Molecular Human Reproduction, Vol. 8, No. 2, 101-108,
February 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive endocrinology |
Androgen receptor mutations causing human androgen insensitivity syndromes show a key role of residue M807 in Helix 8–Helix 10 interactions and in receptor ligand-binding domain stability
1 Department of Obstetrics and Gynecology and 2 Bioinformatics Centre, National University of Singapore, Lower Kent Ridge Road, Republic of Singapore 119074
Abstract
Transactivation activity of the androgen receptor (AR) is induced by the binding of an androgen to its ligand-binding domain (LBD). The tertiary architecture of the AR LBD, in common with other steroid/nuclear receptors, is a sandwich of 12 
-helices (H). We have encountered a missense substitution, M807T, which was associated with partially defective androgen binding in a 46,XY infant with ambiguous genitalia. In contrast, two other substitutions in the same residue 807 to valine and arginine, resulted in almost total abrogation of androgen-binding and complete androgen insensitivity syndrome in two unrelated individuals. We recreated these substitutions in residue 807 and observed that disruption of ligand-binding and transactivation activities was total for M807R and partial for M807V, while the least-affected was M807T. Modelling of the AR LBD indicate that van der Waal interactions between residue 807 (H8) to H9 and H10 were severely disrupted for the arginine mutant, but relatively preserved for the threonine and valine mutants. However, there was a subtle difference between these two variants in that M807T, but not M807V, improved van der Waal contacts with another residue L859 in H10, suggesting the importance of interactions between M807 and L859 for LBD stability. Atomic distances of M807 (H8) to L859 (H10) in corresponding residues of the distantly related ER, RXR, PPAR
and VDR LBD are highly conserved and almost invariant, suggesting that H8/H10 interactions are critical for LBD stability in other members of the steroid/nuclear receptor superfamily.
androgen receptor/helical stability/mutations
Introduction
The physiological androgens, testosterone and dihydrotestosterone (DHT), mediate many differences between man and woman. All androgens act through the androgen receptor (AR) which is encoded by a single copy gene located on the X chromosome. The AR, when activated by the appropriate ligand, translocates to the nucleus and binds to specific response elements in the promoters of androgen-responsive genes, thereby initiating transcription (Quigley et al., 1995
). Like other members of the steroid/nuclear receptor superfamily of transcription factors, the AR has four main functional domains, comprising the N-terminal transactivation domain, a DNA-binding domain, a hinge region and C-terminal ligand-binding (LBD) domain (Figure 1). Transactivation activity of the AR is induced by the binding of an androgen to its LBD, and mutations of the ligand-binding pocket can render the receptor totally unable to bind the ligand, resulting in complete androgen insensitivity syndrome (AIS) and the female phenotype in individuals who are chromosomally male (Gottlieb et al., 1998).
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Although the LBD of various members of the steroid receptor family have only 20–50% homology in general (Wurtz et al., 1996
), the tertiary architecture of the AR (Matias et al., 2000) is very similar to that of other nuclear receptor LBD characterized to date, consisting of a sandwich of 11 to 12 -helices (H). The outer leaves of this sandwich, comprising H1/2, H3 on one side, and H6, H7, H10/11 on the other, envelopes a hydrophobic core. Helix 4/5, H8, and H9 form one-half of this hydrophobic core, the second portion of the core contains an open ligand-binding pocket, which, in the presence of androgen, is closed by repositioning of terminal H12. Twelve hydrophobic residues contact the bound androgen and mutations affecting these androgen-binding residues cause perturbations of ligand-binding dynamics and androgen insensitivity (Poujol et al., 2000). However the structural basis, whereby mutations of residues outside the androgen-binding pocket result in defective ligand-binding and AIS, is not clear. We have shown that such a mutation, involving a missense substitution of methionine by threonine in residue 807, is associated with partially defective androgen binding in a 46,XY infant with ambiguous genitalia, clitoromegaly and partial labio-scrotal fusion (Ong et al., 1999). M807 is a highly conserved residue in H8 of the hydrophobic core (Figure 1
) but does not form part of the androgen-binding pocket (Matias et al., 2000). Nevertheless, administration of DHT, but not testosterone, resulted in markedly improved male genital development in this infant with the M807T mutation, congruent with in-vitro observations indicating that DHT binds and activates the mutant AR more efficiently than testosterone.
