ABSTRACT

Androgens are an important class of C19 steroid hormones that control normal male development and reproductive function. The main circulating androgen is testosterone, which is produced in the Leydig cells of the testis and can also act as a pro-hormone after being metabolized to dihydrotestosterone (DHT) or estradiol (E₂). The biological actions of testosterone and DHT are mediated by the androgen receptor, a member of the nuclear receptor superfamily, which in response to hormone regulates gene expression in target tissues. In this chapter the biosynthesis of androgens, receptor structure/function, and the consequences of genetic changes impacting on receptor expression and signaling in disorders of male development are discussed. For complete coverage of all related areas of Endocrinology, please visit our on-line FREE web-text, WWW.ENDOTEXT.ORG.

INTRODUCTION

Androgens are important hormones for expression of the male phenotype. They have characteristic roles during male sexual differentiation, but also during development and maintenance of secondary male characteristics and during initiation and maintenance of spermatogenesis (1, 2). The two most important androgens in this respect are testosterone and 5α-dihydrotestosterone [Figure 1].

Figure 1.

Structure of testosterone and 5α-dihydrotestosterone and anti-androgens.

While acting through the same androgen receptor, each androgen has its own specific role during male sexual differentiation: testosterone is directly involved in development and differentiation of Wolffian duct derived structures (epididymides, vasa deferentia, seminal vesicles and ejaculatory ducts) [Figure 2A], whereas 5α-dihydrotestosterone, a metabolite of testosterone, is the active ligand in a number of other androgen target tissues, like urogenital sinus and tubercle and their derived structures (prostate gland, scrotum, urethra, penis) [Figure 2B] (3, 4).

Figure 2.

Specific actions of testosterone (T) and 5α-dihydrotestosterone (DHT). A) Testosterone is synthesized in the testis under the control of luteinizing hormone (LH) from the pituitary. After entering target cells in the hypothalamus, pituitary, testis, and Wolffian duct, T binds to the androgen receptor (AR) and the T-AR complex binds to specific DNA sequences and regulates gene transcription, which can result in negative feedback regulation of gonadotrophin synthesis and secretion, in initiation and regulation of spermatogenesis, and in differentiation and development of Wolffian ducts. B) T is synthesized in the testis under the control of LH, enters target cells in urogenital sinus, urogenital tubercle, and several other androgen target tissues and is metabolized to DHT by the enzyme 5α-reductase type 2. DHT binds directly to the AR and the DHT-AR complex interacts with specific DNA sequences and regulates gene transcription resulting in differentiation and development of the prostate, the external genitalia, and during puberty several secondary male sex characteristics.

The interaction of both androgens with the androgen receptor is different. Testosterone has a twofold lower affinity than 5α-dihydrotestosterone for the androgen receptor, while the dissociation rate of testosterone from the receptor is five-fold faster than of 5α-dihydrotestosterone (5). However, testosterone can compensate for this "weaker" androgenic potency during sexual differentiation and development of Wolffian duct structures via high local concentrations due to diffusion from the nearby positioned testis. In more distally located structures, like the urogenital sinus and urogenital tubercle the testosterone signal is amplified via conversion to 5α-dihydrotestosterone.

ANDROGEN BIOSYNTHESIS

Androgens (testosterone and 5α-dihydrotestosterone) belong to the group of steroid hormones. The major circulating androgen is testosterone, which is synthesized from cholesterol in the Leydig cells in the testis. Testosterone production in the fetal human testis starts during the sixth week of pregnancy. Leydig cell differentiation and the initial early testosterone biosynthesis in the fetal testis are independent of luteinizing hormone (LH) (6-8). During testis development production of testosterone comes under the control of LH which is produced by the pituitary gland. Synthesis and release of LH is under control of the hypothalamus through gonadotropin-releasing hormone (GnRH) and inhibited by testosterone via a negative feedback mechanism [Figure 2A] (9).The biosynthetic conversion of cholesterol to testosterone involves several discrete steps, of which the first one includes the transfer of cholesterol from the outer to the inner mitochondrial membrane by the steroidogenic acute regulatory protein (Star) and the subsequent side chain cleavage of cholesterol by the enzyme P450scc (10). This conversion, resulting in the synthesis of pregnenolone, is the rate-limiting step in testosterone biosynthesis. Subsequent steps require several enzymes including, 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase/C17-20-lyase and 17β-hydroxysteroid dehydrogenase type 3 [Figure 3] (11).

