The FXXLF motif mediates androgen receptor-specific interactions with coregulators.
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.
He
Bin
B
Laboratory for Reproductive Biology and the Department of Biochemistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Minges
John T
JT
Lee
Lori W
LW
Wilson
Elizabeth M
EM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D008024:Ligands; D010455:Peptides; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase