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 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.
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Laboratories for Reproductive Biology, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Kemppainen
J A
JA
Wilson
E M
EM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000728:Androgens / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D000882:Haplorhini; D007700:Kinetics; D008024:Ligands; D011944:Receptors, Androgen / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation
D000728:Androgens; D008024:Ligands; D011944:Receptors, Androgen