Sartorius C A, Melville M Y, Hovland A R, Tung L, Takimoto G S, Horwitz K B
Department of Medicine and Pathology, University of Colorado Health Sciences Center, Denver 80262.
Mol Endocrinol. 1994 Oct;8(10):1347-60. doi: 10.1210/mend.8.10.7854352.
Human progesterone target tissues contain two progesterone receptors: B-receptors (hPRB), which are 933 amino acids in length, and A-receptors (hPRA), which lack the N-terminal 164 amino acids. The two isoforms differ functionally when they are occupied by agonists or antagonists. We postulated that the unique 164-amino acid, B-upstream segment (BUS) is in part responsible for the functional differences between the two isoforms and have constructed a series of hPR expression vectors encoding BUS fused to isolated down-stream functional domains of the receptors. These include the two transactivation domains: activation function-1 (AF1), located in a 90-amino acid segment just up-stream of the DNA-binding domain (DBD) and nuclear localization signal (NLS), and AF2, located in the hormone-binding domain. BUS is a highly phosphorylated domain, and contains the serine residues responsible for the hPRB triplet protein structure. The construct containing BUS-DBD-NLS binds tightly to DNA when aided by accessory nuclear factors. In HeLa cells, BUS-DBD-NLS strongly and autonomously activates transcription of chloramphenicol acetyltransferase (CAT) from a promoter containing two progesterone response elements (PRE2-TATAtk-CAT). Transcription levels with BUS-DBD-NLS are equivalent to those seen with full-length hPRB, and are higher than those seen with hPRA. BUS specifically requires an intact hPR DBD to be transcriptionally active. DBD mutants that cannot bind DNA or whose DNA binding specificity has been switched to an estrogen response element cannot cooperate in BUS transcriptional activity. The function of BUS-DBD-NLS is promoter and cell specific. It does not transactivate a CAT reporter driven by the mouse mammary tumor virus promoter in HeLa cells and poorly transactivates PRE2-TATAtk-CAT in PR-negative T47D breast cancer cells. However, in the breast cancer cells, BUS-DBD-NLS transactivation of PRE2-TATAtk-CAT can be reconstituted by either elevating cellular levels of cAMP or linking BUS and DBD to AF1 or AF2 of hPR, each of which alone is also inactive in these cells. We conclude that hPRB contains a unique third activation function (AF3) located within BUS and requiring the functional DBD of hPR. Depending on the promoter or cell tested, AF3 can activate transcription autonomously, or it can functionally synergize with AF1 or AF2. Autonomous AF3 function may explain the unexpected transactivating actions of antiprogestin-occupied hPRB, an issue of importance in hormone-resistant breast cancers and in tissue-specific agonist-like effects of hormone antagonists.
B受体（hPRB），长度为933个氨基酸；A受体（hPRA），缺少N端的164个氨基酸。当这两种异构体被激动剂或拮抗剂占据时，它们在功能上存在差异。我们推测，独特的164个氨基酸的B上游片段（BUS）部分导致了这两种异构体之间的功能差异，并构建了一系列hPR表达载体，这些载体编码与受体分离的下游功能域融合的BUS。这些功能域包括两个反式激活域：激活功能-1（AF1），位于DNA结合域（DBD）和核定位信号（NLS）上游的一个90个氨基酸的片段中；以及AF2，位于激素结合域中。BUS是一个高度磷酸化的结构域，含有负责hPRB三联体蛋白结构的丝氨酸残基。含有BUS-DBD-NLS的构建体在辅助核因子的帮助下能紧密结合DNA。在HeLa细胞中，BUS-DBD-NLS强烈且自主地激活来自含有两个孕酮反应元件（PRE2-TATAtk-CAT）的启动子的氯霉素乙酰转移酶（CAT）转录。BUS-DBD-NLS的转录水平与全长hPRB的相当，且高于hPRA的。BUS特别需要完整的hPR DBD才能具有转录活性。不能结合DNA或其DNA结合特异性已转换为雌激素反应元件的DBD突变体不能在BUS转录活性中发挥协同作用。BUS-DBD-NLS的功能具有启动子和细胞特异性。它在HeLa细胞中不能反式激活由小鼠乳腺肿瘤病毒启动子驱动的CAT报告基因，在PR阴性的T47D乳腺癌细胞中对PRE2-TATAtk-CAT的反式激活作用较弱。然而，在乳腺癌细胞中，通过提高细胞内cAMP水平或将BUS和DBD与hPR的AF1或AF2连接，可恢复BUS-DBD-NLS对PRE2-TATAtk-CAT的反式激活作用，而单独的AF1或AF2在这些细胞中也是无活性的。我们得出结论，hPRB在BUS内含有一个独特的第三激活功能（AF3），且需要hPR的功能性DBD。根据所测试的启动子或细胞的不同，AF3可以自主激活转录，或者与AF1或AF2在功能上协同作用。AF3的自主功能可能解释了抗孕激素占据的hPRB的意外反式激活作用，这在激素抵抗性乳腺癌以及激素拮抗剂的组织特异性激动剂样效应中是一个重要问题。
