Human androgen receptor binding to the androgen response element of prostate specific antigen.
This study examined the in vitro interaction of the human androgen receptor with a putative androgen response element (ARE) in the promoter region of the prostate specific antigen (PSA) gene. To characterize the androgen receptor's interactions with its DNA response elements we expressed the full length human androgen receptor protein in a baculovirus expression system. The receptor was shown to be 110 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and was purified using ion-exchange column chromatography. Binding of the synthetic androgen R1881 to the unpurified recombinant receptor exhibited a kd of 7.6 nM by Scatchard analysis. In DNA gel electromobility shift assays the promoter region from PSA (a 313-bp fragment) was bound by the unpurified recombinant androgen receptor in a sequence-specific manner. An ARE-containing sequence from the promoter region of the PSA gene was synthesized as a 30-bp oligonucleotide and was shown to bind specifically to the human androgen receptor in gel electromobility shift assays by DNA competition and by antibody supershifts of the receptor-ARE complex. The specific binding of the insect cell expressed androgen receptor to its ARE was shown to occur even in the absence of androgen. Androgen receptors purified by ion-exchange chromatography were unable to bind to ARE, suggesting the presence of other factors required for DNA binding.
Luke
M C
MC
Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287.
Coffey
D S
DS
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000032:analysis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009036:Moths; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D017430:Prostate-Specific Antigen / Q000378:metabolism*; D011944:Receptors, Androgen / Q000378:metabolism*
D011944:Receptors, Androgen; D004247:DNA; D017430:Prostate-Specific Antigen