Melanoma antigen gene protein MAGE-11 regulates androgen receptor function by modulating the interdomain interaction

Gene activation by steroid hormone receptors involves the recruitment of the steroid receptor coactivator (SRC)/p160 coactivator LXXLL motifs to activation function 2 (AF2) in the ligand binding domain. For the androgen receptor (AR), AF2 also serves as the interaction site for the AR NH(2)-terminal FXXLF motif in the androgen-dependent NH(2)-terminal and carboxyl-terminal (N/C) interaction. The relative importance of the AR AF2 site has been unclear, since the AR FXXLF motif interferes with coactivator recruitment by competitive inhibition of LXXLL motif binding. In this report, we identified the X chromosome-linked melanoma antigen gene product MAGE-11 as an AR coregulator that specifically binds the AR NH(2)-terminal FXXLF motif. Binding of MAGE-11 to the AR FXXLF alpha-helical region stabilizes the ligand-free AR and, in the presence of an agonist, increases exposure of AF2 to the recruitment and activation by the SRC/p160 coactivators. Intracellular association between AR and MAGE-11 is supported by their coimmunoprecipitation and colocalization in the absence and presence of hormone and by competitive inhibition of the N/C interaction. AR transactivation increases in response to MAGE-11 and the SRC/p160 coactivators through mechanisms that include but are not limited to the AF2 site. MAGE-11 is expressed in androgen-dependent tissues and in prostate cancer cell lines. The results suggest MAGE-11 is a unique AR coregulator that increases AR activity by modulating the AR interdomain interaction.     

Dependence of selective gene activation on the androgen receptor NH2- and COOH-terminal interaction

The agonist-induced androgen receptor NH(2)- and COOH-terminal (N/C) interaction is mediated by the FXXLF and WXXLF NH(2)-terminal motifs. Here we demonstrate that agonist-dependent transactivation of prostate-specific antigen (PSA) and probasin enhancer/promoter regions requires the N/C interaction, whereas the sex-limited protein gene and mouse mammary tumor virus long terminal repeat do not. Transactivation of PSA and probasin response regions also depends on activation function 1 (AF1) in the NH(2)-terminal region but can be increased by binding an overexpressed p160 coactivator to activation function 2 (AF2) in the ligand binding domain. The dependence of the PSA and probasin enhancer/promoters on the N/C interaction for transactivation allowed us to demonstrate that in the presence of androgen, the WXXLF motif with the sequence (433)WHTLF(437) contributes as an inhibitor to AR transactivation. We further show that like the FXXLF and LXXLL motifs, the WXXLF motif interacts in the presence of androgen with AF2 in the ligand binding domain. Sequence comparisons among species indicate greater conservation of the FXXLF motif compared with the WXXLF motif, paralleling the functional significance of these binding motifs. The data provide evidence for promoter-specific differences in the requirement for the androgen receptor N/C interaction and in the contributions of AF1 and AF2 in androgen-induced gene regulation.     

Structural basis for androgen receptor interdomain and coactivator interactions suggests a transition in nuclear receptor activation function dominance

The androgen receptor (AR) is required for male sex development and contributes to prostate cancer cell survival. In contrast to other nuclear receptors that bind the LXXLL motifs of coactivators, the AR ligand binding domain is preferentially engaged in an interdomain interaction with the AR FXXLF motif. Reported here are crystal structures of the ligand-activated AR ligand binding domain with and without bound FXXLF and LXXLL peptides. Key residues that establish motif binding specificity are identified through comparative structure-function and mutagenesis studies. A mechanism in prostate cancer is suggested by a functional AR mutation at a specificity-determining residue that recovers coactivator LXXLL motif binding. An activation function transition hypothesis is proposed in which an evolutionary decline in LXXLL motif binding parallels expansion and functional dominance of the NH(2)-terminal transactivation domain in the steroid receptor subfamily.     

Androgen receptor ligand-binding domain interaction and nuclear receptor specificity of FXXLF and LXXLL motifs as determined by L/F swapping

The androgen receptor (AR) ligand-binding domain (LBD) binds FXXLF motifs, present in the AR N-terminal domain and AR-specific cofactors, and some LXXLL motifs of nuclear receptor coactivators. We demonstrated that in the context of the AR FXXLF motif many different amino acid residues at positions +2 and +3 are compatible with strong AR LBD interaction, although a preference for E at +2 and K or R at +3 was found. Pairwise systematic analysis of F/L swaps at +1 and +5 in FXXLF and LXXLL motifs showed: 1) F to L substitutions in natural FXXLF motifs abolished AR LBD interaction; 2) binding of interacting LXXLL motifs was unchanged or increased upon L to F substitutions; 3) certain noninteracting LXXLL motifs became strongly AR-interacting FXXLF motifs; whereas 4) other nonbinders remained unaffected by L to F substitutions. All FXXLF motifs, but not the corresponding LXXLL motifs, displayed a strong preference for AR LBD. Progesterone receptor LBD interacted with some FXXLF motifs, albeit always less efficiently than corresponding LXXLL motifs. AR LBD interaction of most FXXLF and LXXLL peptides depended on classical charge clamp residue K720, whereas E897 was less important. Other charged residues lining the AR coactivator-binding groove, K717 and R726, modulated optimal peptide binding. Interestingly, these four charged residues affected binding of individual peptides independent of an F or L at +1 and +5 in swap experiments. In conclusion, F residues determine strong and selective peptide interactions with AR. Sequences flanking the core motif determine the specific mode of FXXLF and LXXLL interactions.