ANDROGEN

RECEPTOR

     

Like other nuclear receptors, the androgen receptor is modular in structure and is comprised of the following functional domains labeled A through F: (A/B) N-terminal regulatory domain contains: Activation function 1 (AF-1) between residues 101 and 370 required for full ligand activated transcriptional activity; Activation function 5 (AF-5) between residues 360-485 is responsible for the constitutively activity (activity without bound ligand); Dimerization surface involving residues 1-36 (containing the FXXLF motif where F = phenylalanine, L = leucine, and X = any amino acid residue) and 370-494 which both interact with the LBD in an unusual (for a nuclear receptor) head-to-tail interaction. The AR-NTD transactivation function is highly-modular, with key sequences mapping to amino acids 101 to 370 and 360 to 485 and termed TAU1 and TAU5, respectively.  (C) DNA binding domain (DBD); (D) Hinge region - flexible region that connects the DBD with the LBD; influences subcellular trafficking; (E) Ligand binding domain (LBD) containing Activation function 2 (AF-2), responsible for agonist induced activity (activity in the presence of bound agonist); AF-2 binds both coactivator proteins (containing the LXXLL motif) and/or the AF-1 region of another molecule of androgen receptor (containing the FXXLF motif) to form a head-to-tail dimer; and (F) C-terminal domain.

The testes synthesize testosterone, the principal androgen in the male circulation. The remaining androgens in the bloodstream (~5-10%), including dehydroepiandrosterone (DHEA), androstenediol, and androstenedione are either produced by the adrenal cortex and can be converted into testosterone in peripheral tissues or are derived from peripheral conversion from testosterone (dihydrotestosterone, DHT).  Testosterone (T) is the major circulating androgen and conversion to dihydrotestosterone (DHT) is in a tissue-specific manner by 5α-reductase. Although dehydroepiandrosterone (DHEA) also has weak agonist activity, testosterone and 5-DHT are the major endogenous androgens. Dihydrotestosterone (DHT), the 5α-reduced metabolite of T, is a more potent androgen required for male reproductive tract development.

Together testosterone and dihydrotestosterone (DHT) are responsible for the development of the male phenotype during embryogenesis, male sexual maturation at puberty, maintenance of secondary sexual characteristics, including reproductive function, and support and growth for other tissues, most notably muscle and bone. In addition to these effects, androgens affect a wide variety of tissues including skin, brain, adipose, prostate, and others. The actions of androgen in the reproductive tissues, including prostate, seminal vesicle, testis, and accessory structures are androgenic effects, while the nitrogen retaining effects of androgen in muscle and bone are anabolic effects.

Despite two different ligands, investigations demonstrate the identification and cloning of only one androgen receptor (AR).  Generally accepted is the concept of two hormones and one receptor to explain the different actions of androgens. 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.  Physiologically, functional AR is responsible for male sexual differentiation in utero and for male pubertal changes. In adult males, androgen is mainly responsible for maintaining libido, spermatogenesis, muscle mass, muscle strength, bone mineral density, and assist erythropoiesis. AR occurs in the prostate, skeletal muscle, liver, and central nervous system (CNS), with the highest expression level observed in the prostate, adrenal gland, and epididymis as determined by real-time polymerase chain reaction (PCR).  The binding of endogenous androgens can activate AR, including testosterone and 5-dihydrotestosterone (5-DHT).

Studies at the molecular level demonstrate that androgens function via binding to their cognate nuclear receptors, inducing conformational changes in the occupied androgen receptor (AR). The androgen receptor gene was cloned in 1988 and soon thereafter by several others.  The androgen receptor (AR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors.  The principal action of androgen is to regulate gene expression through the androgen receptor (AR). AR can modulate gene expression directly by interacting with specific elements in the regulatory regions of target genes or indirectly by activating various growth factor signaling pathways.  In addition to the classical mode, AR can modulate the transcription by the direct protein-protein interaction with other factors in a ligand-dependent manner, for which DNA binding by AR is not necessary.  

Steroid hormone receptors (SHR) act as hormone dependent nuclear transcription factors. Upon entering the cell by passive diffusion, the hormone (H) binds the receptor, which subsequently releases heat shock proteins, and translocates to the nucleus. In the nucleus, the receptor dimerizes, binds specific sequences in the DNA called hormone responsive elements (HRE), and recruits a number of coregulators that facilitate gene transcription. Processes include (1) hormone binding, (2) chaperone interaction, (3) nuclear translocation, (4) receptor dimerization, (5) DNA binding, (6) coregulator recruitment, (7) transcription, and (8) proteasomal degradation.  

