He Bin, Minges John T, Lee Lori W, Wilson Elizabeth M
Laboratory for Reproductive Biology and the Department of Biochemistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2002 Mar 22;277(12):10226-35. doi: 10.1074/jbc.M111975200. Epub 2002 Jan 4.
The androgen receptor (AR) activation function 2 region of the ligand binding domain binds the LXXLL motifs of p160 coactivators weakly, engaging instead in an androgen-dependent, interdomain interaction with an FXXLF motif in the AR NH(2) terminus. Here we show that FXXLF motifs are present in previously reported AR coactivators ARA70/RFG, ARA55/Hic-5, and ARA54, which account for their selection in yeast two-hybrid screens. Mammalian two-hybrid assays, ligand dissociation rate studies, and glutathione S-transferase adsorption assays indicate androgen-dependent selective interactions of these FXXLF motifs with the AR ligand binding domain. Mutagenesis of residues within activation function 2 indicates distinct but overlapping binding sites where specificity depends on sequences within and flanking the FXXLF motif. Mutagenesis of the FXXLF motifs eliminated interaction with the ligand binding domain but only modestly reduced AR coactivation in transcription assays. The studies indicate that the FXXLF binding motif is specific for the AR and mediates interactions both within the AR and with coregulatory proteins.
配体结合结构域的雄激素受体（AR）激活功能2区域与p160共激活因子的LXXLL基序结合较弱，而是与AR NH(2)末端的FXXLF基序发生雄激素依赖性的结构域间相互作用。我们在此表明，FXXLF基序存在于先前报道的AR共激活因子ARA70/RFG、ARA55/Hic-5和ARA54中，这解释了它们在酵母双杂交筛选中的入选原因。哺乳动物双杂交试验、配体解离速率研究和谷胱甘肽S-转移酶吸附试验表明，这些FXXLF基序与AR配体结合结构域存在雄激素依赖性的选择性相互作用。激活功能2内残基的诱变表明存在不同但重叠的结合位点，其特异性取决于FXXLF基序内及其侧翼的序列。FXXLF基序的诱变消除了与配体结合结构域的相互作用，但在转录试验中仅适度降低了AR共激活作用。这些研究表明，FXXLF结合基序对AR具有特异性，并介导AR内以及与共调节蛋白之间的相互作用。
