A third transactivation function (AF3) of human progesterone receptors located in the unique N-terminal segment of the B-isoform.

Abstract

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.

An N-terminal Inhibitory Function, IF, Suppresses Transcription by the A-isoform but Not the B-isoform of Human Progesterone Receptors

“…Construction of BUS-DBD, N-terminal B (NT B )-DBD, N-terminal A (NT A )-DBD, DBD-HBD, and BUS-DBD-HBD expression vectors, all containing a nuclear localization signal (NLS), was described in Sartorius et al (5).…”

“…The 933-amino acid B-receptors contain an N-terminal 164-amino acid upstream segment (BUS) that is missing in the truncated 769-amino acid A-receptors (2)(3)(4)(5). The two PR isoforms have AF1 and AF2 in common (5,6). AF1 maps to a 91-amino acid "proline-rich" segment located just upstream of the DBD and AF2 is located in the HBD (6).…”

“…AF1 maps to a 91-amino acid "proline-rich" segment located just upstream of the DBD and AF2 is located in the HBD (6). BUS, restricted to B-receptors, contains AF3 (5). In general, B-receptors are stronger transactivators than A-receptors (5,(7)(8)(9), and only B-receptors can activate transcription in the presence of antiprogestins (9 -11).…”

“…BUS, restricted to B-receptors, contains AF3 (5). In general, B-receptors are stronger transactivators than A-receptors (5,(7)(8)(9), and only B-receptors can activate transcription in the presence of antiprogestins (9 -11). On the other hand, A-receptors can dominantly inhibit B-receptors (9,12,13) as well as other members of the steroid receptor family (14).…”

Crosstalk between steroid receptors and the c-Src-receptor tyrosine kinase pathways: implications for cell proliferation

“…The PR exists as either longer PR-B (120 kDa) or N-terminally truncated PR-A (97 kDa) forms that are transcribed from separate promoters on the same gene, and both proteins appear to be expressed in most target tissues (reviewed in Lange, 2004). PR-B contains an additional activation function (AF3) that is located in the extended Nterminal region, and is generally less transcriptionally active than PR-A (Sartorius et al, 1994;Norris et al, 1997).…”