c-Jun potentiates the functional interaction between the amino and carboxyl termini of the androgen receptor

The transactivation functions of the human androgen receptor (hAR) are regulated by several accessory factors that can be either positive or negative. One factor that has been previously shown to mediate hAR transactivation is the proto-oncoprotein c-Jun. The positive effect is a primary one, can be exerted by both endogenous and exogenous c-Jun, and requires multiple regions of c-Jun. However, the exact mechanism by which c-Jun exerts its enhancing function is unknown. In this study, we have used a mammalian two-hybrid system to ask if c-Jun influences the ligand-dependent amino- to carboxyl-terminal (N-to-C) interaction of hAR, which is thought to be responsible for the homodimerization of this receptor. Our results show that c-Jun enhances both hAR N-to-C terminal interaction and DNA binding in vitro. We have also tested a panel of c-Jun and c-Fos mutants for their activities on the N-to-C interaction, and the data demonstrate that the activities of these mutants parallel their activities on hAR transactivation. A mutation in the hAR activation function-2 (AF-2) abrogates N-to-C interaction, DNA binding, and transactivation, and these activities are not rescued by exogenous c-Jun. Interestingly, the p160 coactivator TIF2 can stimulate hAR N-to-C interaction, a finding consistent with the effect on hAR transactivation. These data strongly suggest that the hAR N-to-C interaction is the target of c-Jun action, and this activity requires a functional receptor AF-2.     

Cyclin D1: mechanism and consequence of androgen receptor co-repressor activity.

Androgen receptor regulation is pivotal for prostate growth and development. Activation of the receptor is dictated by association with androgen (ligand) and through interaction with co-activators and co-repressors. We have shown previously that cyclin D1 functions as a co-repressor to inhibit ligand-dependent androgen receptor activation. We demonstrate that cyclin D1 directly binds the N terminus of the androgen receptor and that this interaction is independent of ligand. Furthermore, we show that the interaction occurs in the nucleus and does not require the LXXLL motif of cyclin D1. Although two distinct transactivation domains exist in the N terminus (AF-1 and AF-5), the data shown support the hypothesis that cyclin D1 targets the AF-1 transactivation function. The constitutively active AF-5 domain was refractory to cyclin D1 inhibition. By contrast, cyclin D1 completely abolished androgen receptor activity, even in the presence of potent androgen receptor co-activators. This action of cyclin D1 at least partially required de-acetylase activity. Finally, we show that transient, ectopic expression of cyclin D1 results in reduced cell cycle progression in androgen-dependent LNCaP cells independent of CDK4 association. Collectively, our data support a model wherein cyclin D1 has a mitogenic (CDK4-dependent) function and an anti-mitogenic function (dependent on regulation of the AF-1 domain) that can collectively control the rate of androgen-dependent cellular proliferation. These findings provide insight into the non-cell cycle functions of cyclin D1 and provide the impetus to study its pleiotropic effects in androgen-dependent cells, especially prostatic adenocarcinomas.     

The amino terminus of the human AR is target for corepressor action and antihormone agonism

Antiandrogens inhibit the ligand-induced transactivation by the androgen receptor (AR) and have a widespread use in the treatment of prostate cancer but their mode of action is not fully understood. Here we show that the ability of the antiandrogen cyproterone acetate (CPA) to inhibit transactivation by the human AR (hAR) involves the corepressor SMRT (silencing mediator for retinoic acid and thyroid hormone receptor). We detect binding of SMRT to hAR when treating with the antiandrogen CPA, but not with the antihormones casodex or hydroxyflutamide. Interestingly, we find that SMRT binds to the N terminus of the hAR. Thereby, SMRT modulates the activity of hAR in receptor-negative CV1 cells. In addition, we have used receptor point mutants that exhibit normal transactivation potential and unchanged partial agonistic activity when treated with CPA, but lack both SMRT binding and SMRT-mediated inhibition of CPA-bound AR. This indicates that mechanisms involved in hAR-mediated transactivation are distinct from antihormone-induced receptor inactivation. Furthermore, we show that treatment of transfected cells with a cAMP analog or coexpression of the catalytic subunit of PKA, known to activate hAR, inhibits the binding of SMRT to the AR. This suggests that the association of SMRT with hAR is regulated at the level of cross-talk mechanisms and that ligand-independent receptor activation is due to corepressor dissociation. Taken together, we provide novel insights in AR regulation, antihormone action, and functional nuclear receptor-corepressor interaction.     

