Disrupted amino- and carboxyl-terminal interactions of the androgen receptor are linked to androgen insensitivity

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.     

Evidence for DNA-binding domain--ligand-binding domain communications in the androgen receptor

DNA binding as well as ligand binding by nuclear receptors has been studied extensively. Both binding functions are attributed to isolated domains of which the structure is known. The crystal structure of a complete receptor in complex with its ligand and DNA-response element, however, has been solved only for the peroxisome proliferator-activated receptor γ (PPARγ)-retinoid X receptor α (RXRα) heterodimer. This structure provided the first indication of direct interactions between the DNA-binding domain (DBD) and ligand-binding domain (LBD). In this study, we investigated whether there is a similar interface between the DNA- and ligand-binding domains for the androgen receptor (AR). Despite the structural differences between the AR- and PPARγ-LBD, a combination of in silico modeling and docking pointed out a putative interface between AR-DBD and AR-LBD. The surfaces were subjected to a point mutation analysis, which was inspired by known AR mutations described in androgen insensitivity syndromes and prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S, and P766A induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-residing mutations K590A, K592A, and E621A lowered the ligand-binding but not the DNA-binding affinity. We therefore propose that these residues are involved in allosteric communications between the AR-DBD and AR-LBD.     

A hybrid ligand method for androgen receptor measurement

Existing techniques for androgen receptor (AR) assay are complicated by cross-reactivity of ligand binding affinities that can lead to incorrect estimation of receptor concentration. Two most frequently used ligands are [3H]dihydrotestosterone [( 3H]DHT) and [3H]methyltrienolone [( 3H]R1881), which in addition to binding to AR also bind to sex hormone binding globulin (SHBG; Kd = 1.5 nM) and progesterone receptors (PgR; Human Kd = 1 nM, rat Kd = 6 nM) respectively. Triamcinolone acetonide (TMA) is commonly used to block binding of [3H]R1881 to PgR, however at high concentrations TMA itself will bind AR (Kd = 7 microM). We have developed a hybrid ligand method for the measurement of AR in the presence of SHBG and PgR. This method used [3H]R1881 as the high specific activity labelled tracer and DHT as the unlabelled competitor of specific AR binding. Using this assay, 20% of human colorectal carcinomas were found to contain AR.     

Isolation and characterization of androgen receptor mutant,AR(M749L), with hypersensitivity to 17-beta estradiol treatment

Estrogens, primarily 17beta-estradiol (E(2)), may play important roles in male physiology via the androgen receptor (AR). It has already been shown that E(2) modulates AR function in LNCaP prostate cancer cells and xenograft CWR22 prostate cancer tissues. Using a molecular model of E(2) bound-AR-ligand binding domain (LBD) and employing site-directed mutagenesis strategies, we screened several AR mutants that were mutated at E(2)-AR contact sites. We found a mutation at amino acid 749, AR(M749L), which confers AR hypersensitivity to E(2). The reporter assays demonstrate that E(2) can function, like androgen, to induce AR(M749L) transactivation. This E(2)-induced AR mutant transactivation is a direct effect of the AR(M749L), because the transactivation was blocked by antiandrogens. The hypersensitivity of AR(M749L) to E(2) is not due to increased affinity of AR(M749L) for E(2), rather it may be due to the existence of the proper conformation necessary to maintain E(2) binding to the AR-LBD long enough to result in E(2)-induced transactivation. AR(M749L) transactivation can be further enhanced in the presence of AR coregulators, such as ARA70 and SRC-1. Therefore, amino acid 749 may represent an important site within the AR-LBD that is involved in interaction with E(2) that, when mutated, allows E(2) induction of AR transactivation.     

L712V mutation in the androgen receptor gene causes complete androgen insensitivity syndrome due to severe loss of androgen function

Inability to respond to the circulating androgens is named as androgen insensitivity syndrome (AIS). Mutations in the androgen receptor (AR) gene are the most common cause of AIS. A cause and effect relationship between some of these mutations and the AIS phenotype has been proven by in vitro studies. Several other mutations have been identified, but need to be functionally validated for pathogenicity. Screening of the AR mutations upon presumptive diagnosis of AIS is recommended. We analyzed a case of complete androgen insensitivity syndrome (CAIS) for mutations in the AR gene. Sequencing of the entire coding region revealed C > G mutation (CTT–GTT) at codon 712 (position according to the NCBI database) in exon 4 of the gene, resulting in replacement of leucine with valine in the ligand-binding domain of the AR protein. No incidence of this mutation was observed in 230 normal male individuals analyzed for comparison. In vitro androgen binding and transactivation assays using mutant clone showed approximately 71% loss of ligand binding and about 76% loss of transactivation function. We conclude that CAIS in this individual was due to L712V substitution in the androgen receptor protein.     

