Ar Structure And Function In The Prostate

The androgen hormone dihydrotestosterone (DHT) is the principal regulator of prostate development and growth during embryonal development and puberty (5). In the adult prostate, it is required for maintenance of tissue homeostasis and secretory function. The circulating steroid hormone, testosterone, diffuses into the cells and is there converted by the 5-a reductase enzymes to the more potent androgen, DHT (6). DHT and other androgen hormones present in the prostate cells bind to a high-affinity binding protein required for mediating their effects, the AR. It is a member of the superfamily of nuclear receptors, its closest relatives are the progesterone, glucocorticoid, and the other steroid hormone receptors (7,8). Binding of androgen hormones triggers activation of the receptor to a transcription factor that interacts with and regulates the activity of gene promoters containing androgen-responsive elements (AREs). Activation involves a cascade of activation processes and the interaction with other components, such as co-regulatory proteins and proteins of the cellular transcription machinery.

The AR is encoded by a single gene on the long arm of the X chromosome spanning approx 180 kb (see gene atlas at: and is inherited in an X-linked fashion. The gene shows the typical structure of the nuclear receptor genes, with eight exons encoding the large N-terminal domain, a central DNA-binding domain composed of two zinc finger elements, a hinge region, and the C-terminal ligand-binding domain (9-11) (Fig. 1). The AR gene promoter is characterized by a short GC-box and a homopurin stretch, but lacks TATA and CAAT boxes typical for many eukaryotic genes (12,13). Binding sites for the transcription factor SP1 and a cAMP-responsive element were characterized (14,15). Two messenger RNA (mRNA) species are transcribed, the major one is 10 kb and the minor one is 7 kb in size (13). The difference is in the large 3' untranslated region that follows the open-reading frame. The receptor protein has 919 amino acids, however, because of two polymorphic triplet repeats in exon 1, one encoding a polyglutamine (CAG) repeat and the second encoding a polyglycine (GCN) repeat with variable lengths, the individual size can vary (16,17). Short triplet repeat regions seem to be associated with a moderately increased risk to develop prostate cancer (18) and extension beyond 40 repeats is the underlying cause of spinal and bulbar muscular atrophy (19). The AR gene is a locus of frequent mutations (http// androgendb). Approximately 300 mutations have been identified, most of them in male pseudoher-maphroditism patients with various degrees of androgen insensitivity caused by loss of AR function (20,21). Approximately 70 AR mutations have been identified in prostate cancer cell lines and specimens. Many cause the AR to acquire promiscuous properties and are described in Chapter 5.

Inactive AR is associated with heat shock proteins, and the binding of the hormone to the ligand-binding domain initiates a cascade transforming the AR into an active transcription factor. Activation includes dissociation of heat shock proteins, hyperphosphorylation, conformational changes, translocation into the nucleus, dimerization, and association with co-modulatory proteins (22-25). Activated AR then interacts with the cellular transcription machinery regulating transcription of genes through binding to AREs (26). Gene regulation may be positive or negative depending on the promoter and cellular context (27-29).

Activated AR interacts with chromatin through its two central zinc finger motifs that are held in place by four conserved cysteine side chains coordinated to a zinc ion (30,31). This domain is the most highly conserved region among the steroid receptors. Nuclear magnetic resonance and X-ray diffraction analysis revealed that the interaction with the DNA occurs through a helix that fits into the major DNA groove (30-32). The N-terminal domain comprises approximately half of the protein and contains the activation function domain (AF)-1 . It brings about most of the transactivation activity, in contrast to other steroid receptors, the AF2 located in the C-terminus is weak in the AR (33-35).

Fig. 1. Androgen receptor (AR) structure. The AR is a ligand-activated transcription factor mediating the effect of androgen hormones. Binding of androgens to the hormone-binding domain in the C-terminus induces a cascade of activation steps resulting in transformation to an active transcription factor. With the central DNA-binding domain built-up of two zinc (Zn) finger motifs, it interacts and binds to AREs in the promoters of genes. Immediately after the second zinc finger, there is a nuclear localization signal (NLS) that is essential for nuclear uptake of the receptor. A short hinge region (H) separates the DNA from the ligand-binding domain at the C-terminus. The large N-terminal transactivation domain contains two polymorphic amino acid repeats encoded by triplet repeats in the AR gene that vary in size among individuals (polyQ and polyG). The N-terminal domain also harbors the main transactivation function, activation function domain (AF)-1. A second transactivation function is localized in the ligand-binding domain, AF2. Both AF units interact with each other and are involved in recruitment of co-modulator proteins and cofactors of the transcription machinery.

