Lessons to be learned from the androgen receptor

ARTICLE

Androgens play a crucial role in several stages of male development. They act via an interaction with the androgen receptor, a ligand dependent transcription factor which belongs to the superfamily of nuclear receptors, including those for the other steroid hormones, the retinoids, the thyroid hormones, and a still growing number of orphan receptors [1, 2]. In the last decade, 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 [3-6]. The tissue specific actions of testosterone and 5alpha-dihydrotestosterone, mediated by the same androgen receptor, suggest a ligand specific recruitment of transcription intermediary factors (TIFs). However, experimental evidence for ligand specific TIFs for the androgen receptor has not been provided as yet. The androgen receptor protein displays a large homology in the DNA-binding domain and in the ligand-binding domain with the other members of the steroid hormone receptor subfamily (e.g. receptors for glucocorticoids, estradiol, progesterone and mineralocorticoids) [5, 7-11].

The aim of the present review is to present some aspects of androgen action unravelled recently. In this overview information will be given on functional domain structure of the human androgen receptor with emphasis on the recent findings of functional interactions between the NH₂-terminal domain and the ligand-binding domain. Post-translational modifications (phosphorylation) of the androgen receptor protein in relation to function will be discussed next. Throughout the text the numbering of the different codons is based on a total number of 919 amino acid residues of the androgen receptor [12].

Conformational changes induced by androgens and antiandrogens

Binding of androgens by the androgen receptor results in two conformational changes of the receptor molecule [13]. Initially, a fragment of 35 kDa, spanning the complete ligand-binding domain and part of the hinge region, is protected by the ligand, but after prolonged incubation times a second conformational change occurs resulting in protection of a smaller fragment of 29 kDa. In the presence of several antiandrogens (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. Further analyses with specific antibodies against different epitopes in the 35 kDa and 29 kDa fragments reveal that only the most COOH-terminal end of the androgen receptor protein is represented by the 29 kDa fragment [13].

Transactivation function in the ligand binding domain

Deletion and mutation studies, as well as mutations found in patients with either the androgen insensitivity syndrome, or prostate cancer have given some insight into which amino acid residues are important for ligand binding [12, 14-17]. The overall picture is that large deletions (> 10 amino acid residues) severely affect hormone binding, but interestingly deletion of the complete ligand binding domain results in a constitutive active molecule [14, 18].

In the ligand-binding domain of the human androgen receptor a transcription activation function (designated as AF-2) has been identified, although it is very weak in comparison with that found in other steroid receptors (e.g. estrogen and glucocorticoid receptors) [19-21]. The AF-2 domain in the androgen receptor can be activated in a hormone dependent way and is strongly enhanced in a promoter dependent way by the co-activators TIF2 and GRIP1 [19-21]. The boundaries of the AF-2 domain in the androgen receptor ligand-binding domain have not been determined as yet, but it contains the core region as defined in the ligand-binding domains of several members of the ligand dependent nuclear receptor family. This AF-2 activation domain (AD) core region contains the conserved sequence 893-Glu-Met-Met-Ala-Glu-897. Mutations in this region can result in a decrease in activation function without affecting the ligand-binding capability. This indicates that the amino acid residues of the AF2-AD core region are not directly involved in ligand binding, but are part of or determine the interaction surface. Recent studies on mutations in this region and the interaction of co-activators confirm this presumption [21, 22]. Interestingly, mutations have not been reported in the AF2-AD core region either of individuals with the androgen insensitivity syndrome or prostate cancer patients, which most likely implies that none of the individual amino acids in the AF-2 AD core region is essential in the full length androgen receptor.

Transactiviation functions in the NH₂-terminal domain

The boundaries of the NH₂-terminal transactivation domain in the androgen receptor (designated as AF1) are not exactly defined, but generally it appears that the region between amino residues 51-211 is essential for transactivation activity in the full length receptor [14]. This region is not involved in the transactivation capacity of the COOH-terminal truncated androgen receptor, which displays constitutive activity [18]. The most important activating region in the constitutive receptor molecule is located in the NH₂-terminal domain between residues 370 and 494. This region is designated as AF-5.