Figure 3.

Biosynthetic pathways for testosterone and DHT synthesis. The classic pathway show testosterone synthesized from cholesterol with further metabolism to DHT. The alternative or “backdoor” pathway shows DHT production without going through testosterone. Note only some of the enzymes are shown for clarity.

METABOLISM OF TESTOSTERONE TO 5ΑΑ-DIHYDRO-TESTOSTERONE

Metabolism of testosterone to 5α-dihydrotestosterone occurs through the classical pathway [Figure 3] and is essential for initiation of the differentiation and development of the urogenital sinus into the prostate [Figure 2B]. Differentiation of male external genitalia (penis, scrotum and urethra) also strongly depends on the conversion of testosterone to 5α-dihydrotestosterone in the urogenital tubercle, labioscrotal swellings, and urogenital folds (1). In recent research there has been considerable interest in the alternative or ‘backdoor’ pathway of DHT production (12 and references therein). This pathway has been found to have a significant role in the normal masculinization of the male fetus (see 13) and abnormal virilization of the female fetus in cases of congenital adrenal hyperplasia resulting from mutations in the enzyme P450 oxidoreductase (14).

The irreversible conversion of testosterone to 5α-dihydrotestosterone is catalyzed by the microsomal enzyme 5α-reductase type 2 (SRD5A2) and is NADPH dependent [Figure 4] (15). The cDNA of the gene for 5α-reductase type 2 codes for a protein of 254 amino acid residues with a predicted molecular mass of 28,398 Dalton (16, 17).

Figure 4.

Metabolism of testosterone to DHT by the enzyme 5α-reductase type 2 (SDR5A2).

The NH₂-terminal part of the protein contains a subdomain proposed to be involved in testosterone binding, while the COOH-terminal region is involved in NADPH-binding (3). The enzyme 5α-reductase type 2 belongs to the 5α-reductase family which is composed of 3 subfamilies with a total of 5 members (18). There are three isozymes: type 1, type 2 and the more recently discovered type 3, which has a role in the conversion of polyprenols to dolichols (important step in protein N-glycosylation) (19, 20). The other members are the proteins glycoprotein synaptic 2 (GPSN2) and glycoprotein synaptic 2 like (GPNS2L) and are most likely involved in double bond reduction during fatty acid elongation (21).

ANDROGEN ACTION

The Androgen Receptor and the Nuclear Receptor Family

Actions of androgens are mediated by the androgen receptor (NR3C4; Nuclear Receptor subfamily 3, group C, gene 4). This ligand-dependent transcription factor belongs to the superfamily of 48 known human nuclear receptors (22). This family includes receptors for steroid hormones, thyroid hormones, all-trans and 9-cis retinoic acid, 1,25 dihydroxy-vitamin D, ecdysone and activators of peroxisome proliferation (23-25). An increasing number of nuclear proteins have been identified with a protein structure homologous with that of nuclear receptors, but without a known ligand. These so-called "orphan" receptors form an important subfamily of transcription factors acting either in the absence of any ligand or with yet unknown endogenous ligands (26). Comparative structural and functional analysis of nuclear hormone receptors has revealed a common structural organization in 4 different functional domains: a NH₂-Terminal Domain, a DNA-Binding Domain, a Hinge Region and a Ligand Binding Domain [Figure 5].

Figure 5.

