Human androgen insensitivity mutation does not alter oligonucleotide recognition by the androgen receptor-dht complex

Terry R. Brown, Stephen W. Rothwell, Claude J. Migeon

Research output: Contribution to journalArticle

Abstract

We studied the binding of dihydrotestosterone-receptor complexes (DHT-R) from human genital skin fibroblasts to oligodeoxyribonucleotides and DNA. Following incubation of fibroblasts with 2 nM [3H]DHT (45 min, 37°C), DHT-R were prepared as total fibroblast sonicates (sonication of cells in 0.5 M KCl), intact fibroblast cytosol (100000 × g supernatant) or intact fibroblast nuclear extract (sonication of nuclei in 0.5 M KC1). DHT-R were also prepared by incubation of fractionated fibroblast cytosol with 4 nM [3H]DHT (4 h, 0°C). Optimal conditions were established for binding of DHT-R from total fibroblast sonicates to oligo-dT cellulose: 60 nun, 0°C, low salt (0.05-0.10 M KC1), linearity with DHT-R concentration, and nucleotide saturation. With total fibroblast sonicates the rank order of DHT-R binding was oligo-dT ≈ -dG > DNA > -dC >- -dA ≈ -dl. Intact fibroblast cytosol displayed a similar preference of DHT-R binding to oligo-dT and -dG but the binding was quantitatively higher than for total fibroblast sonicates, the binding for fractionated fibroblast cytosolic DHT-R formed at 0°C being quantitatively lower. However, binding of DHT-R from eytosol (0°C) to DNA-cellulose was equal to that for DHT-R from cytosol (37°C). Binding of DHT-R from intact fibroblast nuclear extracts was lower than for total fibroblast sonicates. Preparations from cells of patients with receptor-negative, complete androgen insensitivity lacked both DHT-R formation and specific oligonucleotide binding. Binding of oligonucleotides to DHT-R from cells of patients with receptor-positive, complete androgen insensitivity could not be distinguished from that of normal cells. These results suggest: (a) androgen receptor-steroid complexes recognize and bind to certain preferred deoxyribonucleotides; (b) various factors affect the quantitative binding of DHT-R from different cellular preparations to deoxyribonucleotides; and (c) neither qualitative nor quantitative abnormalities for DHT-R of complete androgen-insensitive patients were detectable from oligonucleotide or DNA binding.

Original languageEnglish (US)
Pages (from-to)215-231
Number of pages17
JournalMolecular and Cellular Endocrinology
Volume32
Issue number2-3
DOIs
StatePublished - 1983

Fingerprint

Androgen Receptors
Oligonucleotides
Androgens
Fibroblasts
Mutation
Cytosol
Deoxyribonucleotides
Sonication
DNA
Cells
Oligodeoxyribonucleotides

Keywords

  • androgen receptor
  • DNA binding
  • genital skin fibroblasts

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Human androgen insensitivity mutation does not alter oligonucleotide recognition by the androgen receptor-dht complex. / Brown, Terry R.; Rothwell, Stephen W.; Migeon, Claude J.

In: Molecular and Cellular Endocrinology, Vol. 32, No. 2-3, 1983, p. 215-231.