Ketoconazole binds to the human androgen receptor.

Ketoconazole, an imidazole anti-fungal agent, has often produced features of androgen deficiency including decreased libido, gynecomastia, impotence, oligospermia, and decreased testosterone levels, in men being treated for chronic mycotic infections. Based on these potent effects on gonadal function in vivo as well as previous work in vitro demonstrating affinity of ketoconazole for receptor proteins for glucocorticoids and 1,25(OH)2 vitamin D3 and for sex steroid binding globulin (SSBG), the binding of ketoconazole to human androgen receptors (AR) in vitro was also examined. Ketoconazole competition with [3H]methyltrienolone (R1881) for androgen binding sites in dispersed, intact cultured human skin fibroblasts was determined at 22 degrees C. Fifty percent displacement of [3H]R1881 binding to AR was achieved by 6.4 +/- 1.8 (SE) x 10(-5) M ketoconazole. Additional binding studies performed with ketoconazole in the presence of increasing amounts of [3H]R1881 showed that the interaction of ketoconazole with AR was competitive when the data were analyzed by the Scatchard method. It should be noted, however, that the dose of ketoconazole required for 50% occupancy of the androgen receptor is not likely to be achieved in vivo, at least in plasma. Finally, androgen binding studies performed with other imidazoles, such as clotrimazole, miconazole, and fluconozole, revealed that in this class of compounds only ketoconazole appears to interact with the androgen receptor. Ketoconazole appears to be the first example of a non-steroidal compound which binds competitively to both SSBG and multiple steroid hormone receptors, suggesting that the ligand binding sites of these proteins share some features in common.     

Glutamic acid 709 substitutions highlight the importance of the interaction between androgen receptor helices H3 and H12 for androgen and antiandrogen actions

The mutation of a single amino acid in the ligand binding domain of the human androgen receptor (AR) can induce functional abnormalities; for example, in androgen binding or interactions with coregulators. We report here on the structure/function analysis of the ARE709K substitution that is associated with partial androgen insensitivity syndrome. We introduced several mutations at position 709 and tested the consequences of these changes on AR structure and activity in the presence of androgen and antiandrogens. Our results demonstrate that a strong interaction between helix H12 and residue 709 in H3 is required to obtain a fully functional AR. We show that glutamic acid 709 can be replaced by a bulky tyrosine residue without significant effect on the activation by agonists. In contrast, smaller or linear residues that are unable to maintain a tight interaction with H12 induce a substantial loss of androgen-induced AR activity. We also show that the agonist activity of partial antiandrogens is dependent on the side-chain residue at position 709. Strikingly, the ARE709Y substitution causes the conversion of cyproterone acetate into a pure antiandrogen and bicalutamide into a partial agonist. Together, our structural and functional data reveal the key role of glutamic acid 709 in androgenic and antiandrogenic activities.     

Transient, ligand-dependent arrest of the androgen receptor in subnuclear foci alters phosphorylation and coactivator interactions

Here we report that mutations within the DNA-binding domain of AR, shown previously to inhibit nuclear export to the cytoplasm, cause an androgen-dependent defect in intranuclear trafficking of AR. Mutation of two conserved phenylalanines within the DNA recognition helix (F582, 583A) results in androgen-dependent arrest of AR in multiple subnuclear foci. A point mutation in one of the conserved phenylalanines (DeltaF582, F582Y) is known to cause androgen insensitivity syndrome (AIS). Both AIS mutants (DeltaF582, F582Y) and the export mutant (F582, 583A) displayed androgen-dependent arrest in foci, and all three mutants promoted androgen-dependent accumulation of the histone acetyl transferase CREB binding protein (CBP) in the foci. The foci correspond to a subnuclear compartment that is highly enriched for the steroid receptor coactivator glucocorticoid receptor-interacting protein (GRIP)-1. Agonist-bound wild-type AR induces the redistribution of GRIP-1 from foci to the nucleoplasm. This likely reflects a direct interaction between these proteins because mutation of a conserved residue within the major coactivator binding site on AR (K720A) inhibits AR-dependent dissociation of GRIP-1 from foci. GRIP-1 also remains foci-associated in the presence of agonist-bound F582, 583A, DeltaF582, or F582Y forms of AR. Two-dimensional phospho-peptide mapping and analysis with a phospho-specific antibody revealed that mutant forms of AR that arrest in the subnuclear foci are hypophosphorylated at Ser81, a site that normally undergoes androgen-dependent phosphorylation. Our working model is that the subnuclear foci are sites where AR undergoes ligand-dependent engagement with GRIP-1 and CBP, a recruitment step that occurs before Ser81 phosphorylation and association with promoters of target genes.     