Steroid hormone receptor family. Sequence homologies between the human androgen receptor (hAR), human progesterone receptor (hPR), human glucocorticoid receptor (hGR), human mineralocorticoid receptor (hMR), and the human estrogen receptor alpha (hERα) and beta (hERβ).

The current model for androgen action involves a multi-step mechanism as depicted in Figure 6. Upon entry of testosterone into the androgen target cell, binding occurs to the androgen receptor either directly or after its conversion to 5α-dihydrotestosterone. Binding to the receptor is followed by dissociation of chaperone protein complexes (e.g., heat shock proteins) in the cytoplasm, simultaneously accompanied by a conformational change of the receptor protein resulting in a transformation and a translocation to the nucleus. The complex of chaperone and chaperone-associated proteins is collectively called the ‘foldosome’ and has functions beyond the classical role in the cytosol. The foldosome can for instance affect nuclear translocation and target gene expression (27, 28). Upon binding in the nucleus to specific DNA-sequences the receptor dimerizes with a second molecule and the homodimer entity recruits further additional proteins (e.g., coactivators, general transcription factors, RNA-polymerase II) via specific interaction motifs (29). This finally results in transcriptional activation or suppression of specific androgen responsive genes (30).

Figure 6.

Simplified model of androgen action in an androgen target cell. The androgen receptor (AR) binds testosterone or its active metabolite DHT. After disassociation of heat shock proteins (hsp) the receptor enters the nucleus via an intrinsic nuclear localization signal and binds as a homodimer to specific DNA elements present as enhances upstream of androgen target genes. The next step is recruitment of coactivators, which form the communication bridge between the receptor and several components of the transcription machinery. The direct and indirect binding of the androgen receptor with several components of the transcription machinery (e.g., RNA polymerase II (RNA Pol II), general transcription factors (GTFs)) are key events in nuclear signaling. This communication triggers subsequent mRNA synthesis and consequently protein synthesis resulting in androgen responses. A non-genomic pathway involving the AR via cross-talk with the Src/Raf-1Erk-2 pathway is also known.

Interestingly androgen signaling via the androgen receptor can also occur in a non-genomic, rapid and sex-nonspecific way by crosstalk with the Scr, Raf-1, Erk-2 pathway [Figure 6] (31, 32). The classical androgen receptor is also involved in androgen-mediated induction of Xenopus oocyte maturation via the (MAPK)-signaling cascade in a transcription independent way (33, 34).

Cloning and Structural Organization of the Androgen Receptor Gene

Since cloning of the human androgen receptor cDNA our insights into the mechanism of androgen action have increased tremendously. Only one androgen receptor cDNA has been identified and cloned, despite the two different ligands (35-38). The concept of two hormones and one receptor to explain the different actions of androgens has been generally accepted and, according to the information available from the human genome project, it is very unlikely that additional genes exist coding for a functional nuclear receptor with androgen receptor-like properties (25).

The androgen receptor gene is located on the X-chromosome at Xq11.2 -12. The gene spans 186,587 kilobases (kb) in total and codes for a protein with a molecular mass of approximately 110 kDa [Figure 7] (39, 40). The gene consists of 8 coding exons and the structural organization of the coding exons is essentially identical to those of the genes coding for the other steroid hormone receptors (e.g., exon/intron boundaries are highly conserved) and is characterized by unusually long 5’- and 3’-UTRs [Figure 7] (36, 41-43, 47). As a result of differential splicing in the 3' - untranslated region two androgen receptor mRNA species (of around 7.5 and 10 kb, respectively) have been identified in several human tissues and cell lines (36): only the larger transcript is seen in rodent tissues (36, 43, 47). There is no structural indication in the androgen receptor mRNA for any preferential use of either of the two transcripts or transcript specific functions, but it can be speculated that tissue-specific factors may determine which transcript is present in which androgen target tissue. In the human prostate and in genital skin fibroblasts the 10 kb size mRNA is predominantly expressed (43). It may also be significant that a number of micro-RNAs have been identified and validated that target the 3’-UTR that are likely to contribute to the regulation of receptor levels (44-46) [Figure 7].

