We have developed polyclonal antibodies to two synthetic peptides corresponding to the amino-(N-)terminal or carboxyl-(C-)terminal segments of the human androgen receptor (hAR) protein, as deduced from the nucleic acid sequence of the androgen receptor cDNA. Immunoreactive antisera were identified by solid phase enzyme-linked immunosorbent assay and purified by peptide affinity chromatography. Specific immuno-reactivity with the hAR was confirmed by immunoblotting, using both a fusion protein produced in E. coli that contains the C-terminal 880-amino acid sequence of hAR and the full-length receptor protein produced in COS cells after transfection with a plasmid containing the entire hAR-coding region. Immunohis-tological evaluation of rat and human prostatic tissue using antiC-terminal or anti-N-terminal antibodies demonstrated similar patterns of specific staining of the nuclei of epithelial and stromal cells. Castration resulted in a decrease in the amount of nuclear AR detected in the rat prostate after a short time of exposure to anti-C-terminal antibodies (<4 h), but did not alter the level of specific staining obtained with anti-N-terminal antibodies. This decrease in nuclear staining using anti-C-terminal antibodies could be reversed by treating castrated animals with dihydrotestosterone. When longer times of exposure to the primary antibodies were used, high levels of nuclear staining were obtained with both types of antibodies in prostate specimens from castrate as well as intact rats. This immunohisto-chemical staining pattern contrasts with receptor measurements in rat prostate homogenates that indicate the partition of AR binding into the low salt (cytosolic) fraction in the castrate animal and into the high salt (nuclear) fraction in the intact animal. Our results suggest that the AR is predominantly a nuclear protein even in the absence of ligand and that dihydrotestosterone serves to tighten its association with the nucleus. These data also suggest that the immunoreactivity of anti-C-terminal antibodies is influenced by the presence of dihydrotestosterone, presumably via an alteration in the physical state of the receptor protein.
ASJC Scopus subject areas