The androgen receptor NH2-terminal domain harbors the major transcription activation functions. Within its 537 amino acids, two independent activation domains have been identified: activation function 1 (AF-1) (located between residues 101 and 370) that is essential for transactivity of full length AR, and activation function 5 (AF-5) (located between residues 360-485) that is required for transactivity of a constitutively active androgen receptor, which lacks its LBD [Figure 9] (54). Only large deletions and/or multiple amino acid substitutions within the androgen receptor NH2-terminal domain will affect transcription activity. Single amino acid substitutions within the androgen receptor NH2-terminus have not been observed to significantly impair androgen receptor function.
Figure 9. Functional domain structure of the human androgen receptor. The protein consists of several distinct functional domains: transcription Activation Functions (AF-1; AF-5; in the NH2-terminal domain; and AF-2 in the ligand binding domain); Coactivator interacting domains, Phosphorylation sites, a nuclear localisation signal, the DNA-binding domain, the hinge region and the ligand binding domain.
Another function of the androgen receptor NH2-terminal domain is its binding to the COOH-terminal LBD (55, 56). The NH2-terminal regions required for the binding of the LBD have been mapped to two essential units: the first 36 amino acids and residues 370-494 (57).
The hormone dependent interaction of the NH2-terminal domain with the ligand binding domain can play a role in stabilization of the androgen receptor dimer complex via intermolecular interactions and in stabilization of the ligand receptor complex (58). Several mutations in the ligand binding domain, detected in patients with the syndrome of androgen insensitivity, affect negatively the interaction of the NH2-terminal domain with the ligand binding domain, while androgen binding was impaired, indicating the importance of this interaction (59).
The DNA-binding domain is the best conserved among the members of the receptor superfamily [Figure 5, see above]. It is characterized by a high content of basic amino acids and by nine conserved cysteine residues [Figure 10]. Detailed structural information has been published on the crystal structure of the DNA-binding domain of the glucocorticoid receptor complexed with DNA (60). . More recently also 3D-information became available for AR-DNA interaction at different types of response elements (61).
Figure 10. Structure of the DNA binding domain of the human androgen receptor. The protein structure is represented in the one-letter code. The domain consists of two zinc cluster modules, which are stabilized by the coordination binding of a zinc atom (red dot) by 4 cysteine residues (yellow). The first zinc cluster contains the P-box (proximal box), of which three residues determine androgen response element recognition. The second zinc cluster contains the D-box (distal box) in which amino acids are located that are involved in protein-protein interactions with a second receptor molecule in the homodimer complex.
Briefly, the DNA-binding domain has a compact, globular structure in which two substructures can be distinguished. Both substructures contain centrally one zinc atom which interacts via coordination bonds with four cysteine residues [Figure 10].
The two zinc coordination centers are both C-terminally flanked by an α-helix (60). The two zinc clusters are structurally and functionally different and are encoded by two different exons. The α-helix of the most N-terminal located zinc cluster interacts directly with nucleotides of the hormone response element in the major groove of the DNA. Three amino acid residues at the N-terminus of this α-helix are responsible for the specific recognition of the DNA-sequence of the responsive element [Figure 10].
These three amino acid residues, the so-called P(roximal)-box [Gly; Ser; Val;] are identical in the androgen, progesterone, glucocorticoid and mineralocorticoid receptors, and differ from the residues at homologous positions in the oestradiol receptor. It is not surprising therefore, that the androgen, progesterone, glucocorticoid and mineralocorticoid receptors can recognize the same response element. For the hormone and tissue specific responses of the different receptors additional determinants are needed. Important in this respect are DNA-sequences flanking the hormone response element, receptor interactions with other proteins and receptor concentrations. The second zinc cluster motif is supposed to be involved in protein-protein interactions such as receptor dimerization via the so-called D(istal)-box [Figure 10] (60).