Figure 7.

Human androgen receptor gene was mapped to the long arm of the X chromosome. The human androgen receptor gene consists of coding exons and unusually long 5’- and 3’ UTRs. These have been shown to be important for transcriptional regulation (binding sites for both positive and negative regulatory factors) in the case of the 5’UTR. The 3’UTR region of the mRNA is targeted by a number of microRNAs (miRNAs). The androgen receptor has been shown to downregulate its own mRNA through response elements located in the 5’UTR and exon 2.

ANDROGEN RECEPTOR AMINO ACID NUMBERING

The current sequence of the androgen receptor cDNA and the amino acid numbering of the corresponding protein is based on the NCBI reference sequence NM_000044.3. This is different from the original numbering scheme used over the past 20 years that was based on Gen-Bank mRNA sequence M20132.1 (36).

In order to correctly identify mutations previously published, the following changes should be kept in mind: the variable polyglutamine tract length is two longer (23 instead of 21), whereas the variable polyglycine tract length is one shorter (23 instead of 24) for NM_000044.3 versus M20132.1, respectively. Consequently, the androgen receptor protein of the new reference sequence is one amino acid longer, that is, 920 residues, leading to a +2 shift in amino acid numbering between residues 78 and 449 and to a +1 shift between residues 472 and 919 compared with the previously used standard reference sequence. The +1 shift involves all the amino acid residues in the DNA-binding domain (DBD) and ligand-binding domain (LBD). The new reference numbering is further explained and illustrated in Figure 8 and will be used throughout the text.

Figure 8.

Reference numbering of the androgen receptor (AR) of protein. The numbering of the amino acid residues is according to National Center for Biotechnology Information (NCBI) reference sequence number NM_000044.3, which refers to a gene size of 187,246 nucleotides and an AR protein of 920 amino acid residues with a polyglutamine tract of 23 and a polyglycine tract of 23 (110). Amino acid numbering +2 between 78 and 449; Amino acid numbering +1 between 472 and 919. In addition, a number of splice variants of the AR have been identified in prostate cancer cell lines and patient samples. These splice variants lack most or all of the LBD but retain a functional DBD and NTD with unique C-terminal sequences derived from cryptic exons (CE) (e.g., AR-v7).

ANDROGEN RECEPTOR: FUNCTIONAL DOMAIN STRUCTURE

The DNA-Binding Domain

The DNA-binding domain is the best conserved among the members of the receptor superfamily [Figure 5]. It is characterized by a high content of basic amino acids and by nine conserved cysteine residues [Figure 9A]. Detailed structural information has been published on the crystal structure of the DNA-binding domain of the glucocorticoid receptor complexed with DNA (122). 3D-information is also available for the androgen receptor-DNA interaction on an artificial DNA response element (123) [Figure 9B]. The folding of the DBD is similar to that reported for the glucocorticoid and estrogen receptor DBDs.

Figure 9.

Structure of the DNA binding domain of the androgen receptor. A) 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. B) Structure of the AR-DBD bound to DNA (Pdb 1R41). C) Consensus androgen receptor response element.

Briefly, the DNA-binding domain has a compact, globular structure in which three substructures can be distinguished: two zinc clusters and a more loosely structured carboxy terminal extension (CTE) (124). Both zinc substructures contain centrally one zinc atom which interacts via coordination bonds with four cysteine residues [Figure 9].

