Involvement of three glutamine tracts in human androgen receptor transactivation

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Abstract

The androgen receptor (AR) possesses a polymorphic polyglutamine tract (polyQ), whose length is inversely correlated with its transcriptional activity. Here, we investigated whether 6 and 5 repetitive glutamine tracts (Q6 and Q5, respectively) in the N-terminal domain of AR also have effects on AR transactivation. In a reporter gene assay using two-tandem repeats of an androgen response element, deletion of glutamine tracts significantly increased AR transactivation in the following order: wild-type < a single deletion of polyQ or Q5 < double deletion of polyQ and Q6 < double deletion of polyQ and Q5 < triple deletion. Deletion of polyQ alone or combined deletion of polyQ and Q5 from an AR mutant lacking the ligand-binding domain, which is constitutively active due to activation function-1, increased AR transactivation. However, the glutamine tracts had no influence on activation function-1 activity, suggesting that the glutamine tracts modulate the binding of AR to DNA. Q5, like polyQ, was found to be involved in the interaction between the NH2- and COOH-terminal regions of AR (N–C interaction). These results indicate that the inhibitory effects of polyQ and Q5 on AR transactivation are the due, at least in part, to their negative regulation of N–C interaction.

Introduction

The actions of androgens such as dihydrotestosterone (DHT) are mediated by the androgen receptor (AR) which is a member of the steroid hormone receptor superfamily. After binding to the ligand, AR interacts with an androgen response element (ARE) in the target gene promoters and functions as a transcription factor [1], [2]. Because androgen-dependent AR actions are involved in many physiological processes such as male sexual differentiation and development of skeletal muscle, bone, and prostate gland, the relationship between the structural features and function of AR has been extensively studied [3], [4], [5], [6]. AR and other steroid hormone receptors share a common structure, consisting of an N-terminal domain (NTD), a central DNA-binding domain (DBD), a hinge region, and the ligand-binding domain (LBD) [7]. The NTD is highly divergent among the steroid hormone receptors in primary structure and contains an activation function (AF)-1 region. The LBD, which is moderately conserved, contains a ligand-dependent AF-2 region. A bipartite nuclear localization signal is located in the hinge region. The ligand-independent AF-1 activity of AR is much stronger than the AF-1 activities of other steroid hormone receptors, and the ligand-dependent AF-2 activity of AR is weaker than the AF-2 activities of other steroid hormone receptors. In fact, the AR lacking C-terminal LBD (ARΔC-Nuc) constitutively localizes in the nucleus and exhibits constitutive activity due to its AF-1 activity [8]. In order for full-length AR to exert its proper and maximal transcriptional activity, the ligand induces intramolecular and/or intermolecular interaction between the N-terminal region of NTD and the LBD in the C-terminus of AR (N–C interaction) [9]. In addition, recruitment of coactivators is essential for AR transactivation [1], [2], [10].

The NTD of human AR has a polymorphic polyglutamine (polyQ) tract with an average of 20 repeats. The bias in length of polyQ tract is considered to increase the risk of certain conditions or diseases. For example, in human males, a polyQ length of more than 28 residues greatly increases the risk of infertility resulting from impaired spermatogenesis [11]. A polyQ length of more than 40 residues causes spinal and bulbar muscular atrophy (SBMA/Kennedy's disease) which is a late-onset motor neuron disease [12]. SBMA patients develop a progressive muscle weakness and signs of partial androgen insensitivity such as infertility and gynecomastia [13]. In contrast, a polyQ tract of less than 20 repeats has been associated with increased risk of prostate cancer [14], [15]. Human AR has a tract of 6 glutamine residues (Q6) that is proximal to the C-terminus of polyQ, and a tract of 5 glutamine residues (Q5) 103 residues downstream of Q6. The lengths of the AR glutamine tracts in other mammals differ markedly from those in human AR (Fig. 1). On the other hand, no corresponding repetitive glutamine regions are found in lower vertebrates such as chicken (GenBank/NM_001040090), frog (GenBank/U67129), and goldfish (GenBank/AY090897). In rat AR, the first and third glutamine stretches are much shorter and much longer, respectively, than those in human AR. As shown in Fig. 1, the mammalian AR amino acid sequences, not including the glutamine tracts, are highly conserved. However the functions of Q6 and Q5 in human AR remain unclear. In this study, we investigated the roles of Q6, Q5 and polyQ in the function of human AR by examining the transcriptional activities of a series of deletion mutants.

Section snippets

Cell culture

Human prostate cancer PC-3 cells (AR-negative) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin and maintained at 37 °C in a 5% CO2/95% air atmosphere at 100% humidity unless otherwise indicated. PC-3 cells were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Miyagi, Japan).

Plasmids

Human wild-type AR expression vector pcDNA3.1-AR was described

Role of glutamine regions in AR transactivation

To investigate the roles of Q6, Q5 and polyQ in the transcriptional activity of AR, we constructed a series of deletion mutants of AR (ΔpolyQ, ΔpolyQ + Q6, ΔQ5, ΔpolyQ + Q5, and ΔpolyQ + Q6 + Q5) as shown in Fig. 2A and examined the transcriptional activities of AR mutants using a luciferase reporter assay. To exclude the effect of endogenously expressed AR, we employed AR-negative prostate carcinoma PC-3 cells in this study. In the presence of DHT, the transcriptional activity of ARΔpolyQ was

Discussion

The relationship between the structure and transcriptional activity of AR has been widely studied because ligand-dependent transcriptional activity of AR is involved in many physiological and pathological processes. Mammalian AR has unique glutamine repeat regions with different lengths, and Q6 and Q5 in addition to the polyQ region are located in the NTD of human AR (Fig. 1). In the present study, we demonstrate that the Q6 and Q5 regions are involved in the regulation of AR transactivation,

Acknowledgements

This work was supported by Grants-in-Aid (20580141 and 21780132) for scientific research (to R.Y. and N.H., respectively) from the Japan Society for the Promotion of Science.

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