Mutations in the androgen receptor (AR) are thought to cause complete androgen insensitivity (CAIS) in46, XY human subjects who have a female phenotypedespite normal adult male concentrations of plasmatestosterone. Assays of AR binding in cultured skinfibroblasts from subjects with CAIS show either anapparent absence of AR (AR-) or normal levels ofAR (AR+) binding. In several subjects with CAIS, AR-, no gross AR mutation was detected by Southern blot analyses of genomic DNA and normal sized10 kilobase mRNA was present on Northern blots ofpoly(A+) RNA from cultured genital skin fibroblasts.We have used the polymerase chain reaction toamplify individual exons within the human AR geneof subjects with CAIS and have identified point mutations in three subjects. In one AR- subject(R774C), amino acid 774 was changed from arginine(CGC) to cysteine (TGC), in another AR- subject(R831Q), arginine (CGA) was changed to glutamine(CAA) at position 831, and in an AR+ subject(V866M) a methionine (ATG) was substituted forvaline (GTG) at position 866.Transfection of wild type and mutant AR cDNA clones into COS cells results in detection of ARprotein by immunoblotting. AR ligand binding activityis absent in cells transfected with AR mutants R774Cand R831Q, but present with AR mutant V866M.Androgen binding in cells transfected with AR mutant V866M has a 6-fold lower apparent bindingaffinity than that of wild-type AR. Transcriptionalactivation of the MMTV-CAT reporter gene was androgen dependent and specific and nearly maximal at physiological concentrations (10-10 M) of androgen when wild-type AR was transfected into cells, whereas neither AR mutants R774C nor R831Q were able to stimulate CAT activity even at 10-8 M androgen.AR mutant V866M was able to stimulate CAT activity but the androgen dose dependency was shifted toward pharmacological concentrations of steroid that exceed in vivo levels.The molecular basis of CAIS in humans exhibits genetic heterogeneity. Our study shows that some cases of CAIS are explained by an inability to form a functional AR-steroid complex and hence, the AR is unable to activate transcription of genes essential for male sex differentiation during fetal development.
ASJC Scopus subject areas
- Molecular Biology