A scientific report by Berthold (1849), showing that a testis transplanted into the abdomen of a castrated rooster prevented or reversed the effects of castration.  Before 1960, efforts to determine the mechanism of action of androgens and other steroid hormones often involved studies on the effects of steroids on the enzymes and cofactors of intermediary metabolism. In the early 1960s, two discoveries provided insight into how steroid hormones might work. The first was the observation that 17β estradiol was retained in target tissues apparently containing proteins that act as specific receptors for a given steroid.  The second discovery was that steroid hormones altered the synthesis of RNA in target tissues.  

Published studies demonstrated androgens rapidly enhanced RNA synthesizing activity.  This led to the hypothesis that androgens and other steroid hormones produce their characteristic effects by modulating gene expression. The belief was that testosterone produced by the testes is the most important androgen. However, the predominant androgen retained in the published studies, done on prostatic nuclei, was 5α-dihydrotestosterone and not testosterone.  

In contrast to the prevailing model at that time for the mechanism of action of estrogens, in which 17β-estradiol binds to the estrogen receptor without modification, 5α-dihydrotestosterone, a product of testosterone metabolism, not testosterone, was bound to prostate nuclei. Because the nuclear retention of 5α-dihydrotestosterone was dependent on a protein that bound androgens specifically, and there is inhibition for binding by an antiandrogen, identity of the protein was as an androgen receptor (AR).  The AR gene is on the X chromosome.  To date, there is only a description of one AR gene. The AR gene codes for a protein of 919 amino acids.

Steroid hormones are lipophilic molecules derived from cholesterol and synthesized in the adrenal cortex, the testes, the ovary, and placenta (glucocorticoids, mineralocorticoids, androgens, estrogens, and progestagens).

In addition to natural potent androgens, AR binds a variety of synthetic agonist or antagonist molecules with different affinities. All androgens, natural or chemically designed, exert their action via the AR by binding its unique LBD. However, these various ligands bind AR with very different affinities, their Ki  values ranging from low nanomolar concentrations for the most potent androgens to micromolar concentrations for the weaker ones. It is almost impossible to predict the strength of the interaction between a ligand and a receptor only based on its structure, since steroids with very similar structures can possess markedly different affinities for a given receptor while structurally different ligands could have similar high affinities.

AR ligands can be classified as agonists (androgens) or antagonists (antiandrogens), based on their pharmacological activity (i.e., ability to activate or inhibit the transcription of AR target genes); or as steroidal and nonsteroidal ligands based on structure.

Functional assay methodologies include the treatment of castrated rats with AR ligands possessing anabolic activity results in increased skeletal muscle mass, androgen treatment causes increased expression of sex hormone-binding globulin in the hepatocarcinoma cell line HepG2, and treatment of castrated rats with AR ligands possessing anabolic activity results in increased weight of prostate and seminal vesicles.  

Pharmacologically, androgen actions in reproductive tissues, including the prostate, seminal vesicle, testis, and accessory structures, are commonly referred to as androgenic effects, whereas the growth-promoting effects of androgens in muscle and bone are recognized as anabolic effects.

The concept of a tissue-selective AR modulator (SARM) was first documented in 1999.  

An ideal SARM will have: 1) high specificity for the AR; 2) desirable oral bioavailability and pharmacokinetic profile; and, most importantly, 3) desirable, tissue-selective pharmacological activities.

The major discriminating criterion is tissue selectivity of the ligand in vivo. With much improved tissue selectivity, these ligands should allow previously untenable therapeutic applications. For example, anabolic androgens could be used for the treatment of osteoporosis, muscle wasting conditions such as frailty and those caused by severe burn injury, cancer, and end-stage renal disease and AIDS. They could also be used for hormone replacement therapy in elderly men, and even in women, without concerns related to virilizing effects. On the other hand, tissue-selective antiandrogens could be advantageous for the treatment of BPH and prostate cancer, by specifically blocking androgenic actions in the prostate without abolishing needed effects on muscle, bone, or libido.

Overall, the discovery and development of SARM is at an early stage, with many compounds still under preclinical development and a handful now completing either phase I or phase II clinical trials. No SARM has entered the market to date.  

Most of the SARM identified to date are nonsteroidal anabolic agents, with the first generation [aryl propionamide and quinoline analogues] reported in 1998.  

The aryl propionamide SARM were the first to demonstrate tissue selectivity in vivo in 2003, followed later that year by a tetrahydroquinoline (THQ) SARM, quinoline SARM in 2006, and hydantoin SARM in 2007.

All of these anabolic SARM demonstrate some degree of tissue selectivity in castrated animal models, with stronger agonist activities in anabolic tissues (e.g., levator ani muscle) than in androgenic tissues (e.g., prostate).

The known AR ligands can be classified as steroidal or nonsteroidal based on their structure or as agonist or antagonist based on their ability to activate or inhibit transcription of AR target genes.  Synthetic AR ligands were first developed by modifying the steroidal structure of endogenous androgens. Low oral bioavailability, poor pharmacokinetic properties, and side effects have limited the use of many steroidal AR ligands. Until recently, it was considered impossible to separate the androgenic and anabolic effects of AR ligands due to their reliance on a single AR. However, newly discovered nonsteroidal AR ligands may provide a new strategy to achieve tissue selectivity, as is possible with estrogen receptor ligands.