Interestingly, two other substitutions in the same residue 807, to valine (Murono et al., 1995) and arginine (Adeyemo et al., 1993), have been shown to result in almost total abrogation of androgen-binding and complete AIS in two unrelated XY individuals, in contrast with the partial AIS phenotype observed with the threonine substitution. It is unclear why substitutions in the same residue involving conservative molecules like valine and threonine can result in such differing phenotypes. To elucidate the molecular mechanisms and structural basis whereby the mutations in residue 807 cause differing degrees of receptor dysfunction, we recreated these substitutions and compared their ligand-binding and transactivation properties. These biochemical properties were also examined in conjunction with structural models of the AR mutants. Our method of combinatorial analyses allowed us, for the first time, to appreciate the importance of interactions between M807 in H8 of the hydrophobic core to highly conserved residues in H10 of the outer layer, for the structural integrity of the AR, and of other steroid/nuclear receptor, LBD.
Materials and methods
Construction of M807T, M807V and M807R AR expression plasmids
Mutations in codon 807 were created in cDNA fragments using PCR-based site-directed mutagenesis and cloning into the homologous section of an AR expression vector, pSVhAR (Ghadessy et al., 1999). Briefly, to recreate the M
T (ATG-AGG) base change in codon 807 of the AR cDNA, we designed the following two internal primers (with the mutated codon underlined): sense primer F, 5'-CCTGTGCACGAAAGCACTGC-3' and antisense primer R, 5'-GCAGTGCTTTCGTGCACAGG-3'. The M807V and M807R mutants were similarly created by using primers with the underlined codon appropriately substituted. We used primers B (5'-GTG TCA CACATTGAAGGCTATG-3') in exon 4, and P3' (5'-CACCAACCTTCTCGATAGGCAGC-3') in the ß-globin poly-A tail of the plasmid as outside primers.
Transactivation activity of mutant AR
Mutant or wild type (WT) AR expression plasmids and a luciferase reporter plasmid containing two ARE, ARE-Tata-Luc were co-transfected into HeLa cells, using the lipofection technique (Wang et al., 2001). Transfected cells were exposed to androgens for 48 h before harvesting and quantification of luciferase activity. The androgens used were testosterone, DHT and the non-metabolizable synthetic androgen, mibolerone (MB).
Immunoblotting
Immunoblotting was performed as previously described (Wang et al., 2001). Transfected COS-7 cells were lysed and 30 µg of protein from the cell lysate was resolved on a 7.5% sodium dodecyl sulphate–polyacrylamide gel. Proteins were transferred onto PVDF membranes. AR protein was bound to a human AR monoclonal antibody directed against amino acids 299–315 (mouse anti-hAR; Santa Cruz Biotech. Inc., CA, USA) and subsequently detected with anti-mouse secondary antibody conjugated to horse-radish peroxidase.
Androgen-binding properties of receptors
The ligand-binding properties of mutant and WT AR in transfected COS-7 cells were studied. Confluent monolayer cultures were exposed to increasing doses of tritiated androgens: [1,2,6,7-3H]testosterone, 5-dihydro[1,2,4,5,6,7-3H]testosterone and [17-methyl-3H]MB in serum-free medium for 2 h at 37°C, and specific radiolabelled androgen binding was determined as previously described (Lim et al., 2000). Specific androgen bound, expressed as fmol androgen bound/mg protein, was the difference between total and non-specific binding and each data point was the mean of quadrates.
Computational modelling of AR and other steroid receptor LBD
Homology comparisons and alignments were performed with the ClustalW (v1.6) multiple sequence alignment software. Modelling of structures was performed using LOOK (Molecular Application Group) (Lee and Subbiah, 1991). Although the crystal structure of the AR LBD has been published, the precise coordinates necessary for modelling are not yet available (Matias et al., 2000). The AR is most closely related to the PR, showing a 56% sequence identity with the PR-LBD. Based on the root mean square deviation, the crystal coordinates of AR LBD differ from that of PR-LBD by only 1.16 Å, indicating the high degree of homology between the overall fold of both structures (Poujol et al., 2000). Therefore, a 3-D model of the AR LBD using the PR crystal structure coordinates can reasonably be utilized. Thus the crystal coordinates of ligand-bound progesterone receptor LBD (Williams and Sigler, 1998) was used as a folding template for the AR holo-LBD. The sequence of human AR LBD was initially aligned to the target sequence of PR, the tertiary structure was then modelled using a published algorithm (Lee and Subbiah, 1991). This algorithm uses self-consistent ensemble optimization to calculate the global minimum structure, allowing the positioning of side-chains with high accuracy (Kolatkar et al., 1999). Models of the LBD of the ER (Brzozowski et al., 1997), RXR (Bourguet et al., 1995), PPAR (Nolte et al., 1998) and VDR (Rochel et al., 2000) were constructed using published crystal coordinates, and atomic distances of homologous residues were compared to that of the AR LBD.
