Substitution of Aspartic Acid-686 by Histidine or Asparagine in the Human Androgen Receptor Leads to a Functionally Inactive Protein with Altered Hormone-Binding Characteristics

Abstract

We have identified two different single nucleotide alterations in codon 686 (GAC; aspartic acid) in exon 4 of the human androgen receptor gene in three unrelated families with the complete form of androgen insensitivity. One mutation (G----C) results in an aspartic acid----histidine substitution (with 15-20% of wild-type androgen-binding capacity), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (with normal androgen-binding capacity, but a rapidly dissociating ligand-receptor complex). The mutations eliminate a Hinfl restriction site. Screening for the loss of the Hinfl site in both families with the Asp----Asn mutation resulted in the recognition of heterozygous carriers in successive generations of each. Both mutant androgen receptors were generated in vitro and transiently expressed in COS and HeLa cells. The receptor proteins produced had the same altered binding characteristics as those measured in fibroblasts from the affected subjects. R1881-activated transcription of a GRE-tk-CAT reporter gene construct was strongly diminished by both mutant receptors and was only partially restored using a 100-fold higher concentration of ligand compared with wild-type receptor. Thus, aspartic acid-686 appears essential for normal androgen receptor function. Substitution of this amino acid residue, by either histidine or asparagine, results in androgen insensitivity and lack of androgen-dependent male sexual differentiation.

A Practical Approach to the Detection of Androgen Receptor Gene Mutations and Pedigree Analysis in Families with X-Linked Androgen Insensitivity

“…Immunoprecipitation and Western blot analysis of the AR protein obtained from approximately 5 x 106 genital skin fibroblasts were performed as described previously (8). The AR was immunoprecipitated from whole cell lysates of genital skin fibroblasts with the AR-specific MAb F39.4.1.…”

“…Patient 4 also displayed a normal binding capacity for androgens, but an increased dissociation rate of the ligand-receptor complex was observed (8).…”

Androgen receptor gene mutations in human prostate cancer.

“…Mutated ARs occur naturally as a result of AR gene mutations (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). AR gene mutations were first described in complete androgen insensitivity syndrome (CAIS), an X chromosome-linked inherited disorder that causes XY genotypic males to develop as phenotypic females because of defective AR (13)(14)(15)(16)(17)(18).…”

“…AR gene mutations were first described in complete androgen insensitivity syndrome (CAIS), an X chromosome-linked inherited disorder that causes XY genotypic males to develop as phenotypic females because of defective AR (13)(14)(15)(16)(17)(18). AR gene mutations in CAIS have been found in the N-terminal domain (17,18), the DNA binding domain (17), or the HBD (13)(14)(15)(16)(17). Each of these mutations inactivates AR function, even though some of the mutant AR proteins produced still bind androgen (15)(16)(17).…”

Substitution of arginine-839 by cysteine or histidine in the androgen receptor causes different receptor phenotypes in cultured cells and coordinate degrees of clinical androgen resistance.

“…In 1974 ( 1 ) several laboratories (2,3) began to characterize the androgen-binding activities in the genital skin fibroblasts (GSF) of 46,XY humans with various clinical expressions of androgen resistance (insensitivity), on the presumption that they had mutant ARs. More recently, the cloning of the AR cDNA (4-7) and the application of derivative molecular techniques have permitted us (8)(9)(10)(11)(12)(13)(14) and others (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) to try to correlate specific germline lesions in the androgen-binding region of X-linked AR gene with various dysfunctional behaviors of the AR and with their clinical consequences for the affected subjects. The ultimate aim of such correlative studies is to elucidate the stereochemistry that imparts androgen-binding specificity to an AR and the contribution of an androgenic ligand to the transcriptional regulatory attributes of an A-R complex.…”