Abstract
The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH(2)-terminal region which is not conserved. Recent biochemical and crystallographic studies revealed the molecular basis of AF2 is hormone-dependent recruitment of LXXLL motif-containing coactivators, including the p160 family, to a hydrophobic cleft in the ligand binding domain. Our previous studies demonstrated that AF2 in the androgen receptor (AR) binds only weakly to LXXLL motif-containing coactivators and instead mediates an androgen-dependent interaction with the AR NH(2)-terminal domain required for its physiological function. Here we demonstrate in a mammalian two-hybrid assay, glutathione S-transferase fusion protein binding studies, and functional assays that two predicted alpha-helical regions that are similar, but functionally distinct from the p160 coactivator interaction sequence, mediate the androgen-dependent, NH(2)- and carboxyl-terminal interaction. FXXLF in the AR NH(2)-terminal domain with the sequence (23)FQNLF(27) mediates interaction with AF2 and is the predominant androgen-dependent interaction site. This FXXLF sequence and a second NH(2)-terminal WXXLF sequence (433)WHTLF(437) interact with different regions of the ligand binding domain to stabilize the hormone-receptor complex and may compete with AF2 recruitment of LXXLL motif-containing coactivators. The results suggest a unique mechanism for AR-mediated transcriptional activation.
Highlights
The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH2-terminal region which is not conserved
Because LXXLL motifs mediate the interaction of p160 coactivators with AF2 of nuclear receptors [11,12,13,14,15,16], it raised the possibility that an LXXLL-like motif [13,14,15] in the androgen receptor (AR) NH2-terminal region has a similar function to mediate the N/C interaction
We present evidence that the N/C interaction, referred to in the literature as an interaction between AF1 and the ligand binding domain (LBD) or AF1 and AF2 and initially described for AR [27, 39] and the estrogen receptor [40], is direct, androgen-dependent, and mediated predominantly by the core sequence FXXLF in the AR NH2-terminal region. Both mammalian two-hybrid interaction and Glutathione S-transferase (GST) fusion protein binding studies indicate that NH2-terminal sequence 23FQNLF27 mediates interaction with the AF2 region in the LBD in an androgen-dependent manner
Summary
The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH2-terminal region which is not conserved. This FXXLF sequence and a second NH2-terminal WXXLF sequence 433WHTLF437 interact with different regions of the ligand binding domain to stabilize the hormone-receptor complex and may compete with AF2 recruitment of LXXLL motif-containing coactivators.
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