Results
Functional capacity of M807T, M807V and M807R
To elucidate the molecular mechanism whereby a threonine substitution in residue 807 results in defective AR activity, the M807T mutation was recreated in an AR expression plasmid by site-directed mutagenesis. The WT and mutant AR were co-expressed with an androgen-driven reporter gene, ARE-Tata-Luc, in HeLa cells. No activity of the reporter was detected in the absence of androgen (Figure 2). In the presence of physiological doses of testosterone (1–3 nmol/l), the WT receptor induced a maximal 300-fold increase in luciferase activity compared to replicates not exposed to androgen (Figure 2A). In contrast, the M807T mutant was non-functional, exhibiting <10% of WT activity at physiological doses of testosterone (1–3 nmol/l). However, the function of the mutant AR could be restored by increasing the dose of testosterone to supraphysiological levels (30–100 nmol/l). Thus the dose of testosterone which elicited half-maximal activity of WT AR (0.6 nmol/l) was an order of magnitude smaller than that for the mutant M807T (12 nmol/l). Interestingly, the other physiological androgen, DHT, was a more efficient activator of mutant AR, with half-maximal activity being observed at 1.5 nmol/l of the hormone (Figure 2B). At doses between 1 and 100 nmol/l, the strong synthetic androgen MB was able to fully restore M807T function (Figure 2C). Thus, although the dose–response curves of mutant AR were abnormally shifted to the right for all three androgens, the degree of abnormality was in the order: testosterone > DHT > MB.
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Substitutions of valine (M807V) or arginine (M807R) for methionine in residue 807 have been reported to cause complete androgen insensitivity and an almost non-functional AR in transactivation assays (Adeyemo et al., 1993
; Murono et al., 1995). To elucidate the molecular mechanism whereby a threonine substitution in residue 807 can result in some AR activity and the partial insensitivity syndrome, the transactivation function of the M807T mutant was compared to that of M807V and M807R (Figure 2A). Although all three mutants were transactivation defective with testosterone, there were differences in the degree of AR functional disruption. Thus, M807T had more than half the maximal activity of the WT at a dose of 10 nmol/l testosterone, and greater than maximal activity at 30 nmol/l. In contrast, M807V achieved only 25% of maximal WT activity, whereas M807R displayed no activity even at supraphysiological doses of testosterone. Therefore, the responses of the three AR mutants in vitro to testosterone were consistent with the degree of clinical androgen insensitivity observed, with our patient M807T being the least affected and M807R, the most affected. A similar hierarchical pattern of responses observed for DHT (Figure 2B) and the strong androgen MB (Figure 2C), with the disruption to AR function in the order: M807T < M807V < M807R (Table I). These differences in AR function were not due to differential protein expression, as an immunoblot showed that protein expression was similar for WT and mutant AR in the presence of DHT (Figure 2D).
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Comparative effects of M807T, M807V and M807R substitutions on androgen-binding capacity of AR
The ligand-binding properties of the mutants and WT AR in COS-7 cells were studied using radiolabelled androgens (Table I
). All three mutants M807T, M807V and M807R had strongly reduced testosterone binding (Figure 3). Our ambiguous genitalia associated mutation, M807T, bound only 14.7% of [3H]testosterone compared to WT and the complete AIS-causing mutations, M807V and M807R, bound only 4.9 and 16.9% respectively (Table I). In contrast, using [3H]DHT, the ligand-binding capacity of M807T was nearly restored to normal levels with 93% of WT binding. M807V and M807R also displayed improved DHT binding with 45.3 and 17.3% of WT binding respectively (Table I). Finally, with [3H]MB, M807T binding capacity was restored to near-normal levels, exhibiting 91% of WT binding. In comparison, M807V had 60% while M807R had only 4.6% of normal WT binding affinity with tritiated MB (Table I). Thus, the disruption to the ligand-binding characteristics of the AR mutants were closely related to the disruption to their transactivational function and were in the order: M807T < M807V < M807R.
