Sonia Zoppi

Mutations in the ligand-binding domain of the androgen receptor gene cluster in two regions of the gene

We have analyzed the nucleotide sequence of the androgen receptor from 22 unrelated subjects with substitution mutations of the hormone-binding domain. Eleven had the phenotype of complete testicular feminization, four had incomplete testicular feminization, and seven had Reifenstein syndrome. The underlying functional defect in cultured skin fibroblasts included individuals with absent, qualitative, or quantitative defects in ligand binding. 19 of the 21 substitution mutations (90%) cluster in two regions that account for approximately 35% of the hormone-binding domain, namely, between amino acids 726 and 772 and between amino acids 826 and 864. The fact that one of these regions is homologous to a region of the human thyroid hormone receptor (hTR-beta) which is a known cluster site for mutations that cause thyroid hormone resistance implies that this localization of mutations is not a coincidence. These regions of the androgen receptor may be of particular importance for the formation and function of the hormone-receptor complex.

Complete testicular feminization caused by an amino-terminal truncation of the androgen receptor with downstream initiation.

We have characterized the molecular defect causing androgen resistance in two 46,XY siblings with complete testicular feminization. Although binding studies in genital skin fibroblasts showed a reduced Bmax, an increased dissociation rate of ligand, and an 8S peak of dihydrotestosterone binding on sucrose density gradient centrifugation, no immunoreactive androgen receptor (AR) was detected in immunoblots using anti-NH2-terminal antibodies, suggesting an abnormal amino terminus. Sequence analysis of the AR gene revealed a point mutation CAG-->TAG (Gln-->Stop) at nucleotide 340. In vitro mutagenesis studies suggest the synthesis of the mutant AR is initiated downstream of the termination codon at reduced levels and that each molecule is functionally impaired. These results define a novel mechanism causing androgen resistance: the combination of decreased amount and functional impairment of AR caused by an abnormality within the amino terminus of the receptor. These findings suggest that domains important to the in vivo function of the receptor reside within the amino terminus and that disruption of these domains can occur with only subtle effects on receptor binding. Identification of this mutation made it possible to identify the mutant allele within the family and to ascertain antenatally that it was not present in a 46,XY fetal sibling of the proband at 9 wk gestation.

Amino acid substitutions in the hormone-binding domain of the human androgen receptor alter the stability of the hormone receptor complex.

We have investigated the basis of androgen resistance in seven unrelated individuals with complete testicular feminization or Reifenstein syndrome caused by single amino acid substitutions in the hormone-binding domain of the androgen receptor. Monolayer-binding assays of cultured genital skin fibroblasts demonstrated absent ligand binding, qualitative abnormalities of androgen binding, or a decreased amount of qualitatively normal receptor. The consequences of these mutations were examined by introducing the mutations by site-directed mutagenesis into the androgen receptor cDNA sequence and expressing the mutant cDNAs in mammalian cells. The effects of the amino acid substitutions on the binding of different androgens and on the capacity of the ligand-bound receptors to activate a reporter gene were investigated. Substantial differences were found in the responses of the mutant androgen receptors to incubation with testosterone, 5 alpha-dihydrotestosterone, and mibolerone. In several instances, increased doses of hormone or increased frequency of hormone addition to the incubation medium resulted in normal or near normal activation of a reporter gene by cells expressing the mutant androgen receptors. These studies suggest that the stability of the hormone receptor complex is a major determinant of receptor function in vivo.

Expression and characterization of full-length and partial human androgen receptor fusion proteins. Implications for the production and applications of soluble steroid receptors in Escherichia coli

We have expressed fusion proteins encoding defined segments of the coding segment of the human androgen receptor (hAR) in Escherichia coli using the pGEX-2T expression vector. Large quantities of fusion proteins containing glutathione-S-transferase (GST) linked to the amino or carboxy terminal region of the receptor and a fusion protein containing the complete amino acid sequence of the androgen receptor were produced in soluble form. The GST hAR fusion proteins containing the hormone-binding domain of the androgen receptor exhibit high affinity specific binding for a variety of natural and synthetic androgens. Analysis of the binding properties of the complete and truncated androgen receptor fusion proteins revealed that the amino terminus affects the Kd of the fusion proteins for mibolerone (0.89 vs. 3.43 nM for the truncated and complete fusion proteins, respectively). Despite these differences, both the truncated and complete hAR fusion proteins exhibit a higher affinity for dihydrotestosterone than for testosterone, implying that the preferential affinity for dihydrotestosterone observed in androgen receptor prepared from native sources is a measure of the inherent structure of the hormone-binding domain. Furthermore, the ligand-receptor complex is stable, as the ligand is not easily displaced with unlabelled competitor and is stable to mild heat denaturation. Fusion proteins containing the DNA-binding domain demonstrate specific DNA binding, as evidenced by studies using segments of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) and synthetic glucocorticoid response elements. These studies establish that GST hAR fusion proteins exhibit physical properties similar to those of native androgen receptor. Affinity purification using a glutathione affinity resin and cleavage of the fusion proteins at a thrombin cleavage site permits a marked enrichment using a two-step purification. The use of such methods will facilitate the study of the normal and mutant receptor proteins.