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 (hAR) found in patients with varying degrees of androgen insensitivity syndrome (AIS) were investigated for their effects on receptor dynamics. These were Arg(871)Gly (mild), Ser(814)Asn (partial), Glu(772)Ala (partial), Val(866)Met (complete), and Arg(774)Cys (complete). Previous analysis showed that the mutant receptors exhibited near-normal kinetics, except Arg(774)Cys, which had severely reduced androgen binding, and Val(866)Met, which showed increased equilibrium dissociation constant (K(d)) and elevated dissociation rate (k) values. Ser(814)Asn 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 hAR N/C-terminal interaction, i.e. mild (approximately 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 Ser(814)Asn 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.

A novel mutation F826L in the human androgen receptor in partial androgen insensitivity syndrome; increased NH2-/COOH-terminal domain interaction and TIF2 co-activation

“…An affected NC-TDI and TIF2 interaction is also found for two other mutations close to AF2, G743V (PAIS) and V889M (CAIS) (He et al, 2006; A c c e p t e d M a n u s c r i p t 22 Thompson et al, 2001). In close proximity of G743, three mutations are reported in AIS subjects, R871G, S814N and V866M, which were found to have a defective NC-TDI (Ghali et al, 2003). The M745I mutated residue from a CAIS subject is part of the ligand binding pocket, and causes a defective NC-TDI and a defective interaction with ARA70 (Bonagura et al, 2007).…”

“…Mutations D695N, Y763C, E772A, R774H, R774C and Q798E from AIS subjects are all located on the surface of the LBD at a relatively large distance from AF2, but surprisingly all mutants display a defective NC-TDI (Ghali et al, 2003;Jaaskelainen et al, 2006). Three of these residues (D695, Y763 and R774) together with residues R752 and F754 have been suggested to form a new region for protein-protein interactions, although this is not supported by experimental data (Jaaskelainen et al, 2006).…”

A novel mutation (c.T3816 > C) in the androgen receptor gene in a 46,XY female patient with androgen insensitivity syndrome

“…Disrupted binding of the ligand may also affect the structure of the cofactorbinding groove and inhibit N/C interaction and cofactor interaction [20,21].…”

“…Furthermore, binding of proteins to the cofactor-binding groove might affect binding of the ligand [11]. There is evidence indicating that disrupted N/C interaction can serve as a mechanism for AIS also in cases where ligand binding is normal [12][13][14].…”

Androgen Receptor Exon 1 Mutation Causes Androgen Insensitivity by Creating Phosphorylation Site and Inhibiting Melanoma Antigen-A11 Activation of NH2- and Carboxyl-terminal Interaction-dependent Transactivation

“…MAGE-11 first appeared during the primate lineage in parallel with an AR NH 2 -terminal mutation from Ala-33 in less evolved mammals to Val-33 in human and nonhuman primates. Human AR Val-33 is required for the AR 23 FQNLF 27 FXXLF motif region to interact with MAGE-11 and for competitive inhibitory effects of the AR N/C interaction on AF2 activity that are relieved by MAGE-11 binding the AR FXXLF motif (19). This gain in regulatory control of human AR transactivation by MAGE-11 appears to be critical for normal human male genital development in utero.…”