The use of androgen receptor amino/carboxyl-terminal interaction assays to investigate androgen receptor gene mutations in subjects with varying degrees of androgen insensitivity

Abstract

Five mutations in the ligand-binding domain (LBD) of the human androgen receptor (bAR) found in patients with varying degrees of androgen insensitivity syndrome (A/S) were investigated for their effects on receptor dynamics. These were Arg871Gly (mild), Ser814Asn (partial), Glu772Ala (partial), Val866Met (complete), and Arg774Cys (complete). Previous analysis showed that the mutant receptors exhibited near-normal kinetics, except Arg774Cys, which had severely reduced androgen binding, and Val866Met, which showed increased equilibrium dissociation constant (Kd) and elevated dissociation rate (k) values. Ser814Asn exhibited ligand-selective k values, i.e. increased for dihydrotestosterone and mibolerone, but normal for methyltrenolene. Using mammalian two-hybrid assays, hAR amino/carboxyl (N/C)-terminal interactions of the mutant receptors were analyzed in the presence and absence of the hAR coactivator transcription intermediary factor 2 (TIF2). The mutations conferred decreased bAR N/C-terminal interaction, i.e. mild (∼1.5-fold), partial (2-fold), and complete (10-fold), that mirrored the degree of AIS. All mutant LBDs showed a 2- to 3-fold increase in N/C-terminal interactions when TIF2 was cotransfected, although of a magnitude still less than that of wild-type LBD with TIF2. The ligand-selective properties of the Ser814Asn mutant were also clearly reflected by the N/C-terminal interactions. Thus, measurement of N/C-terminal interactions may assist in the molecular analysis of mutant hARs associated with AIS.