Endotext.com - Endocrinology Of Male Reproduction, Androgen Physiology: Receptor and Metabolic Disorders

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 the initiation and maintenance of spermatogenesis (1). The two most important androgens in this respect are testosterone and 5a-dihydrotestosterone [Figure 1]. While acting through the one androgen receptor, each androgen has its own specific role during male sexual differentiation: testosterone is directly involved in the development and differentiation of wolffian duct derived structures (epididymides, vasa deferentia, seminal vesicles and ejaculatory ducts) [Figure 2A], whereas 5a-dihydrotestosterone, a metabolite of testosterone, is the active ligand in a number of other androgen target tissues, like the urogenital sinus and tubercle and their derived structures (prostate gland, scrotum, urethra, penis) [Figure 2B] (2,3). The interaction of both androgens with the androgen receptor is different. Testosterone has a two fold lower affinity than 5a-dihydrotestosterone for the androgen receptor, while the dissociation rate of testosterone from the receptor is five-fold faster than of 5a-dihydrotestosterone (4). However, testosterone can compensate for this "weaker" androgenic potency during sexual differentiation and development of the wolffian duct structures via high local concentrations due to diffusion from the nearby positioned testis. In the more distally located structures, like the urogenital sinus and urogenital tubercle the testosterone signal is amplified via conversion to 5a-dihydrotestosterone.

Figure 2a. Specific actions of Testosterone (T). T is synthesized in the testis under control of luteinizing hormone (LH) from the pituitary . After entering the target cells (in the hypothalamus, pituitary, testis and wolffian duct) T is directly bound to the androgen receptor (AR) and the complex T-AR binds to specific DNA sequences and regulates the gene transcription, which can result in negative feedback regulation of gonadotrophin synthesis and secretion, in initiation and regulation of spermatogenesis and in the differentiation and development of the wolffian duct.

Figure 2b. Specific actions of 5a-Dihydrotestosterone (DHT). T is synthesized in the testis under control of luteinizing hormone (LH) from the pituitary . After entering the target cells (in the urogenital sinus, urogenital tubercle, and several additional androgen target tissues) T is metablized to 5a-Dihydrotestosterone (DHT) by the enzyme 5a-Reductase type 2. DHT binds subsequently to the androgen receptor (AR) and the complex DHT-AR binds to specific DNA sequences and regulates the gene transcription, which can result in the differentiation and development of the prostate, the external genitalia and at puberty in several secondary male characteristics.

Androgens (testosterone and 5a-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) (5, 6, 7). During testis development the production of testosterone comes under the control of LH which is produced by the pituitary gland. The 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 (8).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 mitochondral membrane by the steroidogenic acute regulatory protein (StAR) and the subsequent side chain cleavage of cholesterol by the enzyme P450scc (9). This conversion, resulting in the synthesis of pregnenolone, is a rate-limiting step in testosterone biosynthesis. Subsequent steps require several enzymes including, 3b-hydroxysteroid dehydrogenase, 17a-hydroxylase/C17-20-lyase and 17b-hydroxysteroid dehydrogenase [Figure 3] (10).

The metabolism of testosterone to 5a-dihydrotestosterone 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 5a-dihydrotestosterone in the urogenital tubercle, labioscrotal swellings and urogenital folds (1). The conversion of testosterone to 5a-dihydrotestosterone is catalyzed by the microsomal enzyme 5a-reductase type 2 (SRD5A2) and is NADPH dependent and irreversible [Figure 4] (11). The cDNA of the gene for 5a-reductase type 2 codes for a protein of 254 amino acid residues with a predicted molecular mass of 28,398 Dalton (12). The NH2-terminal part of the protein contains a subdomain supposedly involved in testosterone binding, while the COOH-terminal region is involved in NADPH-binding (2).

The gene is located on chromosome 2 at locus 2p23. Elucidation of the organization of the 5a-reductase type 2 gene revealed 5 coding exons (12). The enzyme has a pH optimum at pH 5.5 in broken cell preparations, but may function optimally in the native state in intact cells at neutral pH and has an apparent Km for testosterone of 4-50 nM. The apparent Km for NADPH is 3-10 mM. High expression of 5a-reductase type 2 is observed in prostate tissue. Mutations and deletions in the 5a-reductase type 2 gene have been found to be the molecular basis for the syndrome of 5a-Reductase deficiency (2).