Research output: Contribution to journalArticle

Brown, Terry R. ; Rothwell, Stephen W. ; Migeon, Claude J. / Human androgen insensitivity mutation does not alter oligonucleotide recognition by the androgen receptor-dht complex. In: Molecular and Cellular Endocrinology. 1983 ; Vol. 32, No. 2-3. pp. 215-231.
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N2 - We studied the binding of dihydrotestosterone-receptor complexes (DHT-R) from human genital skin fibroblasts to oligodeoxyribonucleotides and DNA. Following incubation of fibroblasts with 2 nM [3H]DHT (45 min, 37°C), DHT-R were prepared as total fibroblast sonicates (sonication of cells in 0.5 M KCl), intact fibroblast cytosol (100000 × g supernatant) or intact fibroblast nuclear extract (sonication of nuclei in 0.5 M KC1). DHT-R were also prepared by incubation of fractionated fibroblast cytosol with 4 nM [3H]DHT (4 h, 0°C). Optimal conditions were established for binding of DHT-R from total fibroblast sonicates to oligo-dT cellulose: 60 nun, 0°C, low salt (0.05-0.10 M KC1), linearity with DHT-R concentration, and nucleotide saturation. With total fibroblast sonicates the rank order of DHT-R binding was oligo-dT ≈ -dG > DNA > -dC >- -dA ≈ -dl. Intact fibroblast cytosol displayed a similar preference of DHT-R binding to oligo-dT and -dG but the binding was quantitatively higher than for total fibroblast sonicates, the binding for fractionated fibroblast cytosolic DHT-R formed at 0°C being quantitatively lower. However, binding of DHT-R from eytosol (0°C) to DNA-cellulose was equal to that for DHT-R from cytosol (37°C). Binding of DHT-R from intact fibroblast nuclear extracts was lower than for total fibroblast sonicates. Preparations from cells of patients with receptor-negative, complete androgen insensitivity lacked both DHT-R formation and specific oligonucleotide binding. Binding of oligonucleotides to DHT-R from cells of patients with receptor-positive, complete androgen insensitivity could not be distinguished from that of normal cells. These results suggest: (a) androgen receptor-steroid complexes recognize and bind to certain preferred deoxyribonucleotides; (b) various factors affect the quantitative binding of DHT-R from different cellular preparations to deoxyribonucleotides; and (c) neither qualitative nor quantitative abnormalities for DHT-R of complete androgen-insensitive patients were detectable from oligonucleotide or DNA binding.

AB - We studied the binding of dihydrotestosterone-receptor complexes (DHT-R) from human genital skin fibroblasts to oligodeoxyribonucleotides and DNA. Following incubation of fibroblasts with 2 nM [3H]DHT (45 min, 37°C), DHT-R were prepared as total fibroblast sonicates (sonication of cells in 0.5 M KCl), intact fibroblast cytosol (100000 × g supernatant) or intact fibroblast nuclear extract (sonication of nuclei in 0.5 M KC1). DHT-R were also prepared by incubation of fractionated fibroblast cytosol with 4 nM [3H]DHT (4 h, 0°C). Optimal conditions were established for binding of DHT-R from total fibroblast sonicates to oligo-dT cellulose: 60 nun, 0°C, low salt (0.05-0.10 M KC1), linearity with DHT-R concentration, and nucleotide saturation. With total fibroblast sonicates the rank order of DHT-R binding was oligo-dT ≈ -dG > DNA > -dC >- -dA ≈ -dl. Intact fibroblast cytosol displayed a similar preference of DHT-R binding to oligo-dT and -dG but the binding was quantitatively higher than for total fibroblast sonicates, the binding for fractionated fibroblast cytosolic DHT-R formed at 0°C being quantitatively lower. However, binding of DHT-R from eytosol (0°C) to DNA-cellulose was equal to that for DHT-R from cytosol (37°C). Binding of DHT-R from intact fibroblast nuclear extracts was lower than for total fibroblast sonicates. Preparations from cells of patients with receptor-negative, complete androgen insensitivity lacked both DHT-R formation and specific oligonucleotide binding. Binding of oligonucleotides to DHT-R from cells of patients with receptor-positive, complete androgen insensitivity could not be distinguished from that of normal cells. These results suggest: (a) androgen receptor-steroid complexes recognize and bind to certain preferred deoxyribonucleotides; (b) various factors affect the quantitative binding of DHT-R from different cellular preparations to deoxyribonucleotides; and (c) neither qualitative nor quantitative abnormalities for DHT-R of complete androgen-insensitive patients were detectable from oligonucleotide or DNA binding.

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