Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate

Douglas A. Husmann, Carol M. Wilson, Michael J. Mcphaul, Wayne D. Tilley, Jean D. Wilson

Research output: Contribution to journalArticle

139 Citations (Scopus)

Abstract

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 immunoreactivity 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. Immunohistological evaluation of rat and human prostatic tissue using anti-C-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 immunohistochemical 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.

Original languageEnglish (US)
Pages (from-to)2359-2368
Number of pages10
JournalEndocrinology
Volume126
Issue number5
StatePublished - May 1990

Fingerprint

Androgen Receptors
Cell Nucleus
Prostate
Antibodies
Staining and Labeling
Dihydrotestosterone
Proteins
Salts
Peptides
COS Cells
Castration
Stromal Cells
Nuclear Proteins
Affinity Chromatography
Immunoblotting
Nucleic Acids
Transfection
Immune Sera
Amino Acid Sequence
Plasmids

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. / Husmann, Douglas A.; Wilson, Carol M.; Mcphaul, Michael J.; Tilley, Wayne D.; Wilson, Jean D.

In: Endocrinology, Vol. 126, No. 5, 05.1990, p. 2359-2368.

Research output: Contribution to journalArticle

Husmann, Douglas A. ; Wilson, Carol M. ; Mcphaul, Michael J. ; Tilley, Wayne D. ; Wilson, Jean D. / Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. In: Endocrinology. 1990 ; Vol. 126, No. 5. pp. 2359-2368.
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abstract = "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 immunoreactivity 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. Immunohistological evaluation of rat and human prostatic tissue using anti-C-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 immunohistochemical 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.",
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