The two zinc coordination centers are both C-terminally flanked by an α-helix (122, 123). The two zinc clusters are structurally and functionally different and are encoded by two different exons [see Figures 7 and 8]. 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 9A]. These three amino acid residues, the so-called P(proximal)-box [Gly; Ser; Val;] are identical in the androgen, progesterone, glucocorticoid and mineralocorticoid receptors, and differ from the residues at homologous positions in the estradiol receptor. It is not surprising therefore, that the androgen, progesterone, glucocorticoid and mineralocorticoid receptors can recognize the same response element. The receptor DNA binding domain requires a CTE of minimally four residues (amino acids 626 – TLGA – 629) for proper binding to an ARE (androgen response element) with an inverted repeat of high affinity ARE-half sites and a CTE of at least twelve residues (amino acids 626 – TLGARKLKKLGN – 637) for binding to an ARE with one high and one low affinity half site (125). 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 involved in protein-protein interactions such as receptor dimerization via the so-called D(distal)-box [Figure 9A and B] (122, 123).

The Ligand Binding Domain

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] (37, 60-63, 134). 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 [Figure 10A and B] (135, 136).

Figure 10.

Structure of the ligand binding domain of the human androgen receptor. A) The crystal structure of the LBD with DHT bound (pdb 1137). Specific amino acid- hormone interactions are illustrated in the right-hand panel. B) The LBD structure with the synthetic agonist R1881 and a coactivator peptide with an FxxLF motif bound to AF2 region (pink oval) (pdb 1XOW). C) Structure of the LBD showing the location of the BF3 pocket (blue oval) with triiodothyroacetic acid/TRIAC bound (pdb 2PKL).

The 3-dimensional structure has the typical nuclear receptor ligand binding domain fold (59). Interestingly the ligand binding pocket consists of 18 amino acid residues interacting more or less directly with the bound ligand, with a relatively small number of specific hydrogen-bonds and hydrophobic interactions determining hormone-selectivity [Figure 10A] (135). The ligand binding pocket is somewhat flexible and can accommodate ligands with different structures. The structural data are being used in designing optimized selective androgen receptor modulators (SARMs) (137, 138). Several AR mutations found in prostate tumors have been investigated functionally, including T878S, T878A, H875T, V716M, W742C, and L702H as a single mutation or in combination with T878A. Similar to T878A these AR mutations have a broadened ligand specificity and are activated by different low affinity ligands like estradiol, progesterone, glucocorticoids and different partial and full antagonists (139-146).

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. Upon hormone binding the fold of the ligand binding domain results in a globular structure with an interaction surface for binding of interacting proteins like co-activators (AF2) [Figure 10B]. In this way the androgen receptor selectively recruits a number of proteins and can communicate with other partners of the transcription initiation complex. Crystallization studies of wild type androgen receptor ligand binding domain with antagonists have not been reported so far. However, the structural consequences of surface modulatory compounds on the receptor LBD crystals complexed with DHT are promising for future developments of new androgen receptor modulators including a new type of androgen receptor antagonists (147).

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 8, see above] (103). The AF2 function in the ligand binding domain is strongly dependent on the presence of nuclear receptor coactivators. In vivo experiments favor a ligand-dependent functional interaction between the AF-2 region in the ligand-binding domain with the NH₂-terminal domain (113, 115). In fact, the AF2 surface demonstrates a preference for more bulky hydrophobic amino acids over the LxxLL motif and the structural basis for this has been described (148-150). Thus, the receptor NTD FxxLF motif [Figure 10B] is more effective at forming a charge clamp with Glu898 and Lys721 and burying the phenylalanine residues into the AF2 pocket, whereas peptides containing the sequence LxxLL make weaker and fewer contacts with the LBD.