The 5a-reductase type 1 (SRD5A1) isozyme, also catalyzing the conversion of testosterone to 5a-dihydrotestosterone, differs from the type 2 isozyme in its amino acid composition, kinetics, biochemical properties, substrate specificity, tissue distribution and pH optimum (11, 13). The type 1 enzyme is not involved in androgen dependent male sexual differentiation. The 5a-reductase type 1 cDNA codes for a protein of 259 amino acid residues with a predicted molecular mass of 29,462 Dalton (13). The gene is located on chromosome 5 at locus 5p15 (11). The apparent Km for testosterone is 1-5 mM and for NADPH 3-10 mM. The expression of 5a-reductase type 1 in prostate tissue is relatively low. The difference in pH optima between 5a-reductase type 1 and type 2 enzymes can be used diagnostically for the differential assessment of the individual isoenzymes in genital skin fibroblasts of patients with the syndrome of 5a-reductase deficiency (2, 11).

Male mice with a disruption of the 5a-reductase type 1 gene have a normal phenotype and are fertile. However, female mice with a null allele, have a severe parturition defect, suggesting a role of 5a-reduced androgens synthesized by the type 1 isozyme in normal female physiology (14)

The actions of androgens are mediated by the androgen receptor. This ligand dependent transcription factor belongs to the superfamily of nuclear receptors. This family includes receptors for steroid hormones, thyroid hormones, all-trans and 9-cis retinoic acid, 1,25 dihydroxy-vitamin D, ecdysone and peroxisome proliferator-activated receptors (15, 16, 17). In addition 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 as yet unknown endogenous ligands (18). 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].

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 5a-dihydrotestosterone. Binding to the receptor is followed by dissociation of 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. 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) resulting in specific activation of transcription at discrete sites on the chromatin.

Figure 6. Simplified model of androgen action in an androgen target cell. The key protein is the androgen receptor, which binds testosterone directly or its active metabolite 5a-dihydrotestosterone (DHT). After dissociation of heat shock proteins (hsp) the receptor enters the nucleus via an intrinsic nuclear localization signal. Upon steroid hormone binding, which may occur either in the cytoplasm or in the nucleus, the androgen receptor binds as dimers to specific DNA elements present as enhancers in upstream promoter sequences of androgen target genes. Direct and indirect communication of the androgen receptor complex with the several components of the transcription machinery (e.g. RNA-polymerase II [RNA-Pol II], TATA box binding protein [TBP], TBP associating factors [TAF's], general transcription factors [GTF's]) are key events in signal transduction. This communication via socalled coactivators triggers mRNA synthesis and consequently protein synthesis, which finally results in an androgen response. A non-genomic pathway involving the classical androgen receptor via cross-talk with the Src/Raf-1/Erk-2 pathway is also known.

Interestingly androgen signalling 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] (19, 20). The classical androgen receptor is also involved in androgen-mediated induction of Xenopus oocyte maturation via the (MAPK)-signalling cascade in a transcription independent way (21).

Since the cloning of the human androgen receptor cDNA our insights into the mechanism of androgen action have been increased tremendously. Only one androgen receptor cDNA has been identified and cloned, despite the two different ligands (22, 23, 24, 25). 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 (17).

The androgen receptor gene is located on the X-chromosome at Xq11-12 and codes for a protein with a molecular mass of approx. 110 kDa [Figure 7] (26, 27). The gene consists of 8 coding exons and the structural organization is essentially identical to those of the genes coding for the other steroid hormone receptors (e.g. exon/intron boundaries are highly conserved) [Figure 7] (28, 29). As a result of differential splicing in the 3' - untranslated region two androgen receptor mRNA species (8.5 and 11 kb, respectively) have been identified in several cell lines (30). There is no structural indication in the androgen receptor mRNA for any preferential use of either of the two transcripts and neither for a specific function, 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 predominantly the 11 kb size mRNA is expressed.

