Characterization of alternative amino acid substitutions at arginine 830 of the androgen receptor that cause complete androgen insensitivity in three families

We have studied two different missense mutations at arginine-830 in exon 7 of the human androgen receptor (hAR) gene that cause complete androgen insensitivity (CAIS) in three families. These substitutions result from point mutations at nucleotide 2489: a G→T transversion causes Arg830Leu and a G→A transition causes Arg830Gln. Genital skin fibroblasts of the patients have negligible androgen-binding capacity. The mutations were recreated in an hAR cDNA expression vector that was transiently transfected into COS-1 cells. Both mutant androgen receptors have increased dissociation rate constants and apparent equilibrium rate constants when measured with 5α-dihydrotestosterone or the synthetic, nonmetabolizable androgens, miboterone or methyltrienolone. The mutant androgen-binding activities share a distinctive thermal misbehavior. At 37°C R830Q and R830L are about 40% and 10% of normal, respectively. At 22°C both mutants gain androgen binding while the normal decreases by 20% for R830Q the augmented value approaches 60% of the normal. During prolonged 18 h incubation at 37°C, androgen binding of the normal AR is stable while that of both mutants decreases by at least 85%. Both mutants have a very reduced ability to transactivate a cotransfected androgen-responsive reporter gene, but R830Q is better than R830L. We conclude that arginine-830 is important for A–R complex stability, and that its replacement by glutamine or leucine yields distinctive functional aberrations. The importance of arginine at residue 830 in the hAR is reflected in the fact that it occupies the homologous position in the hormone-binding domains of the receptors for progesterone, glucocorticoid, and mineralocorticoid; furthermore, in the estrogen receptor it is conservatively replaced by lysine.