So, the androgen receptor can use different transactivation domains (AF1 and AF5, respectively, in the NH₂-terminal domain and AF2-AD in the COOH-terminal domain) depending on the "form" of the receptor protein (Fig. 1). Two AF functions are ligand dependent (AF1 and AF2), whereas AF5 operates in a ligand independent way. The ligand dependency of AF1 in the full length androgen receptor and the switch to AF5 in the COOH-terminal truncated androgen receptor strongly suggests a functional inhibitory action of the ligand-binding domain on AF1 in the absence of ligand and on AF5 in the presence of ligand. The AF2 function is strongly dependent on the presence of ligand and androgen receptor coactivators.

Functional interaction of the NH₂-terminal domain and the COOH-terminal domain

In the previous section evidence is presented for a possible interaction between the ligand-binding domain and the AF functions in the NH₂-terminal domain. Investigating this NH₂-terminal domain ­ COOH-terminal domain (N/C) interaction in more detail reveals that only certain regions in the NH2-terminal domain are involved in the interaction [21, 23-25]. Interestingly, the AF1 core region is not involved in this interaction; amino acid residues 3-36 as well as amino acid residues 370-494 are necessary for a proper functional interaction. In the COOH-terminal domain the AF2-AD core region (amino acid residues: 893-Glu-Met-Met-Ala-Glu-897) is involved in the interaction as was established by substituting an essential amino acid residue (Glu 897) by a glutamine residue. A similar mutation also affects the functional interaction of the androgen receptor ligand-binding domain with TIF2, suggesting that both the NH₂-terminal domain and TIF2 are recognising the same interaction surface of the ligand-binding domain upon hormone binding [21].

Androgen receptor phosphorylation

The newly synthesized androgen receptor protein migrates as a 110 kDa protein during SDS-PAGE and becomes phosphorylated within 10 minutes upon synthesis, resulting in an additional protein band at 112 kDa [26, 27]. This rapid post-translational modification is important for the acquisition of the hormone binding properties of the androgen receptor [28].

A second important phosphorylation step of the androgen receptor occurs upon hormone binding resulting in a third isoform migrating at 114 kDa during SDS-PAGE [27, 29]. All three isoforms (e.g. 110, 112 and 114 kDa) exist in several androgen responsive cell lines in the presence of androgens and migrate as a triplet. The presence of the triplet correlates very well with DNA-binding by the androgen receptor, because mutational analysis of certain amino acid residues in the DNA-binding domain, which severely affects the DNA-binding properties of the androgen receptor, also simultaneously displays a defective hormone induced phosphorylation [27]. Recently the absence of the androgen receptor triplet in genital skin fibroblasts from a patient with the androgen insensitivity syndrome has been used as indicator for a androgen receptor defective in DNA-binding [29]. In the androgen receptor gene of this patient a mutation was found in the splice acceptor site of intron 2, resulting in a defective splicing of the androgen receptor mRNA. The mature transcript contained an additional 69 nucleotides between exon 2 and exon 3 sequences. The translation of this altered splice product is a protein with an insertion of 23 amino acid residues between the first and the second zinc cluster of the DNA-binding domain. Additional protein analysis experiments revealed that in genital skin fibroblasts of the index patient the extended protein was expressed in large quantities. Tight nuclear binding of this mutated receptor protein could not be observed, corresponding with the absence of the triplet isoforms normally seen for wild type androgen receptors. Only a doublet of 110-112 kDa was expressed, indicating a defective DNA dependent phosphorylation of the human androgen receptor.

The experiments described above indicate that post-translational modification (e.g. phosphorylation) of the androgen receptor protein might be important at two different steps of receptor activation: 1) acquisition of ligand binding capabilities and 2) during transformation to the DNA-binding/transcription activation form.

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