The human androgen receptor: Structure/function relationship in normal and pathological situations

Discrete functions have been attributed to precise regions of the human androgen receptor (hAR) by expression of deletion mutants in COS and HeLa cells. A large C-terminal domain constitutes the hormone-binding region and a central basis, cysteine-rich domain is responsible for DNA binding. In addition, separate domains responsible for transactivation and nuclear translocation have been identified. In LNCaP cells (a prostate tumor cell line) the hAR is a heterogeneous protein which is synthesized as a single 110 kDa protein, but becomes rapidly phosphorylated to a 112 kDa protein. Metabolic labeling experiments using radioactive orthophosphate also indicated that the hAR is a phosphoprotein. Structural analysis of the AR gene in LNCaP cells and in 46, XY-individuals displaying androgen insensitivity (AIS) has revealed several different point mutations. In LNCaP cells the mutation affects both binding specificity and transactivation by different steroids. In a person with complete AIS a point mutation was identified in the splice donor site of intron 4, which prevents normal splicing and activates a cryptic splice donor site in exon 4. The consequence is a functionally inactive AR protein due to an in-frame deletion in the steroid-binding domain. In two unrelated individuals with complete AIS, two different single nucleotide alterations in codon 686 (Asp) were found. Both mutations resulted in functionally inactive ARs due to rapidly dissociating hormone-AR complexes. It is concluded that the hAR is a heterogeneous phosphoprotein in which functional errors have a dramatic impact on phenotype and fertility of 46, XY-individuals.     

Suppression of androgen receptor signaling in prostate cancer cells by an inhibitory receptor variant

There is increasing evidence that sensitization of the androgen receptor (AR) signaling pathway contributes to the failure of androgen ablation therapy for prostate cancer, and that direct targeting of the AR may be a useful therapeutic approach. To better understand how AR function could be abrogated in prostate cancer cells, we have developed a series of putative dominant-negative variants of the human AR, containing deletions or mutations in activation functions AF-1, AF-5, and/or AF-2. One construct, AR inhibitor (ARi)-410, containing a deletion of AF-1 and part of AF-5 of the AR, had no intrinsic transactivation activity but inhibited wild-type AR (wtAR) in a ligand-dependent manner by at least 95% when transfected at a 4:1 molar ratio. ARi-410 was an equally potent inhibitor of gain-of-function AR variants. Ectopic expression of ARi-410 inhibited the proliferation of AR-positive LNCaP cells, but not AR-negative PC-3 cells. Whereas ARi-410 also marginally inhibited progesterone receptor activity, this was far less pronounced than the effect on AR (50% vs. 95% maximal inhibition, respectively), and there was no inhibition of either vitamin D or estrogen receptor activity. In the presence of ligand, ARi-410 interacted with wtAR, and both receptors translocated into the nucleus. Whereas the amino-carboxy terminal interaction was not necessary for optimal dominant-negative activity, disruption of dimerization through the ligand binding domain reduced the efficacy of ARi-410. In addition, although inhibition of AR function by ARi-410 was not dependent on DNA binding, the DNA binding domain was required for dominant-negative activity. Taken together, our results suggest that interaction between ARi-410 and the endogenous AR in prostate cancer cells, potentially through the DNA binding and ligand binding domains, results in a functionally significant reduction in AR signaling and AR-dependent cell growth.     

Structural characterization of the human androgen receptor ligand-binding domain complexed with EM5744, a rationally designed steroidal ligand bearing a bulky chain directed toward helix 12

Antiandrogens are commonly used to treat androgen-dependent disorders. The currently used drugs unfortunately possess very weak affinity for the human AR (hAR), thus indicating the need to develop new high-affinity steroidal antiandrogens. Our compounds are specially designed to impede repositioning of the mobile carboxyl-terminal helix 12, which blocks the ligand-dependent transactivation function (AF-2) located in the AR ligand-binding domain (ARLBD). Using crystal structures of the hARLBD, we first found that H12 could be directly reached from the ligand-binding pocket (LBP) by a chain positioned on the C18 atom of an androgen steroid nucleus. A set of 5alpha-dihydrotestosterone-derived molecules bearing various C18 chains were thus synthesized and tested for their capacity to bind hAR and act as antagonists. Although most of those having very high affinity for hAR were agonists, several very potent antagonists were obtained, confirming the structural importance of the C18 chain. To understand the role of the C18 chain in their agonistic/antagonistic properties, the structure of the hARLBD complexed with one of these agonists, EM5744, was determined at a 1.65-A resolution. We have identified new interactions involving Gln(738), Met(742), and His(874) that explain both the high affinity of this compound and the inability of its bulky chain to prevent the repositioning of H12. This structural information will be helpful to refine the structure of the chains placed on the C18 atom to obtain efficient H12-directed steroidal antiandrogens.