The Androgen Receptor
The androgen receptor is a member of the steroid receptor family of nuclear transcription factors. This family is a group of structurally related nuclear transcription factors that mediate the action of steroid hormones. The steroid receptor family includes three other receptors including the glucocorticoid receptor, the mineralocorticoid receptor, and the progesterone receptor (Beato 1989). Although there are several regions of each receptor that are heterologous, the ligand-binding and DNA-binding domains are surprisingly highly conserved (Sheffeild-Moore 2000). In addition to their structural homology, these receptors also are related by their ability to activate gene transcription via the same DNA hormone response element (Quigley et al 1995).
There are two characterized forms of the androgen receptor. The first, and predominant form, is a 110-114 kDa protein of 910-919 amino acids (Jenster et al 1991, Wilson et al 1992, Liao et al 1989). The second is a smaller 87 kDa protein of about 720-729 amino acids in length that makes up only about 4-26% of the detectible androgen receptors located in varying tissues (Wilson and McPhaul 1996). The relevance of this second form of receptor is unknown, but the full-length receptor has been well-characterized. The isolation and characterization of this form of the human androgen receptor cDNA has allowed for sequencing of its amino acid constituents (Chang et al 1989).
The human androgen receptor is a single polypeptide comprised of four discrete functional domains (Quigley 1998).
The A/B region is the N-terminal domain of the AR and comprises over half of the receptor protein (residues 1-537). Within this domain is a transcription activation region and several regions of homopolymeric amino acid stretches that may be important in transcriptional regulation. These amino acid stretches may also be important in interactions with other regions of the receptor protein and in determining the three-dimensional structure of the receptor. Among the four members of the steroid receptor family, this region is poorly conserved both in length and sequence similarity (Evans 1988). The C region of the AR (residues 559-624) is the DNA binding domain. This region is composed of two folded "zinc fingers" which each binding one zinc ion. The first zinc finger is responsible for recognition of the target DNA sequence while the second stabilizes DNA-receptor interaction by contact with the DNA phosphate backbone (Freedman 1992, Berg 1989). Between members of the steroid receptor family, this region is the most highly conserved. At the overlap between the between the C and D regions, there is a nuclear targeting sequence (amino acids 617-633) that is responsible for androgen dependent translocation from the cytosol to the nucleus (Jenster 1993). The D region, or hinge region (residues 625-669), seems to be responsible for androgen dependent conformational changes of the AR. In addition, one of the AR phosphorylation sites is located in this region (Zhou et al 1995). Finally, the E region is the C-terminal domain of the androgen receptor and is responsible for ligand binding. This region consists of about 250 amino acids (residues 670-920) and functions in specific, high affinity binding of androgens. This region is also thought to be the binding site for inhibitory proteins such as the 90 kDa heat shock protein that resides on the inactivated AR. Transcriptional co-activators may also reside here (Jenster 1991).
The AR, as described above, has been identified in a vast array of genital and non-genital tissues using several techniques including northern and western blot analysis as well as immunohistochemical techniques. Most recently, immunohistochemical techniques have become the predominant method of characterization of both cellular and subcellular distribution of the AR due to the sensitivity, specificity, and ease of the method (Ruizeveld De Winter et al 1991, Kadi et al 1999, Sar et al 1990). Using immunohistochemical techniques, the AR has been clearly demonstrated in nearly all tissues (Janssen et al 1994, Kimura et al 1993, Takeda et al 1990). As mentioned earlier, despite characterization in both the cytosol and nucleus, the exact location of ligand binding is not yet clear. The predominant model (shown above in figure 1) however suggests that the cytosolic AR is inactive until it binds ligand. At this point it it undergoes an appropriate conformational change and it is transolcated to the nucleus via the nuclear targeting sequence (residues 617-633; Grino et al 1987, Jenster et al 1993, Zhou et al 1994). At this point, it is able to undergo binding to the androgen response element.
Based on early studies, the AR has been identified as a high affinity, low capacity receptor. Saturation binding analyses done using the androgen receptor radiolabelled ligands [3H]-T, [3H]-methyltrienolone (MT), and [3H]-DHT have shown saturability and therefore have been analyzed using scatchard plots corrected for nonspecific binding. The results of such investigations have been difficult to interpret however due to several confounding variables. First, since the amount of AR protein seems to be very low, small experimental errors will translate into large statistical errors. In addition, a significant amount of non-specific binding is found in AR experiments (Snochowski et al 1979). If this binding is not corrected for, then experimental errors could again be large. Also, androgens (especially T and DHT but not MT) are subject to several metabolic enzyme systems that differ between tissues. If uncontrolled, these metabolic pathways could also confound results. For example, measurements of apparent DHT binding to the AR may be underestimated due to metabolic conversion to androstanediols that do not bind to the receptor while the measures for apparent T binding could be overestimated due to formation of DHT which will bind strongly to the receptor (Michel and Baulieu 1980). Finally, since Kd values and Bmax values for the AR are variable with species, sex, age, and androgen levels, controlling all variables relevant to the AR presents a difficult proposition (Stahl et al 1978). As a result of the potential confounds discussed, Kd values from 0.074nM up to 6.4nM and Bmax values from 1-30 fmol/mg protein have been reported for the AR in various tissues using different radiolabelled ligands. With this said, close inspection of the published literature reveals that since different radiolabelled ligands have been used in the research, since different cytosolic preparations have been employed, and since different populations and species have been used, it only stands to reason that variable results have been obtained.
