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Abstract
Progesterone receptors (PR) contain three activation functions (AFs) that together define the extent to which they regulate transcription. AF1 and AF2 are common to the two isoforms of PR, PR-A and PR-B, whereas AF3 lies within the N-terminal 164 amino acids unique to PR-B, termed the "B-upstream segment" (BUS). To define the BUS regions that contribute to AF3 function, we generated a series of deletion and amino acid substitution mutants and tested them in three backgrounds as follows: BUS alone fused to the PR DNA binding domain (BUS-DBD), the entire PR-B N terminus linked to its DBD (NT-B), and full-length PR-B. Analyses of these mutants identified two regions in BUS whose loss reduces AF3 activity by more than 90%. These are associated with amino acids 54-90 (R1) and 120-154 (R2). R1 contains a consensus (55)LXXLL(59) motif (L1) identical to ones found in nuclear receptor co-activators. R2 is adjacent to a second nuclear receptor box (L2) at (115)LXXLL(119) and contains a conserved tryptophan (Trp-140). Their mutation completely disrupts AF3 activity in a promoter and cell type-independent manner. Critical mutations elicited similar effects on all three B-receptor backgrounds. This underscores the probability that these mutations alter a process linking BUS structure to the function of full-length PR-B in a fundamental way.
Highlights
Progesterone receptors (PR) contain three activation functions (AFs) that together define the extent to which they regulate transcription
To define the B-upstream segment” (BUS) regions that contribute to AF3 function, we generated a series of deletion and amino acid substitution mutants and tested them in three backgrounds as follows: BUS alone fused to the PR DNA binding domain (BUS-DNA binding domains (DBD)), the entire PR-B N terminus linked to its DBD (NT-B), and full-length PR-B
Transcription by the Wild-type BUS Constructs—The present studies analyze the activity of BUS, amino acids 1–164 of human PR-B, in three different backgrounds (Fig. 1A) as follows: BUS-DBD, which contains BUS linked directly upstream of the PR DBD and NLS; NT-B, which contains the entire N terminus of PR-B including the DBD and NLS; and full-length PR-B
Summary
We have postulated that AF3 in BUS plays an important role, and we have compared the structures of N-terminal A (NT-A) and N-terminal B (NT-B), which are constitutively active forms of PR lacking only the HBD of the full-length receptors [23, 24], in an effort to explain their functional differences. These studies indicate that the N termini exist in a non-globular, extended conforma-. We discuss the implications of this for BUS structure, and we speculate that BUS is involved in protein-protein interactions that generate AF3 function
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