Mapping the unique activation function 3 in the progesterone B-receptor upstream segment -: Two LXXLL motifs and a tryptophan residue are required for activity

Progesterone receptors (PR) contain three activation functions (AFs) that together define the extent to which they regulate transcription. AF I 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 (RI) and 120-154 (R2). RI contains a consensus (LXXLL59)-L-55 motif (LI) identical to ones found in nuclear receptor co-activators. R2 is adjacent to a second nuclear receptor box (L2) at (LXXLL119)-L-115 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.