Figure 7. Human androgen receptor gene was mapped to the long arm of the X-chromosome. The human androgen receptor protein (919 amino acid residues) is encoded by 8 exons. Analogous to other nuclear receptors the protein consists of several distinct functional domains: the NH2-terminal domain containing two polymorphic stretches, the DNA-binding domain, the hinge region and the ligand binding domain.

The androgen receptor DNA - and ligand-binding domains have a high homology with the corresponding domains of the other members of the steroid receptor subfamily.

There is a remarkably low homology between the androgen receptor NH2-terminal domain and that of the other steroid receptors [Figure 5, see above] (31, 32, 33, 34, 35, 36). A poly-glutamine stretch, encoded by a polymorphic (CAG)nCAA - repeat is present in the NH2-terminal domain [Figure 8] (37). The length of the repeat has been used for identification of X-chromosomes for carrier detection in pedigree analyses (38, 39).

Figure 8. Variation in the polyglutamine stretch encoded by the (CAG)nCAA repeat in exon 1. The normal range of this polystretch is 9 - 38 glutamine residues, while in the motor neuron disease spinal/bulbar muscular atrophy (also called Kennedy's disease) the (CAG)nCAA repeat in exon 1 is expanded and codes for more than 40 Glutamine residues.

Variation in length (9 - 38 glutamine residues) is observed in the normal population and has been suggested to be associated with a very mild modulation of androgen receptor activity (40). This assumption is based on in vitro experiments after transient transfection of androgen receptor cDNA's containing (CAG)nCAA - repeats of different lengths (41, 42). Translating this finding to the in vivo situation it can be envisaged that either shorter or longer repeat lengths can result in a relevant biologic effect during lifetime. This concept has been explored in epidemiological studies of men with prostate cancer or infertility. With respect to the prostate cancer, a clear picture has not emerged and controversy persists. In several studies, shortening of the (CAG)nCAA repeat length in exon 1 of the androgen receptor gene was found to correlate with an earlier age of onset of prostate cancer, and a higher tumor grade and aggressiveness (43, 44, 45). However, in other epidemiological studies in prostate cancer patients these associations were not confirmed (46, 47).

In several investigations in male infertile patients an association was found between a longer (CAG)nCAA repeat and the risk of defective spermatogenesis (48, 49, 50). This suggests that a less active androgen receptor, due to a moderate expanded repeat length, may be a factor in the etiology of male infertility.

The (CAG)nCAA - repeat in exon 1 of the androgen receptor gene is expanded in patients with spinal and bulbar muscular atrophy (SBMA) and varies between 38 and 75 repeat units [Figure 8] (40, 51). Spinal and bulbar muscular atrophy is characterized by progressive muscle weakness and atrophy. Clinical symptoms usually manifest in the third to fifth decade and result from severe depletion of lower motornuclei in the spinal cord and brainstem (40, 52, 53). In addition, SBMA patients frequently exhibit endocrinological abnormalities including testicular atrophy, infertility, gynecomastia, and elevated LH, FSH and estradiol levels. Sex differentiation proceeds normally and characteristics of mild androgen insensitivity appear later in life.

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 the 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 the stabilization of the androgen receptor dimer complex via intermolecular interactions and in the 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 not 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). This structural information might also be representative of the other members of the steroid hormone receptor family.

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 a-helix (60). The two zinc clusters are structurally and functionally different and are encoded by two different exons. The a-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 a-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 the 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 the 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. It contains also the nuclear localization signal.

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, 61). Recently the crystal structure of the human androgen receptor ligand binding in complex with the synthetic ligand methyltrienolone (R1881) and 5a-dihydrotestosterone, respectively, have been determined (62, 63). 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 (62).

Crystallographic data on the ligand binding domain complexed with agonist predict 11 helices (no helix 2) with two anti-parallel b-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 (64). 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 (64). 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 (65).

Immediately after translation, the androgen receptor becomes phosphorylated resulting in the appearance of two isoforms separable by SDS-polyacrylamide gel electrophoresis (66). 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, 67). 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 (67, 68). 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, 67).