Extracellular correction of the androgen-receptor transformation defect in two families with complete androgen resistance

We have characterized the cellular and extracellular phenotype of the mutant androgen receptor (AR) from two families who have complete androgen resistance despite a normal androgen-binding capacity (Bmax) in their genital skin fibroblasts (GSF). The cellular receptors fail to up-regulate their basal AR activity in response to prolonged incubation with 5 alpha-dihydrotestosterone (DHT), or with two synthetic androgens, methyltrienolone (MT) and mibolerone (MB), and form A-R complexes with increased equilibrium (Kd) and non-equilibrium (k) dissociation constants. In addition, they are thermolabile when recently dissociated, but not in their native state. A-R complexes made in normal or mutant cytosol at 4 degrees C elute from DEAE-Sephacel at approximately 0.25 M KCl (untransformed), with or without prior passage through Sephadex G-25; when made in cells at 37 degrees C, extracted with 0.4 M KCl in a buffer containing 10 mM Na2MoO4, and desalted by G-25, they elute at less than or equal to 0.1 M KCl. Normal KCl-extracted DHT- and MB-R complexes dissociate (37 degrees C) at the same slow, linear rate as their in-cell counterparts (transformed); the mutant ones dissociated more slowly than their rapidly-dissociating in-cell counterparts and, to a variable extent, nonlinearly-an early faster phase, a later slower (transformed). Thus, as judged by two conventional criteria of steroid-R complex transformation, the mutant A-R complexes can transform, possibly in two steps, under certain cell-free conditions. This behavior differentiates a class of structural AR mutations whose molecular definition awaits application of recombinant DNA techniques to the X-linked AR locus.     

Study of the impact of the T877A mutation on ligand‐induced helix‐12 positioning of the androgen receptor resulted in design and synthesis of novel antiandrogens

Antiandrogen flutamide, an antagonist of the wild‐type androgen receptor (AR), is used in the clinics for treating metastatic prostate cancer. However, the T877A mutated AR is paradoxically activated by hydroxyflutamide, an active form of flutamide. Despite of crystallographic studies, how the T877A mutation results in antagonist‐agonist conversion of hydroxyflutamide remains a puzzle. Here, started from a structural model of the apo form of AR ligand‐binding domain (AR‐LBD), we have investigated the impact of the T877A mutation on ligand‐induced helix‐12 positioning by replica‐exchange molecular dynamics (REMD) simulations with an unique protocol, which is capable of simulating the H12 dynamics and keeping the main body of AR‐LBD unchanged. Specifically, (i) we have computationally demonstrated that on the binding of hydroxyflutamide, the apo form of H12 rearranges into the agonistic form in the T877A mutant, but into the antagonistic forms in the wild‐type receptor, shedding light on hydroxyflutamide agonism/antagonism; (ii) By REMD simulations, we have predicted antiandrogen SC184 is a non‐agonist of the T877A mutant. This was confirmed by luciferase assays; and (iii) on the basis of the binding modes of hydroxyflutamide and SC184 from the simulations, we designed a novel flutamide derivative called SC333, which was subsequently predicted to be a pure antagonist of the T877A mutant. We then synthesized and experimentally confirmed SC333 is a pan‐antiandrogen effective against the wild‐type and the T877A and W741C mutated ARs, showing low micromolar cytotoxicity in LNCaP cells. Importantly, we demonstrated that distribution of the H12 conformations from REMD simulations is correlated with ligand agonist/antagonist activity. Proteins 2010. © 2009 Wiley‐Liss, Inc.