Interestingly, a previously unknown regulatory surface cleft, named BF-3, has been identified in the receptor LBD (147) [Figure 10C]. BF-3 comprises of Ile-673, Phe-674, Pro-724, Gly-725, Asn-728, Phe-827, Glu-830, Asn-834, Glu-838 and Arg-841. The androgen receptor transcriptional activity and co-activator binding can be decreased by binding of thyroid hormones triiodothyronine (T3) and TRIAC and three non-steroidal anti-inflammatory drugs to the BF-3 pocket. In addition, several mutations of the amino acid residues of BF-3 have been found in subjects with either androgen insensitivity syndrome (AIS, loss of function mutation) or in prostate cancer (gain of function mutation) (151). Mutational analyses have shown the requirement of several of these amino acid residues for receptor-dependent transcriptional activity. However, these analyses have been performed only in the presence of DHT (147). The influence of each of these residues in the presence of T3, TRIAC or other nonsteroidal anti-inflammatory drugs is therefore unknown.

A long-standing question in the field concerning dimerization of the AR-LBD has recently been resolved with new crystallographic studies (152, 153). The work from the Estébanez-Perpiñá group identified sequences in helix 5 as novel dimerization interface. Significantly, a number of point mutations associated with androgen insensitivity or prostate cancer map to this region emphasizing its functional importance for AR signaling (152).

ANDROGEN RECEPTOR POSTTRANSLATIONAL MODIFICATIONS

Methylation, Acetylation, Ubiquitination and SUMOylation

The androgen receptor protein can be extensively covalently modified either by methylation, acetylation, ubiquitination, SUMOylation or phosphorylation [Figure 11] (132, 133, 167-171).

Figure 11.

Post-translational modifications of the human androgen receptor. AC, acetylation of lysine residues (631, 633, and 634); CH₃, methylation of lysine (633); P, phosphorylation of serines (16, 83, 96, 215, 258, 310, 426, 516, 651, and 792); SUMO-1, sumoylation on lysines (388 and 521); Ub, ubiquitination of lysines (846 and 848).

All these reactions are reversible and consequently enzymes that mediate dephosphorylation, deacetylation, deubiquitination, demethylation and de-SUMOylation are also potential regulators of androgen receptor activity. A total of 23 sites in the androgen receptor protein have been identified undergoing direct modification (170). These posttranslational modifications can contribute significantly to androgen receptor structure, activity and stability. It has been shown for instance that the histone methyltransferase SET9 is able to methylate the receptor in the hinge region at the Lysine residues 631 and 633 resulting in enhancement of transcriptional activity of the receptor (172, 173). The same Lysine residues together with Lysine 634 can be acetylated and the acetylation-deficient mutants have a decreased transcriptional activity, while the acetylation-mimetic mutations showed an enhanced transcriptional activity (132, 174). Recently, phosphorylation of serine 83 was observed to result in recruitment of the histone acetyltransferase p300, acetylation of the receptor and enhanced receptor stabilization and transcriptional activity (175).

Conversely, disruption of acetylation, through mutating the lysine residues or knock-down of p300 resulted in receptor ubiquitination and degradation. This study elegantly demonstrates how different post-translational modifications of the androgen receptor can work in concert to regulate receptor expression and activity. RNF6 dependent ubiquitination of Lysine residues 846 and 848 in the receptor protein results in recruitment of the coregulator ARA54 by the androgen receptor and directly determines promoter selectivity and specificity of the receptor (176).

SUMOylation of the androgen receptor occurs at two sites Lysine residues 388 and 521, but SUMOylation only at Lysine 388 results in a significant reduction of transcriptional activity (177). However, recent, whole genome analysis revealed that SUMOylation regulated both receptor recruitment to DNA and target gene selection (178). Significantly, the physiological importance of SUMOylation has been demonstrated in a knock-in mouse model, ARKI, where the SUMOylation sites were mutated to arginine (179). Male animals developed normally but were found to be infertile due to defects in epididymal sperm maturation. Crucially, In the ARKI animals the AR-dependent transcriptional activity was impaired in the epididymis and there was an absence of receptor SUMOylation linking this PTM to normal male reproduction and fertility (179).