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 (69). 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 (68, 70). Furthermore phosphorylation of the androgen receptor can play an essential role in the hormone independent activation of the androgen receptor. Protein kinases, active in the MAPK and AKT (protein kinase B) signalling pathways, and which can be activated either through HER-2/neu or growthfactors, are important in this respect (71, 72).

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 (73, 74, 75).

In contrast to the full antagonists hydroxyflutamide and bicalutamide, CPA and RU486 can partially activate the androgen receptor with respect to transcription activation (76, 77). 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 (76, 77). 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) (78). 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, 5a-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.

It has been known for quite some time that defects in male sexual differentiation in 46, XY individuals have an X-linked pattern of inheritance. It was Reifenstein who reported in 1947 on families with severe hypospadias, infertility and gynecomastia (79). The end-organ resistance to androgens has been designated as androgen insensitivity syndrome (AIS) and is distinct from other forms of male pseudohermaphroditism like 17b-hydroxy-steroiddehydrogenase type 3 deficiency or 5a-reductase type 2 deficiency (2, 80, 81). It is generally accepted that defects in the androgen receptor gene can prevent the normal development of both internal and external male structures in 46, XY individuals and information on the molecular structure of the human androgen receptor gene has facilitated the study of molecular defects associated with androgen insensitivity. Due to the X-linked character of the syndrome, only 46, XY individuals are affected, while in female carriers only sporadic reports are available on delayed menarche (82). Naturally occurring mutations in the androgen receptor gene are an interesting source for the investigation of receptor structure-function relationships. In addition, the variation in clinical phenotypes provides the opportunity to correlate a mutation in the androgen receptor structure with the impairment of a specific physiological function.

The main phenotypic characteristics of individuals with the complete androgen insensitivity syndrome (CAIS) are: female external genitalia, a short, blind ending vagina, absence of wolffian duct derived structures like epididymides, vasa deferentia and seminal vesicles, the absence of a prostate, the absence of pubic and axillary hair and the development of gynecomastia (83, 84). M�llerian duct derived structures are usually absent because anti-m�llerian hormone action is normal due to the presence of both testes in the abdomen or in the inguinal canals. Usually, testosterone levels are within the normal range ( 10 - 40 nmol/L) or elevated, while elevated luteinizing hormone (LH) levels (> 10 IU/L) are also found indicating androgen resistance at the hypothalamic-pituitary level. The high testosterone levels are also substrate for aromatase activity, resulting in substantial amounts of estrogens, which are responsible for further feminisation in CAIS individuals.

In the partial androgen insensitivity syndrome (PAIS) several phenotypes ranging from individuals with predominantly a female appearance (e.g. external female genitalia and pubic hair at puberty, or with mild cliteromegaly, and some fusion of the labia) to persons with ambiguous genitalia or individuals with a predominantly male phenotype (also called Reifenstein syndrome) (83, 84). Patients from this latter group can present with a micropenis, perineal hypospadias, and cryptorchidism. In the group of PAIS individuals, wolffian duct derived structures can be partially to fully developed, depending on the biochemical phenotype of the androgen receptor mutation. At puberty, elevated luteinizing hormone, testosterone, and estradiol levels are observed, but in general, the degree of feminization is less as compared to individuals with CAIS. Individuals with mild symptoms of undervirilization (mild androgen insensitivity syndrome) and infertility have been described as well.Phenotypic variation between individuals in different families has been described for several mutations (84, 85, 86, 87). However, in cases of CAIS no phenotypic variation has been described within one single family, in contrast to families with individuals with PAIS (88).

Since the cloning of the androgen receptor cDNA in 1988 and the subsequent elucidation of the genomic organization of the androgen receptor gene, molecular biology tools have been available for the molecular analysis of the androgen receptor gene in individuals with AIS [Figure 7, see above] (28, 29). In addition to endocrinological data such as levels of testosterone, luteinizing hormone, androstenedione, and 5a-dihydrotestosterone, which can vary widely in AIS individuals, the most reliable approach is the sequencing of each individual androgen receptor exon and the flanking intron sequences. In general, AIS can be routinely analyzed and separated from entirely different syndromes presenting with similar phenotypes including testicular enzyme deficiencies, 5a-reductase type 2 deficiency, Leydig cell hypoplasia due to inactivating luteinizing hormone receptor mutations. Furthermore, in pedigree analysis intragenic polymorphisms like the highly polymorphic (CAG)nCAA repeat encoding a poly-glutamine stretch, the polymorphic GGN repeat encoding a poly-glycine stretch, the HindIII polymorphism [Figure 8, see above] (26) and the StuI polymorphism (89), can be used as X-chromosomal markers (38, 90, 91).

