title: Androgen receptor in human skin fibroblasts. Characterization of a specific 17beta-hydroxy-5alpha-androstan-3-one-protein complex in cell sonicates and nuclei.
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 1,2-3-H-testosterone, 1,2-3-H17beta-hydroxy-5alpha-androstan-3-one (dihydrotestosterone, DHT) and 1,2-3-H-5alpha-androstane-3alpha, 17beta-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 3-H-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 degrees and 37 degrees. Saturation analysis indicated that there were 1250-18,600 binding sites per whole cell. By sucrose gradient centrifugation 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 of 3-H-DHT binding. Within 15 hours puromycin (20 mug/ml) in serum-free medium caused a 40-60 percent decrease in binding for the same cell lines. Although the highest levels of 3-H-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.
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D002850:Chromatography, Gel; D013196:Dihydrotestosterone / Q000378:metabolism*; D004355:Drug Stability; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008297:Male; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D011956:Receptors, Cell Surface; D012867:Skin / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013696:Temperature; D013739:Testosterone / Q000378:metabolism*
chemical_list: D011506:Proteins; D011956:Receptors, Cell Surface; D013196:Dihydrotestosterone; D013739:Testosterone