Between the DNA-binding domain and the ligand binding domain a non-conserved hinge region is located, which is also variable in size in different steroid receptors [Figure 9, see above]. The hinge region can be considered as a flexible linker between the ligand binding domain and the rest of the receptor molecule. The hinge region is important for nuclear localization and contains a bipartite nuclear localization signal. Also co-repressor binding can occur via the hinge region. In some nuclear receptors, including the AR, an acetylation can occur in the hinge region at a highly conserved acetylation consensus site [KLLKK] (62).
Finally the second-best conserved region is the hormone binding domain. This domain is encoded by approximately 250 amino acid residues in the C-terminal end of the molecule [Figure 5, see above] (24, 31, 32, 33, 34, 63). The crystal structure of the human androgen receptor ligand binding in complex with the synthetic ligand methyltrienolone (R1881) and 5α-dihydrotestosterone, respectively, have been determined (64, 65 ). The 3-dimensional structure has the typical nuclear receptor ligand binding domain fold. Interestingly the ligand binding pocket consists of 18 amino acid residues interacting more or less directly with the bound ligand (64 ).
Crystallographic data on the ligand binding domain complexed with agonist predict 11 helices (no helix 2) with two anti-parallel β-sheets arranged in a so-called helical sandwich pattern. In the agonist-bound conformation the carboxy-terminal helix 12 is positioned in an orientation allowing a closure of the ligand binding pocket. The fold of the ligand binding domain upon hormone binding results in a globular structure with an interaction surface for binding of interacting proteins like co-activators. In this way the androgen receptor recruits selectively a number of proteins and can communicate with other partners of the transcription initiation complex.
The androgen receptor can use different transactivation domains (AF1 and AF5, respectively, in the NH2-terminal domain and AF2 in the COOH-terminal domain) depending on the "form" of the receptor protein [Figure 9, see above] (54). The AF2 function in the ligand binding domain is strongly dependent on the presence of nuclear receptor coactivators. In vivo experiments favour a ligand dependent functional interaction between the AF-2 region in the ligand-binding domain with the NH2-terminal domain (55, 57).
Deletions in the ligand binding domain abolish hormone binding completely (66 ). Deletions in the N-terminal domain and DNA-binding domain do not affect hormone binding. Deletion of the ligand binding domain leads to a constitutively active androgen receptor protein with trans-activation capacity comparable to the full length androgen receptor (66 ). Thus it appears that the hormone binding domain acts as a repressor of the trans-activation function in the absence of hormone. This regulatory function of the androgen receptor ligand binding domain in the absence of hormone, is not unique for the androgen receptor and has been reported also for the glucocorticoid receptor (67 ).
Immediately after translation, the androgen receptor becomes phosphorylated resulting in the appearance of two isoforms separable by SDS-polyacrylamide gel electrophoresis (68 ). The non-phosphorylated faster migrating 110 kDa isoform is converted into a 112 kDa phospho-isoform. Mutational analysis of serine 81 or serine 94 in the androgen receptor NH2-terminal domain abolishes this up-shift indicating that phosphorylation of these serine residues likely contributes to the phosphorylation of the 112 kDa androgen receptor isoform (41, 69). Three other androgen receptor phosphorylation sites have been identified using mutational analysis and trypsin-digestion of 32P-labelled androgen receptor followed by HPLC analysis and Edman degradation (69, 70, 71 ). These include the serine residues at position 515, 650, and 662. Substitution of serine 650 reduced androgen receptor activity by up to 30%. Mutation of serines 81 and 94 had little or no effect on androgen receptor function (41, 69).
