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.