Human Androgen Receptor
Created by Hilary Whitworth
The androgen receptor (AR) is a steroid receptor critical to the development and maintenance of the male phenotype throughout the stages of human life including embryogenesis, virilization and homeostasis (1). The androgen receptor is able to have such a dramatic effect on and control over the male phenotype because it acts as a transcription factor. After binding hormone, the AR translocates into the nucleus where it dimerizes with itself and proceeds to interacts with DNA at androgen response elements on regulatory regions of target genes (5). The androgen receptor is in the nuclear receptor family of proteins (6). These are receptor proteins for ligands including steroid, thyroid hormone, vitamin D3, and retinoic acid (6).
The androgen receptor is similar to other nuclear receptors. The progesterone receptor (PDB ID 1E3K) is also a steroid nuclear receptor and therefore has a very similar structure and can bind some of the same ligands, including R1881 (4). The similarity of the AR Ligand binding domain with other receptors can also be seen by comparing the sequence of the human AR with the sequences of the human progesterone receptor, the human mineralcorticoid receptor, and the human glucocorticoid receptor [see image] (6). The image also includes sequences of androgen receptors from other organisms (6). This structure can be divided into four domains: the N-terminal domain (NTD), the DNA binding domain (DBD), the hinge region, and the ligand binding domain (LBD) (3). Other researchers have described only three functional domains, leaving out the hinge region (5). The four regions will be outlined here with a detailed description of the LBD because it, when complexed with R1881, is the chosen domain for the project.
The NTD is a flexible and disordered region which contains a major activation region, the AF-1. When the AF-1 region is separated from the ligand binding domain, the protein becomes constitutively active. The AF-1 region interacts with the LBD causing an N to C terminal inter or intra molecular interaction that is potentially necessary for the transcriptional activation of some AR target genes. The NTD also contains homopolymeric repeats, most importantly a polyglutamine repeat that when extended or shortened too much leads to clinically relevant phenotypic changes. (3)
The DNA binding domain (PDB ID 1R4I) is much more conserved than the NTD and has therefore been easier to describe. Shaffer et al. was able to crystallize this domain binding to androgen response elements on DNA (7). There are two zinc fingers formed and a C terminal extension in this domain. The first zinc finger has been found to regulate the specification of DNA binding and the second to be involved in the dimerization of the AR. The AR homodimer is composed of the AR monomers bound in a head to head conformation. The C terminal extension is important in the overall structure and also works with the first zinc finger on DNA binding specificity. (3)
The hinge region serves to link the DBD and the LBD. While it is not always considered a functional domain, Heemers et al. described it as aiding in many functions, including: DNA binding, AR dimerization, and nuclear translocation. This is because the nuclear translocation signal is partially located on this linking region. (3)
The ligand binding domain is a conserved region located in the C terminus (5). It's structure resembles that of ligand binding domains in other nuclear receptors, containing 12 alpha helices(H1-H12) in an alpha helical sandwich conformation (3). There are also beta strand components to the secondary structure (5). None of the residues in the LBD lies outside the allowed regions of the Ramashcandran plot (4). Binding of an agonist stabilizes H12 and this forms a hydrophobic groove so called AF-2 (3). More specifically, when agonist is bound, H12 closes the ligand binding pocket and when antagonist is bound, H12 has a different conformation and opens the entrance to the ligand binding pocket (4). The ligand binding pocket is composed H3, H4, H5 and H11 as well as the beta strand. It is mostly hydrophobic amino acids with side chains that can adopt various positions so that they can fit the bound ligand (5). This nonspecific, non-polar region uses van der Waals forces to interact with the hydrophobic regions of the steroid ligand and this lack of specificity allows the protein to interact with ligands of various structures (5). Conversely, there are some polar amino acids that interact through hydrogen bonding with the hydrophilic oxygen molecules at either end of the steroid and this increases the stability of the interaction (5).
The ligand R1881 is the chemically modified steroid metribolone (5). When the LBD of the AR is complexed with this specific ligand, as in the chosen protein, the secondary structure includes 9 alpha and two 3-10 helices and four beta strands. (4). Some notable structural elements include a disulfide bridge forms between Cys-669 and Cys-844 and a cis peptide bond occurs at position Pro 849 (4). In this form, there are 18 residues involved in the binding: F-891, M-895, N-705, L-701, L-880, T-877, F-876, Q-783, M-787, M-742, F-764, L-744, M-749, R-752, Q-711, M-745, L-707, L-704, which together form the ligand binding pocket (4). Arg-752 forms a hydrogen bond with the O-3 of R1881 and a water molecule near O-3 (4). That water molecule also interacts via hydrogen bonds with Met-745 and Gln-711 (4). A different water molecule also hydrogen bonds with Gln-711, Val-685 and Phe-764 (4). The 17-Beta hydroxyl group of R1881 forms hydrogen bonds with Asn-705 and Thr-877 (4).
The binding of androgen to LBD of the AR leads to the eventual function of the AR by causing a series of events including homodimerization, nuclear translocation, and the binding of the AR to androgen response elements on the regulatory regions of target gene DNA (5). Therefore, mutations in the ligand binding domain can have drastic effects on the transcription of the AR dependent genes and this may lead to clinically relevant phenotypes. Two main clinical effects of AR mutation are androgen insensitivity and prostate cancer (4). Androgen insensitivity syndrome is a disorder causing a range of phenotypes including a complete female phenotype and infertility, correlated with mutations in the androgen receptor gene (6). Similarly, prostate cancer correlates with androgen receptor mutations. As prostate cancer advances, the androgen receptor becomes increasingly important. Despite low levels of androgen in late stage prostate cancer, the androgen receptor remains present and inhibition of the androgen receptor leads to a decrease in prostate cancer cell growth (2).
Many of the known mutated residues in the LBD (50%) are involved in these two diseases. Those involved in prostate cancer tend to occur near the R1881 17-beta hydroxyl group, except for the mutation of Met-749 which is implicated in prostate cancer and is near the O-3 of R1881. Mutated residues in androgen insensitivity syndrome are located throughout the LBD. Three mutations are important in complete androgen insensitivity syndrome (CAIS) and all three are substitutions that change the size of the amino acid side chain. N-705 normally hydrogen bonds to R1881 and helps stabilize the H11 H12 loop. In CAIS, N mutates to S and can no longer form this stabilizing bond. The second mutation is from L-707 to R-707. L-707 contacts the A ring of R1881 and five residues within the protein: V685, A687, Gln711, F764, L768. These amino acids are in a loop between H2 and H3, within H3 and in S1 and S2. The mutation disrupts the LBD and the folding of the protein itself because it does not have these interactions. The third mutation is M749 to V749, again changing the size of the side chain and affecting the ligand binding pocket. With these mutations there is no detectable R1881 bound and therefore no transcriptional activity. (4)
Additional mutations implicated in less severe forms of androgen insensitivity syndrome include the substitution of R-752 to N-752 which disrupts a hydrogen bond between the A ring of the steroid and the LBD, therefore disrupting the stability of binding. Also, with the mutation of F-764 to S-764, R1881 binds with similar affinity but dissociates much faster, decreasing the effect of the hormone. Similar to the previous mutation, F764 is involved in stabilization of the A ring as shown in the previous hydrogen bond. (4)
The androgen receptor's biological role as a nuclear receptor and transcription factor has important phenotypic effects that become evident upon disruption of normal processes. The serious clinical consequences of mutation of the androgen receptor indicate the close correlation between the structure of the molecule, especially the ligand binding domain, and its overall function.