A 3RD TRANSACTIVATION FUNCTION (AF3) OF HUMAN PROGESTERONE RECEPTORS LOCATED IN THE UNIQUE N-TERMINAL SEGMENT OF THE B-ISOFORM

Human progesterone target tissues contain two progesterone receptors: B-receptors (hPR(B)), which are 933 amino acids in length, and A-recep tors (hPR(A)), which lack the N-terminal 164 amino acids. The two isof orms differ functionally when they are occupied by agonists or antagon ists. We postulated that the unique 164-amino acid, B-upstream segment (BUS) is in part responsible for the functional differences between t he two isoforms and have constructed a series of hPR expression vector s 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), a nd AF2, located in the hormone-binding domain. BUS is a highly phospho rylated domain, and contains the serine residues responsible for the h PR(B) 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 o f chloramphenicol acetyltransferase (CAT) from a promoter containing t wo progesterone response elements (PRE(2)-TATA(tk)-CAT). Transcription levels with BUS-DBD-NLS are equivalent to those seen with full length hPR(B), and are higher than those seen with hPR(A). BUS specifically requires an intact hPR DBD to be transcriptionally active. DBD mutants that cannot bind DNA or whose DNA binding specificity has been switch ed to an estrogen response element cannot cooperate in BUS transcripti onal activity. The function of BUS-DBD-NLS is promoter and cell specif ic. It does not transactivate a CAT reporter driven by the mouse mamma ry tumor virus promoter in HeLa cells and poorly transactivates PRE(2) -TATA(tk)-CAT in PR-negative T47D breast cancer cells. However, in the breast cancer cells, BUS-DBD-NLS transactivation of PRE(2)-TATA(tk)-C AT 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 hPR(B) contains a unique th ird activation function (AF3) located within BUS and requiring the fun ctional 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 tr ansactivating actions of antiprogestin-occupied hPR(B), an issue of im portance in hormone-resistant breast cancers and in tissue-specific ag onist-like effects of hormone antagonists.