In this review, I will discuss binding affinity and capacity in one tissue (skeletal muscle) to simplify the discussion. Since skeletal muscle does not possess significant amounts of the enzyme responsible for the conversion of T to DHT (5alpha reductase), T is the predominant AR ligand. In research examining the skeletal muscle AR using labeled T, the lack of metabolic conversion to DHT helps to eliminate one potential confound. Other metabolic conversions using the 3alpha- and 3beta-hydroxysteroid dehydrogenases remain and can only be controlled by the addition of ammonium sulfate to the cytosolic preparation to eliminate the activity of these enzymes (Michel and Baulieu 1980). These two precautionary measures, however are incomplete and the synthesis of methyltrienolone (17 alpha-methyl-3-oxo-estra-4,9,11-trien-17 beta-ol), which binds to the AR with the same affinity as T yet is not metabolizable, was of greta value to AR binding studies. The use of labeled MT therefore has added another element of control to AR studies.
Preliminary work by Michel and Baulieu using [3H]-T and [3H]-androstanolone (DHT) ligands in enzyme-free preparations of castrate male and female rat quadriceps femoris yielded similar Kd values for both ligands of approximately 0.70nM (Michel and Baulieu 1980). These values are similar to several other reports of T affinity in skeletal muscle. In another investigation using rat thigh homogenates prepared with [3H]-T, Kd values of approximately 1nM and Bmax values of 15-30 fmol/mg protein were found (Michel and Baulieu 1974). The Bmax values in this experiment are higher than those reported anywhere else in the literature for skeletal muscle. Krieg et al, using [3H]-androstanolone (DHT) in rat muscle homogenates found a Kd range of 1.4nM to 6.4nM and 0.8-4.2 fmol/mg protein (Krieg 1976). Krieg et al could not explain the reason for differences between their work and the work of Michel and Baulieu but hypothesized that such errors could potentially arise due to the very small amount of receptors in the cytosol as well as the fact that these receptors are very difficult to isolate. Later work by Saatok et al, using the nonmetabolizable [3H]- MT in rabbit skeletal muscle cytosol preparations, found Kd values of 1.25-1.66nM and Bmax values of 2.76-5.18 fmol/mg protein, potentially confirming the work of Krieg (Saatok 1984). Finally, work by Snochowski et al also using [3H]- MT in male and female human skeletal muscle cytosolic preparations has indicated that Kd values for the AR were approximately 0.074-0.7nM (mean of 0.28nM) while Bmax values were 1-4fmol/mg protein (Snochowski et al 1981). From these variable data it is obvious that although the AR is clearly a high affinity and low capacity receptor, useful and consistent quantitative data have not been obtained regarding affinity and capacity in muscle. The differences in this literature again could be due to the different radioligands used in the studies, different ages and androgen levels in the subject populations, and amplified experimental errors due to such small levels of detectible AR protein. To discuss skeletal muscle in relation to the prostate, although variable results have been obtained for prostate tissue as well, Bmax values seem to be about 10fold lower (per mg protein) while Kd values are of a similar magnitude (Ekman et al 1979).
Androgen Metabolism and AR Binding
To further elaborate on the importance of androgen metabolism in the determination of AR levels and, more importantly, in the mechanism of action of androgens, a brief discussion of this subject is in order. T and DHT are the two most potent androgens in the body however their relative importance in different tissues varies. It is known that although the prostate AR can bind both T and DHT, the affinity of the AR for T is only 33% of that for DHT (Grover et al 1975). In contrast, in skeletal muscle and kidney fractions, the binding affinity for T is greater than that for DHT despite affinity for both ligands (Gloyna and Wilson 1969, Bullock et al 1974). T itself is extensively metabolized in most androgen-sensitive tissues. The predominant metabolite in the prostate, for example, is DHT. Important in this metabolic pathway are the relative levels of 5alpha reductase and 3alpha- and 3-beta hydroxysteroid dehydrogenases. Since DHT is the active metabolite in most tissues, high conversion of T to DHT via the 5 alpha reductase pathway and low metabolism of DHT via the 3 hydroxysteroid dehydrogenase pathways is necessary for optimal androgen action (Rommerts 1999). In skeletal musle, on the other hand, 5alpha reductase activity is low while 3alpha- and 3-beta hydroxysteroid dehydrogenase activity is higher leading to a predomination of T and other inactive metabolites. In fact, in skeletal muscle, less than 5% of T is metabolized (Minguell and Sierralta 1975). This is optimal due to the fact that in skeletal muscle, the AR has a higher affinity for T than for DHT. Interestingly, despite the differences between the metabolic pathways for T as well as the differences in affinities for various T metabolites, the AR appears to be nearly identical in structure across androgen sensitive tissues. The differences in androgen action between tissues is now thought to be due to DNA response elements as well as specific co-activators or repressors present in the different tissues.
Conclusions
In conclusion, this review has discussed several relevant topics in androgen pharmacology, physiology, and receptor theory. Although much is yet to be discovered regarding androgen mechanism of action, the androgen receptor, regulation of androgen receptor mediated transcription, and control of the androgenic and anabolic effects of testosterone and its metabolites, new discoveries are rapidly being reported. With investigations currently being conducted to examine androgen receptor co-activator proteins as well as androgen response element functions in a wide spectrum of tissues, manipulation of some of the diverse actions of androgens can be postulated. And these discoveries may contribute to the holy grail of androgen research; successful dissociation of the anabolic and androgenic affects of androgens.
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