In the androgen receptor gene, 4 different types of mutations have been detected in 46, XY individuals with AIS: single point mutations resulting in amino acid substitutions or premature stop codons, nucleotide insertions or deletions most often leading to a frame shift and premature termination, complete or partial gene deletions (>10 nucleotides), and intronic mutations in either splice donor or splice acceptor sites which affect the splicing of androgen receptor RNA (92). The most recent update on androgen receptor gene mutations is available at http://www.mcgill.ca/androgendb/

Mutations in the NH2-terminal domain (exon 1 of the gene) do not occur frequently and the vast majority of the mutations result directly in a stop codon or in premature termination due to frameshifts caused by nucleotide insertions or deletions (http://www.mcgill.ca/androgendb/). Mutations in 36 different codons have been reported in the NH2-terminal domain, which is in approx. 7% of all codons in exon 1.

An interesting mutation is described in the fourth nucleotide, which results in a decreased translational efficiency of the androgen receptor mRNA in an individual with PAIS (93). Three other missense mutations were reported in combination with mosaicism or with a mutation in an other region of the gene.In a family with PAIS associated with severe hypospadias, the length of the androgen receptor NH2-terminal poly-glutamine repeat has been reported to be shortened to only 12 glutamine residues (94). The shortened glutamine stretch as such, is not the cause for the androgen resistance but seems to increase the thermolability of the androgen receptor in combination with a point mutation in exon 5 (Y763C) in the ligand binding domain. This point mutation causes rapid dissociation but no thermolability. These data support a functional interaction of the two separated regions in the androgen receptor and indicates further that the defect becomes critical in only part of the androgen target tissues because of the partial character of the androgen resistance found in this family (94).

In general, mutations in the DNA binding domain (e.g. single nucleotide substitutions) result in a normal hormone-binding protein, which is defective in DNA-binding/dimerization and consequently in transcription activation. In total 42 different mutations have been reported in 29 different codons in the DNA-binding domain, which is in approx. 27% of all codons in exons 2 and 3 (http://www.mcgill.ca/androgendb/). Twenty-one mutations were observed in the first zinc cluster and twenty-one in the second zinc cluster. Since the 3D structures of the DNA-binding domain of several nuclear receptors have been published, the consequence of mutations in the androgen receptor DNA-binding domain can predicted on basis of the structure of the glucocorticoid receptor DNA-binding domain (60). This is illustrated in two studies in which 3D-modelling of the mutated DNA binding domain of the androgen receptor predicts the functional activity of mutant receptors (95, 96). A mutation (G577R) in the so-called P-box [Figure 10, see above], which is involved in androgen response element recognition, was found in a PAIS individual. This mutation affected differentially transactivation of several natural and synthetic promoters, suggesting that androgen target genes may be differentially affected by this mutation (97). An interesting observation was made with respect to the second zinc cluster in which either one of two adjacent arginine residues (Arg607 & Arg608) were found to be mutated in PAIS individuals who developed breast cancer [Figure 10, see above] (98, 99). It is speculated that a decrease in androgen action within the breast cells could account for the development of male breast cancer by the loss of a protective effect of androgens. However, the same mutations in several other PAIS individuals did not result in breast cancer development.

The mutation Ala596Thr in the second zinc cluster in the so-called D-box resulted in abolishment of dimerization in a PAIS individual [Figure 10, see above] (100). A similar mutation at an identical position in the second zinc cluster of the glucocorticoid receptor DNA-binding domain has been created to discriminate between dimerization/DNA binding of the glucocorticoid receptor and protein-protein interactions with other transcription factors such as the AP-1 transcription complex (101). It appeared that the dimerization mutant did not affect the cross-talk with other transcription factors. In this way, a tissue specific response can be influenced by a single amino acid change and if this is also true for the mutant androgen receptor then the partial phenotype can be explained.