ANTI-ANDROGENS AND SELECTIVE ANDROGEN RECEPTOR MODULATORS

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 also display partial progestational and glucocorticoid actions and are therefore not considered to be pure anti-androgens. The non-steroidal anti-androgens hydroxyflutamide, nilutamide and bicalutamide [see Figure 1] are pure antiandrogens (207-209). Recent developments have led to the generation and marketing of second-generation non-steroidal antiandrogens, such as enzalutamide (formerly called MDV3100) [Figure 1], which have been reported to be more effective at blocking receptor nuclear translocation and activity (210). Recently, two new anti-androgens apalutamide and darolutamide have received FDA approval for treatment of non-metastatic castrate resistant prostate cancer (see 211, 212). However, resistance to enzalutamide has now also been identified as a result of an Phe876Leu point mutation in the LBD (213) and the expression of NH₂-terminal domain splice variants (163) in CRPC, emphasizing the need for continued research and development of strategies to switch off androgen receptor signaling.

TISSUE-SPECIFIC ANDROGEN RECEPTOR MEDIATED ACTIONS IN MOUSE MODELS

Genetic mouse models in which the androgen receptor gene has been inactivated (so-called ARKO [androgen receptor knock-out] mouse models) are valuable tools to understand in detail the role of receptor-mediated pathways in male and female reproductive functions. For this purpose several different mouse models have been developed for studying androgen receptor mediated tissue-specific action in almost all known androgen target tissues, although the application of the mouse findings to the human situation has its limitation (233-238). Furthermore, the development of a mouse model for imaging of luciferase activity under control of endogenous androgen receptor activity has contributed to a further elucidation of tissue-specific receptor action (239).

ANDROGEN RECEPTOR DISORDERS

There is growing evidence for the involvement of the androgen receptor in the gender biases seen in a wide range of pathological conditions, from cancers of non-reproductive tissues (i.e., bladder, liver) (see 240, 241) to cardiovascular and metabolic disease (see 242-244). However, in this review we will focus on receptor mutations leading to defects of male development and fertility.

ANDROGEN METABOLISM DISORDERS

The metabolism of testosterone to 5α-dihydrotestosterone by the enzyme 5α-reductase type 2 (SRD5A2) is essential for the initiation of the differentiation and development of the urogenital sinus into the prostate. The differentiation of the male external genitalia (penis, scrotum and urethra) also strongly depends on the conversion of testosterone to 5α-dihydrotestosterone in the urogenital tubercle, labioscrotal swellings and urogenital folds, respectively [Figure 2B, see above] (3, 4). Interestingly in the SPARKI mouse expression of Srd5α2 gene is significantly impaired in the epididymis and the androgen-regulation of the gene was demonstrated to involve three selective AREs (304).

Molecular Basis for the Syndrome of 5α-Reductase Type 2 Deficiency

A reflection of defective or absence 5α-reductase type 2 enzyme activity can be obtained in patients’ serum and urine samples by measuring testosterone levels (elevated), 5α-dihydrotestosterone levels (decreased) and by measuring the ratio of testosterone/5α-dihydrotestosterone (increased after hCG stimulation) (3). Furthermore, in cultured genital skin fibroblasts (if available) the conversion of testosterone to 5α-dihydrotestosterone can be assessed and is an option for establishing a defective enzyme. In broken cell preparations at pH 5.5, the type 2 isozyme activity is measured more specifically and can be compared with a preparation from a normal person (3).

Genetic analysis of 5α-reductase type 2 deficiency has become possible since the cloning of the cDNA (17). The gene is located on chromosome 2 at locus 2p23. The enzyme is encoded by 5 exons and the most reliable approach to detect gene mutations is the sequencing of each individual exon and the flanking intron sequences [Figure 12]. A relatively large number of loss of function mutations in the type 2 steroid 5α-reductase has been identified in 46XY individuals with this rare autosomal recessive disorder of sex development (46XY, DSD).

Figure 12.

