Specific sex steroid-binding sites are associated with the salt-insoluble nuclear matrix from which lipids, histones, and chromatin have been extracted. In intact cultured normal human genital skin fibroblasts incubated for 1 h at 37 C with a saturating concentration (2 nM) of [3H]dihydrotestoster one ([3H]DHT), approximately 50% of the total intracellular androgen receptor-steroid complexes were found in the nucleus. Within isolated nuclei from such cells, 28-49% of the specific androgen receptor binding was associated with the nuclear matrix. The antiandrogen cyproterone acetate inhibited DHT binding within the nuclear matrix. Cultured genital skin fibroblasts from two unrelated patients with receptor-positive complete androgen insensitivity (CAIS, AR+), had normal (∼50%) nuclear binding of DHT, and 35% and 45% of it was localized to the nuclear matrix. Genital skin fibroblasts from a patient with receptor-negative complete androgen insensitivity (CAIS, AR-) had no specific DHT binding in isolated nuclei or nuclear matrix. Scatchard analysis of specific DHT binding in the nuclear matrix isolated from cells of normal subjects after an in vitro exchange assay (0 C; 24 h) revealed the presence of saturable (maximum binding, ≅200 fmol/mg nuclear DNA), high affinity (Kd ≅ 1.0 nM) binding sites. By contrast, in the nuclear matrix isolated from cells of a patient CAIS, AR+, the binding affinity forDHT was 3- fold lower (Kd ≅ 3.0 nM). When cytosolic androgen receptor-DHT complexes prepared from cells preincubated at 37 C for 1 h with [3H]DHT were incubated at 0 C for 1 h with isolated nuclei and nuclear matrix in the presence of 0.15 M KC1, 40-60% of specific nuclear binding was associated with the nuclear matrix. In these cell-free in vitro experiments, radiolabeled DHT-receptor complexes prepared from normal or mutant cells were mixed with isolated nuclei and nuclear matrix prepared from cells of normal subjects or patients with CAIS, AR+ orCAIS, AR-. Under these conditions, specificDHT binding in nuclei and nuclear matrix was quantitatively similar in the presence of a mutant (CAIS, AR+) receptorsteroid complex or in the presence of nuclei or nuclear matrixfrom the mutant cells (CAIS, AR- or AR+) when compared simultaneously with the same subcellular fractions prepared from the cells of normal subjects. When the cytosol DHT receptor complexes formed in vitro at 0 C (4 h) were added inincreasing amounts to the isolated nuclear matrix and binding was determined after 1 h at 0 C, the binding affinity of the cytosolic receptor from cells of the patient with CAIS, AR+ was approximately one third that of the DHT-receptor complex from cells of normal subjects. In summary, the nuclear matrix of cultured genital skin fibroblasts contains specific, high affinity, saturable sites for androgen receptor binding, and high affinity occupancy of nuclear matrix binding sites by the intact steroid-receptor complex may be required for androgen biological activity. However, the experimental design did not allow us to differentiate between an alteration in the binding affinity of the receptor for the steroid or a defect in the binding affinity of the DHT-receptor complex for nuclear matrix-associated acceptor sites.
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Clinical Biochemistry
- Biochemistry, medical