In the so-called hinge region, located between amino acid residues 622 and 670 [Figure 9, see above], only five mutations have been reported. The relatively low number of mutations in the hinge region (only in 8% of all codons) indicates that this region might be very flexible and that some variation in composition and length of this region is not detrimental for androgen receptor function http://www.mcgill.ca/androgendb/.

Two amino acid substitutions within the hinge region have been described that resulted in CAIS and three in PAIS. The I664N substitution on the border of the hinge region and ligand-binding domain, resulted in a decreased hormone binding (102).

It can be expected that mutations in the ligand binding domain might affect different functional aspects (eg. loss of ligand binding, changes in ligand binding affinity and specificity, changes in co-activator receptor interactions, changes in receptor stability and thermolability). A large number of mutations (199 different mutations in 124 codons, which is in 50% of all codons of the ligand binding domain) in the ligand binding domain have been reported in all 5 exons in individuals with either CAIS or PAIS (http://www.mcgill.ca/androgendb/). However, it appears that most mutations are located in exon 4 (16 mutations in helix 3), in exon 5 (25 mutations in helices 4 and 5) and in exon 7 (27 mutations in helices 9 and 10). Interestingly mutations are found in 13 of the 18 amino acid residues considered to interact with the ligand directly (62). For some mutations (in total 12, distributed over the whole ligand binding domain) both a complete and a partial phenotype has been described, indicating that phenotype does not always match with genotype. In the AF-2 core region (893-EMMAEIIS-900) of the androgen receptor ligand-binding domain a relatively low number of mutations have been reported [Figure 9, see above]. Only at positions M895 and Ile898 mutations have been described in individuals with the complete syndrome (103, 104). It can be speculated that in this part of helix 12 mutations in the androgen receptor ligand-binding domain are less deleterious for androgen receptor function as compared to those in helix 5 and in the b-turn, where almost every amino acid residue has been found to be mutated in AIS individuals.

Only a few cases have been reported on partial or complete androgen receptor gene deletions, indicating the relatively low frequency of this type of androgen receptor defect (105, 106, 107, 108, 109, 110, 111) ( http://www.mcgill.ca/androgendb/). All cases reported are found in CAIS individuals, with the exception of two cases, one in which an exon 4 deletion was found in a person with azoospermia (106) and another one in which a large intron 2 deletion of at least 6 kb was reported involving a branch point site, which resulted in a partial exon 3 skipping during the splicing process (110).

Deletion of either exon 3 or exon 4 occur both in-frame and result in a non-functional protein lacking either the second zinc cluster or the hinge region and the NH2-terminal part of the ligand-binding domain [Figure 7, see above]. In case of an exon 3 deletion, an intact and functional ligand-binding domain is present [Figure 7, see above]. So far, functionally significant mutations in the androgen receptor promoter region or in the 5'- and 3'- untranslated regions of the gene have not been reported.

A special group of interesting, but rare mutations are the splice donor and splice acceptor site mutations in the androgen receptor gene in AIS individuals (http://www.mcgill.ca/androgendb/). For all splice donor sites in the gene, the consensus splice donor site sequence GTAAG/A is present. The 8 reported mutations in donor splice sites are all substitutions either at position +1 (G --> A or G --> T), position +3 (A --> T), position + 4 (A --> T) or position + 5 (G --> A) and result in defective splicing with the consequence of one or more exons spliced out, or the use of a cryptic splice donor site within the preceding exon (102, 103, 112, 113, 114, 115). In 7 of the reported cases, the phenotype is complete androgen insensitivity. In one case, an insertion of one nucleotide (T) at position + 4 in the splice donor site of intron 6 has been reported, resulting in a partial androgen insensitive phenotype (116). Only 3 mutations have been reported in splice acceptor sites, which all affect the splicing of the androgen receptor RNA. Interestingly, a substitution at position -11 (T>G) has been found in the pyrimidine-rich region of the splice acceptor site of intron 2, resulting in the activation of a cryptic splice acceptor site at position -70/-69 and consequently in the insertion of 69 nucleotides (corresponding to 23 additional amino acid residues) in the mRNA between exons 2 and 3 (70). The corresponding protein is defective in DNA-binding because the insertion has occurred between the first and second zinc cluster [Figure 7, see above].