Besides the basal phosphorylation resulting in the 110-112 kDa doublet, addition of androgen induces another shift and the generation of a 110-112-114 kDa androgen receptor triplet (41). This triplet is the result of both an addition and a redistribution of phosphorylated sites, however, it is unknown which exact residues are involved (72 ). Interestingly, mutations that inactivate androgen receptor function, such as mutations resulting in loss of DNA binding or transactivation, inhibit the formation of the 114 kDa isoform. This suggests that part of the androgen - induced phosphorylation occurs during or after androgen receptor transcription regulation (41). In conclusion phosphorylation of the androgen receptor can be linked to activation of hormone binding and to modulation of DNA binding (70, 73, 70). Furthermore phosphorylation of the androgen receptor can play an essential role in the hormone independent activation of the androgen receptor by protein kinases in the MAPK and AKT (protein kinase B) signalling pathways, activated either through HER-2/neu or growthfactors (74, 75 ).
Androgen receptor antagonists are compounds that interfere in some way in the biological effects of androgens and are frequently used in the treatment of androgen-based pathologies. The steroidal anti-androgens, cyproterone acetate (CPA) and RU38486 (RU486; mifepristone), have partial agonistic and antagonistic actions. Interestingly both compounds display also partial progestational and glucocorticoid action and are therefore considered not to be pure anti-androgens. The non-steroidal anti-androgens hydroxyflutamide, nilutamide and bicalutamide are pure antiandrogens (76, 77, 78 ).
In contrast to the full antagonists hydroxyflutamide and bicalutamide, CPA and RU486 can partially activate the androgen receptor with respect to transcription activation (79, 80). Binding of androgens by the androgen receptor results in two consecutive conformational changes of the receptor molecule. Initially, a fragment of 35 kDa, spanning the complete ligand binding domain and part of the hinge region, is protected by the ligand. After prolonged incubation times a second conformational change occurs resulting in protection of a smaller fragment of 29 kDa (79, 80). In the presence of several anti-androgens (e.g. cyproterone acetate, hydroxyflutamide and bicalutamide) only the 35 kDa fragment is protected, and no smaller fragments are detectable upon longer incubations. Obviously, the 35 kDa fragment is correlated with an inactive conformation, whereas the second conformational change, only inducible by agonists and considered as the necessary step for transcription activation, is lacking upon binding of anti-androgens.
The term SARM (= Selective Androgen Receptor Modulator) was introduced in 1999 in analogy of the term SERM (Selective Estrogen Receptor Modulator) (81). A SARM can be defined as a molecule that targets the androgen receptor, and elicits a biological response, in a tissue specific way. In a sense, anti-androgens (molecules that target specifically the androgen receptor pathway resulting in inhibition of the biological effects of androgens) can be considered as a special subtype of SARMs.Recently information became available on androgen signalling via the androgen receptor in a non-genomic, rapid and sex-nonspecific way by crosstalk with the Scr, Raf-1, Erk-2 pathway [Figure 6, see above] (19, 20). The anti-apoptotic action via the androgen receptor in bone cells (osteocytes, osteoblasts), and also in HeLa cells, could be induced by androgens and estrogens and inhibited by antiandrogens as well as anti-estrogens. The anti-apoptotic action appeared to be dissociated from the genomic action of the androgen receptor. Also the progesterone induced oocyte maturation in Xenopus laevis appeared to be mediated in a non-genomic way by androgens and the androgen receptor via activating the MAPK pathway after the rapid conversion of progesterone to androstenedione and testosterone (21). These interesting findings stimulate the development of new compounds which can selectively activate the androgen receptor either in a non-genomic pathway or in a genotropic transcriptional activation pathway.
Based on the conformational changes of the AR ligand binding domain, induced by androgens or anti-androgens, it can be concluded that the different transcriptional activities displayed by either full agonists (testosterone, 5α-dihydrotestosterone , methyltrienolone), partial agonists (RU486 and CPA) or full antagonists (hydroxyflutamide, bicalutamide) are the result of recruitment of a different repertoire of co-regulators (coactivators or corepressors) as a consequence of these conformational changes. The differential recruitment of co-regulators can be considered as special form of ligand selective modulation of the androgen receptor ligand binding domain and can be applied in broader sense also to the tissue selective modulation of androgen action, where levels of co-activators and co-repressors may determine ultimately the final activity(82).