Mutations in the5α-reductase type 2 gene (SDR5A2) reported in patients with the syndrome of 5α-reductase deficiency. The 5α-reductase type 2 enzyme is encoded by 5 different exons and mutations have been reported in all 5 exons, as well as a complete gene deletion, small deletions of nucleotides, and splice site mutations.

Interestingly worldwide 87 different mutations have been detected in the 5α-reductase type 2 gene in patients with the syndrome of 5α-reductase type 2 deficiency in several different ethnic groups [Figure 12] (3, 4, 285, 305-307, 311-339). Identical mutations have been reported in different ethnic groups and some of them can be considered to be due to a founder effect and some to have occurred de novo (340-342). The mutations were found in all five exons of the gene, although the majority of the mutations are reported in exons 1 and 4 [Figure 12].

The mutations comprise of 57 amino acid substitutions (65.5%), one complete gene deletion (3, 306), one complete exon 1 deletion (16), one substitution at stop codon 255 resulting in a Serine residue (336), ten small deletions resulting in either a premature stop codon or in an in-frame amino acid residue deletion, four small insertions (335), nine nonsense mutations and four splice site mutations, resulting in aberrant splicing [Figure 12]. The majority of the reported patients are homozygous for one of the mutations. A smaller number of patients appeared to be compound heterozygous, while a small group of patients are heterozygous (331, 340, 341).

In general male carriers of a single mutant allele have normal fertility as is the case for female carriers. The largest investigated kindreds were found in the Dominican Republic, in Turkey and in New Guinea (3, 305, 333). In all three kindreds the high incidence can be directly related to a founder affect in geographical isolated populations with a high degree of inbreeding. For other cases also a large incidence of proven consanguinity is reported (3, 305).

In prostate cancer de novo mutations in the 5α-reductase type 2 have been reported, resulting in increased 5α-reductase activity (317, 333, 343, 344). This finding indicates a role for increased 5α-dihydrotestosterone levels in the prostate, during prostate cancer progression in a subset of patients. The V89L mutant significantly reduced SRD5A2 enzymatic activity by almost 30% (316, 342, 343). The rare allele frequency of the V89L variant is 22%, 23,5%, and 46,1% for African Americans, Caucasians, and Asians, respectively, paralleling a substantial racial/ethnic variation in prostate cancer risk, indicating that this polymorphism might be implicated in prostate cancer carcinogenesis (343-346).

CONCLUSIONS-KEY POINTS

Androgenic steroids are important for normal development and function of male reproductive tissues and for anabolic actions in muscle and bone. The multiple actions of the main circulating androgen testosterone and the more potent metabolite DHT are mediated by a single intracellular receptor protein, the androgen receptor. The hormone-bound receptor acts primarily to differentially regulate gene expression in target tissues and its encoding gene is located on the X chromosome, making it a single-copy gene in males. Thus, genetic changes affecting expression or structure/function of the receptor protein will lead to a range of diseases associated with loss or impaired androgen signaling, including disruption of male development, infertility or a late onset neurodegenerative disease (SBMA). Furthermore, altered expression and genetic changes in the receptor are also key drivers in progression of prostate cancer to a therapy-resistant stage.

Since the first cloning of the androgen receptor cDNA, over thirty years ago, considerable progress has been made in our understanding of receptor structure and function. Advances include: the availability of 3D-structures of the isolated LBD with different ligands bound and the isolated DBD; structural characterization of the intrinsically disordered NH₂-terminal domain; first glimpse of the structure of full-length AR transcriptional complex on DNA; the identification of a plethora of co-regulatory proteins binding to the ligand- and NH₂-terminal domains; identification of gene regulatory pathways in target cells; and a better understanding of the impact of genetic changes affecting receptor structure/function. Future research will likely focus on the mechanisms determining cell/tissue-selective expression and function of the androgen receptor in both normal and pathophysiological conditions and a more complete structural descriptions of the full-length receptor bound to different DNA response elements and co-regulatory proteins.

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