The metabolism of testosterone to 5a-dihydrotestosterone by the enzyme 5a-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 5a-dihydrotestosterone in the urogenital tubercle, labioscrotal swellings and urogenital folds, respectively [Figure 2B, see above] (2, 3).

46, XY individuals with impairment of 5a-reductase type 2 have normally virilized wolffian duct derived structures, with seminal vesicles (although small seminal vesicles have been reported as well), with vasa deferentia, epididymides and ejaculatory ducts and no m�llerian duct derived structures (2, 117, 118). However, differentiation of the urogenital sinus and genital tubercle is not observed, resulting in absence of the prostate and in ambiguous or in female external genitalia at birth (2, 117, 118). Affected 46, XY individuals are therefore often raised as girls. At puberty all affected individuals show some or a severe degree of virilization often resulting in deepening of the voice, an increased muscle mass, growth of the penis, scrotal development, testicular descent and sometimes leading to a gender change (2, 119).

Gynecomastia in adulthood does not occur. The additional virilization may result from the action of testosterone, because testosterone is available at high levels during puberty. In addition, some testosterone may be converted to 5a-dihydrotestosterone by some residual 5a-reductase activity and by the action of 5a-reductase type 1, which is expressed in non-genital skin, pubic skin, liver and certain brain regions. In the affected 46, XY individuals a typical female pubic hair pattern develops, while the facial and body hair amount is absent or reduced (3). This last observation points to a role for 5a-reductase type 2 in the normal development of this type of body hair. Male pattern baldness has never been observed. 5a-reductase type 2 deficient patients are usually infertile due to the absence or underdevelopment of the prostate and seminal vesicles, in addition to oligospermia or azoospermia due to maldescent of the testes. However, fertile patients have also been reported (2, 117). 46, XX female carriers have normal fertility, decreased body hair and delayed menarche, normal sebum production but no history of acne (2, 117). This suggests a role of 5a-reductase type 2 enzyme in females in the physiology and pathophysiology of body hair growth, menarche and follicular development (117).

A reflection of defective or absence 5a-reductase type 2 enzyme activity can be obtained in patients serum and urine samples by measuring testosterone levels (elevated), 5a-dihydrotestosterone levels (decreased) and by measuring the ratio of testosterone/5a-dihydrotestosterone (increased after hCG stimulation) (2). Furthermore in cultured genital skin fibroblasts (if available) the conversion of testosterone to 5a-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 (2).

Genetic analysis of 5a-reductase type 2 deficiency has become possible since the cloning of the cDNA (12). 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 11].

Figure 11. Mutations in the human 5a-reductase type 2 gene (SDR5A2) reported in patients with the syndrome of 5a-reductase deficiency. The 5a-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 42 different mutations have been detected in the 5a-reductase type 2 gene in patients with the syndrome of 5a-reductase type 2 deficiency in several different ethnic groups [Figure 11] (2, 3, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129). 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 (2, 117). The mutations were found in all five exons of the gene and comprise of 33 amino acid substitutions (78.5%), one complete gene deletion (12), two small deletions resulting in either a premature stopcodon or in an inframe amino acid residue deletion, 5 nonsense mutations and one splice donor site mutation in intron 4, resulting in aberrant splicing [Figure 11]. The majority of the reported patients are homozygous for one of the mutations. A smaller number of patients appeared to be compound heterozygous, while also a small group of patients are heterozygous (2, 117).

In general male carriers of a single mutant allele have a 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 (2, 117). 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.


	ANDROGEN PHYSIOLOGY: RECEPTOR AND METABOLIC DISORDERS Chapter 3 - Albert O. Brinkmann, Ph.D. August 4, 2003	Index Contributors Search