A point mutation in the second zinc finger of the DNA-binding domain of the androgen receptor gene causes complete androgen insensitivity in two siblings with receptor-positive androgen resistance.

Abstract

We have analyzed the nucleotide sequence of complementary and genomic DNAs of the human androgen receptor (AR) gene in two siblings (patients 9006 and 9030) with receptor-positive complete androgen insensitivity (Rec(+)-CAI). Northern analysis indicated that mRNA of the AR was normal in size. However, its expression was relatively reduced in both patients. Consistent with the normal androgen-binding capacity (496 and 552 fmol/mg DNA for patients 9006 and 9030, respectively) but decreased DNA-binding ability (168 fmol/mg DNA) measured in genital skin fibroblasts, no mutation was found in both N-terminal and ligand-binding domains of the AR. However, a single base substitution (G-->A) was found in the second zinc finger of the DNA-binding domain at nucleotide 2372 of the AR cDNA in both cases. This resulted in the replacement of a highly conserved arginine residue (amino acid 614) by a histidine. When the mutated receptor plasmid was cotransfected into PC-3 cells together with the reporter chloramphenicol acetyltransferase gene, chloramphenicol acetyltransferase activity was not induced by 5 alpha-dihydrotestosterone treatment, confirming that the mutation renders the AR nonfunctional and can, therefore, be held responsible for the clinical features in these patients. These results highlight the importance of Arginine-614 in the second zinc finger of the DNA-binding domain of the AR in the protein-DNA interaction.

Increase of androgen-induced cell death and androgen receptor transactivation by BRCA1 in prostate cancer cells

“…1A, addition of the BRCA1 enhances the activity of wild-type AR (lane 2 vs. lane 4). Notably, BRCA1 cannot potentiate the transactivation of mutant ARR614H, which has lost the DNA binding capacity by an arginine-to-histidine substitution at amino acid residue 614 (lane 6 vs. lane 8) (11). Moreover, p53, the universal tumor suppressor (32), showed no effect on the AR transactivation (lane 2 vs. lane 3).…”

“…The N-terminal region with 555 amino acid residues contains the domain involved in the transcriptional activation of AR. The AR plays important roles in male sexual differentiation, prostate cell proliferation, and the progression of prostate cancer (9)(10)(11). The findings of transcriptional interference͞squelching of steroid receptors provided the concept of the existence of transcriptional coregulators to mediate steroid receptor function (12,13).…”

Structure-Function Analysis of Bag1 Proteins

“…The reporter pLCI plasmid contains the full-length mouse mammary tumor virus long terminal repeat sequence linked with the chloramphenicol acetyltransferase (CAT) gene (28,29). The pSG5-AR plasmid contains the cDNA for the wild-type AR (28).…”

Suppression of the Human Erythropoietin Gene Expression by the TR2 Orphan Receptor, a Member of the Steroid Receptor Superfamily

“…Cells were cultured in a humidified 5% CO 2 , 95% air incubator at 37°C. Transient transfection of the HepG2 cells plated at an initial density of 3 ϫ 10 5 /60-mm dish was carried out using a calcium phosphate-DNA precipitation method as described previously (44,45). To normalize the transfection efficiency, the ␤-galactosidase expression plasmid was cotransfected.…”