Disrupted Amino- and Carboxyl-Terminal Interactions of the Androgen Receptor Are Linked to Androgen Insensitivity

Abstract

We have compared the functional consequences of seven single-point mutations in the ligand-binding domain (LBD) of the androgen receptor (AR). The mutations span helices 3 to 11 and are present in patients suffering from androgen insensitivity syndromes (AIS) and other male-specific disorders. The mutants, except M742V, bound to androgen response elements in vivo and in vitro and showed a testosterone-dependent conformational change. With regard to functional activity, the mutant M742V had severely blunted ability to transactivate or exhibit the androgen-dependent amino/carboxyl-terminal (N/C) interaction; mutants F725L, G743V, and F754L showed reduced transactivation potential and attenuated N/C interaction; and mutants V715M, R726L, and M886V had minor functional impairments. The mutants belonging to the first two groups also displayed reduced response to coexpressed GRIP1. In addition, mutations of amino acids M894 and A896 in the putative core activation domain 2 (AF2) in helix 12 confirmed that this helix is important for N/C interactions. Thus, amino acids located between helices 3 and 4 (F725 and R726), in helix 5 (M742, G743, and F754), and in helix 12 (M894 and A896) play critical roles in mediating the N/C interaction of AR. The data also show that disrupted N/C interaction is a potential molecular abnormality in AIS cases in which LBD mutations have not resulted in markedly impaired ability to bind androgen.

Androgen Receptor Alterations in Prostate Cancer Relapsed during a Combined Androgen Blockade by Orchiectomy and Bicalutamide

“…This observation indicates that different types of AR variants are developed and selected for during bicalutamide and flutamide treatments and supports the data by Han et al (2001), who suggested that changes in the hormonal environment may drive the selection of spontaneous somatic mutations that provide a growth advantage for CaP. Although the consequences of the detected mutations for AR function in vivo is beyond the scope of the present study and remains an issue for further study, based on previous reports on the function of AR variants found in prostate cancer Culig et al, 1993;McDonald et al, 2000;Sack et al, 2001;Taplin et al, 1995;Thompson et al, 2001;Wang et al, 2000;Zhao et al, 2000), it is tempting to predict that the amino acid changes in the ligand binding domain detected here alter the specificity of hormone binding, such that activation of transcription can take place in the presence of not only androgens, but also of other steroid hormones and antiandrogens used in therapy.…”

Androgen Receptor Mutations in High-Grade Prostate Cancer before Hormonal Therapy

“…In agreement with our previous results (Thompson et al, 2001), only minimal binding of AR to DNA was detectable in the absence of androgen in the culture medium, whereas extracts derived from cells grown in the presence of testosterone displayed strong AR-DNA complex formation (Fig. 1C).…”

“…All cell culture and transfections using PC-3 and COS-1 cells were performed essentially as described in Hyytinen et al (2002) and Thompson et al (2001).…”

“…Preparation of whole cell extracts for EMSA and immunoblot analysis of ARs were performed as described (Thompson et al, 2001). …”

Amino acids 3–13 and amino acids in and flanking the 23FxxLF27 motif modulate the interaction between the N‐terminal and ligand‐binding domain of the androgen receptor

“…Disruption of the N/C interaction by mutation of the 23 FxxLF 27 motif has a limited effect on full length AR transactivation function [20,Steketee,unpublished observation]. However, several AR LBD mutants with reduced or completely abolished N/C interaction have been found in androgen insensitivity patients [11,56,57]. Additionally, both N/C interaction and the transactivating function of the AR prostate cancer mutant T877A can be induced by natural low affinity ligands like progesterone or E 2 or the AR antagonist cyproterone acetate [18].…”