - LTR PRE的报告基因存在RU486的情况下未观察到转录激活。与hPR B型不同，在RU486存在的情况下，A型不能激活来自PRE/GRE - tk - CAT的转录。hPR/RU486与cPR/R5020或人糖皮质激素受体/地塞米松（hGR/Dex）复合物之间的体内竞争进一步支持了hPR/RU486在体内与其同源反应元件结合。实际上，观察到的转录抑制被证明是由于对MMTV PRE的竞争，因为未观察到hPR/RU486的转录干扰，并且hPR/RU486与cPR/R5020或hGR/Dex之间未形成异二聚体。然而，无配体的hPR无法竞争，这表明配体结合是hPR在体内与DNA结合的前提条件。
