FXXLF and WXXLF sequences mediate the NH2-terminal interaction with the ligand binding domain of the androgen receptor

The nuclear receptor superfamily members of eukaryotic transcriptional regu lators contain a highly conserved activation function 2 (AF2) in the hormon e binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH2-terminal region which is not conserved. Recent bioche mical and crystallographic studies revealed the molecular basis of AF2 is h ormone-dependent recruitment of LXXLL motif-containing coactivators, includ ing the p160 family, to a hydrophobic cleft in the ligand binding domain. O ur previous studies demonstrated that AF2 in the androgen receptor (AR) bin ds only weakly to LXXLL motif-containing coactivators and instead mediates an androgen-dependent interaction with the AR NH2-terminal domain required for its physiological function, Here we demonstrate in a mammalian two-hybr id assay, glutathione S-transferase fusion protein binding studies, and fun ctional assays that two predicted cu-helical regions that are similar, but functionally distinct from the p160 coactivator interaction sequence, media te the androgen-dependent, NH2- and carboxyl-terminal interaction. FXXLF in the AR NH2-terminal domain with the sequence (23)FQNLF(27) mediates intera ction with AF2 and is the predominant androgen-dependent interaction site. This FXXLF sequence and a second NH2-terminal WXYLF sequence (WHTLF437)-W-4 33 interact with different regions of the ligand binding domain to stabiliz e the hormone-receptor complex and may compete with AF2 recruitment of LXXL L motif-containing coactivators. The results suggest a unique mechanism for AR-mediated transcriptional activation.