The encoding gene is located on the long arm of the X-chromosome, and malfunction is associated with three diseases: syndromes of male sex ambiguity caused by partial or complete loss of AR function; prostate cancer, in which escape from hormone-ablation therapy is associated with AR alterations that result in an autonomous AR activation; and, finally, spinal and bulbar muscular atrophy, which is characterized by late onset and progressive weakening of skeletal muscles caused by an extension of the CAG triplet repeat.

More than 70 mutations have been detected in prostate cancer tissue, xenografts, and cell lines, the vast majority of them missense mutations. Several of the mutations located in the ligand-binding domain were shown to generate promiscuous receptors. Mutations located in the N-terminus of the receptor seem to alter the interaction with co-regulator proteins.

However, for full transactivation, both domains are required, and there is a physical interaction between the AF1 region in the N-terminus and the AF2 region in the C-terminus, which is important for recruitment of some coactivator proteins (36,37).

Between the DNA-binding domain and the ligand-binding domain, the so-called hinge region provides a nuclear translocation signal, the function of which is essential for nuclear localization (38). In addition, this region also provides the interface for interacting proteins, such as filamin A or p21-activated kinase 6 (PAK) (39,40). The ligand-binding domain at the C-terminus of the receptor protein is structured in 12 a-helices and a P-sheet, and has a 3D structure very similar to the ligand-binding domains of other steroid receptors. The orientation of helix 12 differs depending on agonist or antagonist binding. It closes the ligand-binding pocket in case of an agonist and leaves the pocket open when an antagonist is bond (41). In mutant receptors that are activated by nonandrogenic steroids or anti-androgens, the orientation of helix 12 is the same as after androgen binding, thus, providing a structural explanation for the promiscuous behavior (42).

The ARE consensus element is made up of two imperfect palindrome 6 bp elements separated by a spacer of 3 nucleotides (Fig. 1) (43). This element is not specific for the AR, related steroid receptors such as progesterone and glucocorticoid receptors also bind to and activate transcription through this element. Androgen-specific regulation of these promoters requires additional cofactors acting in a cell- and tissue-specific manner (44,45). In several androgen target genes, the AREs contain direct hexamere repeats of the canonical sequence 5'-TGTTCT-3' (46-48).


Induction of programmed cell death induced by blockade of AR signaling is the fundamental treatment for non-organ-confined prostate cancer. This can be achieved through androgen withdrawal by surgical or chemical castration or interruption of androgen signal transmission by anti-androgens (49). Often, a combination of androgen withdrawal and receptor inhibition by anti-androgens is also applied to achieve total androgen blockade. Although initially effective in the vast majority of treated patients, essentially all tumors develop resistance after a mean time of approx 2 years. Research into the basics of this progression to a hormone-insensitive tumor state provided mounting evidence in the last decade that changes in AR signaling are crucially involved (50,51). The underlying mechanisms can be grouped into four major categories, increase of AR protein levels, gain of function mutations, ligand-independent receptor activation through signaling crosstalk, and changes in the interaction with transactivation co-modulators. These mechanisms are discussed in Chapters 4-7.


Escape from androgen ablation therapy is associated with AR gene amplification in approx 20 to 30% of patients (52,53). Moreover, increased AR protein levels were also found in tumors without gene amplification (54). Amplification and overexpression is the result of a selection process observed after escape from hormone therapy, but only very rarely in untreated tumors. Not surprisingly, increase of the receptor protein seems to enable tumor cells to survive in the condition of androgen deprivation and adapt to it. Upregulation of AR expression is the only gene expression change that is consistently found in xenograft tumor progression models (2). Interestingly, patients with an amplified AR gene showed a better response to second-line androgen ablation treatment, although this did not seem to bring about an advantage with regard to survival (55).


The second mechanism used by prostate cancer cells to escape therapy is mutating the AR. Contrary to AR mutations found in male pseudohermaphroditism patients, which cause partial or complete loss of function, mutations occurring in prostate cancer provide a gain of function. Approximately 80 mutations have been identified (56). In most cases, prostate cancer AR mutations result in exchange of a single amino acid, only few deletions or mutations that introduce premature stop codons or affect noncoding regions of the AR gene have been found. With only few exceptions, AR mutations in prostate cancer are somatic (57-59).

AR mutations have been found in advanced and metastatic tumors as well as in primary, untreated tumor tissue and also in cases of latent carcinoma (57,60-67). The predominant properties of mutant receptors found in prostate tumors are loss of androgen specificity and increased agonistic activation through nonandrogen steroid hormones and antiandrogens (Table 1). The best-studied example of this kind of AR mutation is AR T877A, initially detected in the LNCaP cell line and later also found in a number of prostate tumor specimens. In addition to androgens, the steroid hormones, estradiol

Table 1

Androgen Receptor Mutations in Prostate Cancer"

Amino acid position





Zinc finger 2

Duplication of exon 3

CWR22 xenograft

Decreased AR transcriptional activity

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