Domain interactions between coregulator ARA(70) and the androgen receptor (AR).
The coregulator function of AR-associated protein 70 (ARA(70)) was investigated to further characterize its interaction with the AR. Using a yeast two-hybrid assay, androgen-dependent binding of ARA(70) deletion mutants to the AR ligand-binding domain (LBD) was strongest with ARA(70) amino acids 321-441 of the 614 amino acid ARA(70) protein. Mutations adjacent to or within an FxxLF motif in this 120-amino acid region abolished androgen-dependent binding to the AR-LBD both in yeast and in glutathione-S-transferase affinity matrix assays. Yeast one-hybrid assays revealed an intrinsic ARA(70) transcriptional activation domain within amino acids 296-441. In yeast assays the ARA(70) domains for transcriptional activation and for binding to the AR-LBD were inhibited by the C-terminal region of ARA(70). Full-length ARA(70) increased androgen-dependent AR transactivation in transient cotransfection assays using a mouse mammary tumor virus-luciferase reporter in CV1 cells. ARA(70) also increased constitutive transcriptional activity of an AR NH(2)-terminal-DNA binding domain fragment and bound this region in glutathione-S-transferase affinity matrix assays. Binding was independent of the ARA(70) FxxLF motif. The results identify an ARA(70) motif required for androgen-dependent interaction with the AR-LBD and demonstrate that ARA(70) can interact with the NH(2)-terminal and carboxyl-terminal regions of AR.
Zhou
Zhong-xun
ZX
Department of Pediatrics, The Laboratories for Reproductive Biology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7500, USA.
He
Bin
B
Hall
Susan H
SH
Wilson
Elizabeth M
EM
French
Frank S
FS
D020816:Amino Acid Motifs; D000728:Androgens / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D015723:Gene Library; D006801:Humans; D008297:Male; D056918:Nuclear Receptor Coactivators; D015513:Oncogene Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D020798:Two-Hybrid System Techniques; D015003:Yeasts
D000728:Androgens; C111382:NCOA4 protein, human; D056918:Nuclear Receptor Coactivators; D015513:Oncogene Proteins; D011944:Receptors, Androgen; D011965:Receptors, Glucocorticoid; D011980:Receptors, Progesterone; D015534:Trans-Activators; D014157:Transcription Factors