Androgen receptor in human skin fibroblasts characterization of a specific 17β-hydroxy-5α-androstan-3-one-protein complex in cell sonicates and nuclei

Bruce S. Keenan, Walter Meyer, Arthur J. Hadjian, Claude J. Migeon

Research output: Contribution to journalArticle

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Abstract

Cultured human skin fibroblasts were shown to contain an androgen binding activity (receptor) which was heat-labile and destroyed by trypsin. Specific binding was seen after incubations of these cells with 1,2-3H-testosterone, 1,2-3 H-17β-hydroxy-5α-androstan-3-one (dihydrotestosterone,DHT) and 1,2-3H-5α-androstane-Sα, 17β-diol. This receptor had a high affinity (Kd = 0.2-1.6 nM) and a high degree of specificity for DHT, It was measured as a 3H-DHT-protein complex by gel filtration chromatography using a method which distinguishes specific from nonspecific binding. Receptor activity was distributed about equally between nuclear and extranuclear components at all times studied and was present in both compartments when cell incubations were carried out at 4° and 37°. Saturation analysis indicated that there were 1250-18,600 binding sites per whole cell. By sucrose gradient centrifugatlon the receptor had a sedimentation coefficient (S20, w) of about 4. Cells grown for 8 days without serum in the medium maintained the same levels of3H-DHT binding. Within 15 hours puromycin (20 μg/ml) in serum-free medium caused a 40-60 percent decrease in binding for the same cell lines. Although the highest levels of 3H-DHT binding were observed in fibroblasts from newborn foreskin, appreciable cytosol and nuclear binding were seen in cells from forearm, neck and abdominal skin. Receptor activity was stable during prolonged culture. Fibroblasts from several skin sites from patients with the androgen insensitivity syndrome (testicular feminization) had no detectable specific DHT binding. In this study it was demonstrated that skin fibroblasts can rapidly convert testosterone to its active form, DHT, bind DHT to a specific receptor protein and transport this complex to their nuclei. Therefore this may prove to be a convenient system for studying androgen action in vitro.

Original languageEnglish (US)
Pages (from-to)535-552
Number of pages18
JournalSteroids
Volume25
Issue number4
DOIs
StatePublished - 1975
Externally publishedYes

Fingerprint

Dihydrotestosterone
Androgen Receptors
Fibroblasts
Cell Nucleus
Skin
Proteins
Androgen-Insensitivity Syndrome
Androgens
Cells
Foreskin
Puromycin
human AR protein
androstan-3-one
Serum-Free Culture Media
Protein Transport
Chromatography
Sedimentation
Cell culture
Forearm
Cytosol

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Molecular Biology

Cite this

Androgen receptor in human skin fibroblasts characterization of a specific 17β-hydroxy-5α-androstan-3-one-protein complex in cell sonicates and nuclei. / Keenan, Bruce S.; Meyer, Walter; Hadjian, Arthur J.; Migeon, Claude J.

In: Steroids, Vol. 25, No. 4, 1975, p. 535-552.

Research output: Contribution to journalArticle

Keenan, Bruce S. ; Meyer, Walter ; Hadjian, Arthur J. ; Migeon, Claude J. / Androgen receptor in human skin fibroblasts characterization of a specific 17β-hydroxy-5α-androstan-3-one-protein complex in cell sonicates and nuclei. In: Steroids. 1975 ; Vol. 25, No. 4. pp. 535-552.
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