MECHANISMS OF ACTION

The current model for androgen action involves a multi step mechanism. Upon entry of testosterone into the androgen target cell, binding occurs to the androgen receptor (AR) either directly or after its conversion to 5α-dihydrotestosterone. Androgens exert their effects by binding to the highly specific androgen receptor (AR). Like other steroid receptors, AR is a soluble protein that functions as an intracellular transcriptional factor. AR function regulation is by the binding of androgens, which initiates sequential conformational changes of the receptor that affect receptor-protein interactions and receptor-DNA interactions. Ligand binding initiates a series of events leading to the regulation of target genes by the receptor. Initiation of these complicated processes is by the ligand-induced conformational changes in the ligand-binding domain.

In its basal, unliganded state, the AR resides primarily in the cytoplasmic compartment where it exists in a complex with heat shock proteins (Hsp) and immunophilin chaperones through interactions with the ligand-binding domain.  The entry of ligands into the cell results in receptor binding causing a conformational change that releases heat shock proteins and allows translocation of the steroid hormone receptor to the nucleus.  

Inside the nucleus, the activated AR binds to specific recognition sequences known as androgen response elements (ARE) in the promoter and enhancer regions of target genes. Translocated receptor binds to the androgen response element (ARE), characterized by the six-nucleotide half-site consensus sequence 5'-TGTTCT-3' spaced by three random nucleotides located in the promoter or enhancer region of AR gene targets.

Upon binding to a hormone response element (HRE), the receptor dimer recruits coregulators (including coactivators and corepressors) to form an active preinitiation complex.  The preinitiation complex recruitment of coregulators occurs through the ligand-dependent transactivation function (AF-2) located in the LBD and hence controls transcription of specific genes.  The direct and indirect communications of the androgen receptor complex with several components of the transcription machinery are key events in nuclear signaling. This communication triggers subsequently mRNA synthesis and consequently protein synthesis, which finally results in an androgen response. Interestingly, androgen signaling via the androgen receptor can also occur in a nongenomic, rapid, and sex-nonspecific way by crosstalk.

Genomic organization of the AR gene: The genome spans more than 80 kb that includes the exonic organization shown in the second panel. Location of three codon repeat regions in the first exon that codes for the N-terminal domain is in the third panel. The diagram of the protein structure demonstrates how the exon organization translates into discrete functional regions of the receptor.  

AR is a member of the steroid hormone receptor family of genes. Like the other members of this family of transcription factors, the exons of the AR gene code for functionally distinct regions of the protein similar to the modular structure of other steroid hormone receptor genes. Like other members of the nuclear receptor superfamily, AR has four major functional regions: the N-terminal transactivation domain (NTD), a central DNA-binding domain (DBD), a carboxyl terminal ligand-binding domain (LBD), and a hinge region connecting the DBD and LBD.  

ANDROGEN RECEPTOR

The AR genomic organization is conserved throughout mammalian evolution from rodents to man. The human androgen receptor gene maps to the long arm of the X-chromosome at Xq11.2-12 [approx. 186 kb].  The gene is oriented with the 5' end toward the centromere and spans ~90 kb of DNA containing eight exons  that code for a ~2,757-base pair open reading frame within a 10.6-kb mRNA.  The human androgen receptor protein includes 919 amino acid residues with a molecular mass of approximately 110 kDa.  

Because of differential splicing in the 3'-untranslated region, studies show two AR mRNA species (8.5 kb and 11 kb, respectively) in several cell lines.  There is no structural indication in the AR mRNA for any preferential use of either of the two transcripts and neither for a specific function and 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.

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).  Encoding the N-terminal domain (NTD), the transcriptional regulatory region of the protein, is exon 1 (1586 base pairs), exons 2 and 3 (152 and 117 base pairs, respectively) encode the DNA-binding domain (DBD), exons 4 to 8 code for the C-terminal ligand-binding domain (LBD), which vary from 131 to 288 base pairs in size, and there is a small hinge region between the DNA-binding domain and ligand-binding domain.

Specific interaction with androgenic ligands results in the conformational activation of AR. This allows the binding of the occupied AR with additional nuclear proteins (i.e., coactivator proteins and general transcription factors) to produce transcriptional complexes that can activate or repress specific gene expression by binding to the androgen-responsive elements present in the promoter regions in a series of androgen regulated genes. It does this by forming an active transcriptional complex, resulting in site-directed chromatin remodeling and enhancement of target gene expression.

Hormone binding induces a transconformation of the receptor and allows its translocation into the nucleus where it initiates transcription through specific interactions with the transcription machinery.  

 

 

 

 

 

 

 

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