Dominant negative regulation of trans-activation by the rat androgen receptor: roles of the N-terminal domain and heterodimer formation.
A series of deletion mutants was constructed for the rat androgen receptor (AR) to delineate sequences involved in transcriptional activation. Using transient expression conditions in CV-1 cells and in vitro DNA-binding studies, the amino-terminal domain of the receptor was shown to contain a region (residues 147-296) that is mandatory for trans-activation. Receptors with deletions (residues 147-408) in the N-terminal domain, but with intact DNA- and ligand-binding domains, interacted in vitro with androgen-responsive elements albeit with affinities lower than that of the wild type receptor. Coexpression of N-terminal deletion mutants (delta 46-408 and delta 38-296) with the wild type AR blunted trans-activation by the latter protein in a dominant fashion. By contrast, a hormone-binding-deficient receptor (delta 788-902) that had poor intrinsic activity potentiated the trans-activation by the native receptor. Mechanisms by which deletion mutants in the N-terminal region abolish the function of the wild type protein appear to involve heterodimer formation during interaction with DNA and direct competition for available binding sites on DNA, rather than squelching of accessory proteins. In contrast to impaired trans-activation, binding of the ligand to N-terminal deletion mutants brought about conformational changes that were comparable in wild type and mutant forms, as judged by electrophoretic mobility shift assays. Taken together, these data have specified a region in the N-terminal domain of the AR that plays a decisive role in transcriptional regulation.
Palvimo
J J
JJ
Department of Physiology, University of Helsinki, Finland.
Kallio
P J
PJ
Ikonen
T
T
Mehto
M
M
Jänne
O A
OA
D000818:Animals; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D013196:Dihydrotestosterone / Q000378:metabolism; D005799:Genes, Dominant; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats / Q000235:genetics*; D011944:Receptors, Androgen / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D013196:Dihydrotestosterone; D004247:DNA