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

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.