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Molecular distances of M807 from neighbouring residues
To understand the structural basis for the varying functional defects in our mutants, we performed molecular modelling of the AR LBD using crystal coordinates from the closely related progesterone receptor (Williams and Sigler, 1998
). The 12 helices of the AR LBD are folded into a three-layered sandwich. Helices 1/2, H3, and H7, H10/11 form the two outer layers, while the inner layer consists of a ligand-binding pocket and a non ligand-binding hydrophobic core (Figure 4). The hydrophobic core is formed by H4/5, H8 and H9, the axes of which are oriented at about right-angles to the helices in the outer layers. Adjacent to this hydrophobic core is a cavity, the ligand-binding pocket, which on androgen binding is closed by H12, resulting in a stable holo-receptor. M807 lies in the middle of H8, the central helix in the hydrophobic core of the AR LBD. Our model predicts that residue 807 has van der Waal contacts with H7 and H10 from the outer layer of the sandwich, and with adjacent H5 and H9 of the hydrophobic core. Thus M807 is 4.13 and 3.81 Å from L859 and L863 respectively on H10, 3.59 and 4.39 Å from I799 and F794 respectively on H7, 3.95 Å from F747 on H5, and 3.82 Å from L838 on H9 (Figure 5). These spatial relationships suggest that M807 is a focal residue that anchors the outer layer (H7, H10/11) of the AR LBD to its hydrophobic core (H5, H8, H9).
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Molecular modelling of mutations in residue 807
Arginine
Generation of M807R showed the presence of a positively charged side-chain protruding into the hydrophobic cavity that surrounded the R807 focal point. The coordinates of the highly polar amide groups carried on R807 in the vicinity of the non-polar carbonyl groups of the neighbouring side-chains were as follows: L859 at 3.13 Å to R807, I799 at 2.44 Å to R807 and F747 at 3.02 Å to R807 (Table II
). The close proximity of these side-groups will result in phase antagonism between the surrounding hydrophobic alkyl side-chain clusters and the hydrophilic R807 side-chain, causing active side-chain disruptions, thereby completely destabilizing receptor structure and function, consistent with the complete AIS phenotype.
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Valine
Although the M807V mutation increased the distance to L859 to beyond van der Waal range, contact with H10 was still maintained through interactions with L863. Both interactions (F794, I799) with H7 were disrupted, while interactions with H5 and H9, through F747 and L838 respectively, were preserved (Table II
).
Threonine
Interestingly, the M807T mutant maintained links with H10 through an enhanced interaction, not with L863, but with L859 whose distance of 3.68 Å was optimal for van der Waal forces. Like the M807V mutant, M807T abrogated interactions with H7, while maintaining contact with L838 in H9 (Table II).
Although threonine and valine mutants retained partial AR function in ligand binding and transactivation assays, M807T preserved the AR function consistently better than did M807V. Furthermore, M807T was associated with a less severe AIS phenotype compared to M807V. It was interesting to note that the key difference between the threonine and valine mutants was that M809T improved van der Waal contact with L859 (3.68 Å), whereas M807V exerted a strong negative effect by increasing this distance to 5.54 Å (Table II). Since M807T preserved AR function more than M807V in vitro and in vivo, our data suggested the importance of interactions between residues 807 in H8, and 859 in H10.
Structural comparisons with ER, RXR, PPAR and VDR
Interestingly, residues in H10 (L859, L863), H9 (L838), and H5 (F747) whose van der Waal interactions were relatively preserved in our threonine and valine mutants, are conserved even in distantly related receptors like ER, RXR, PPAR and VDR (Figure 1). In contrast, the residues in H7 (I799, F794), which were disrupted by the M807V and M807T mutations, are poorly conserved. In particular, the residues homologous to L859 in H10 were almost invariably leucine (Figure 1), suggesting that the interactions between H10 and H8 evident in the AR may generally apply to other steroid receptors. This led us to model and examine the crystal structures of other steroid receptor LBD. Intriguingly, the atomic distances between residues corresponding to M807 in H8, and to L859 and L863 in H10 in the ER, RXR, PPAR and VDR were similar and highly conserved. Thus the means ± SD of distances from 807 to 859 and to 863 were 4.12 ± 0.23 and 4.18 ± 0.61 Å respectively (Table III). In comparison, distances from M807 to other residues (I799, F794, F747) were longer and more variable. The conservation of key residues and atomic distances in H8 and H10 supports the concept that H8–H10 interactions are critical for stability of the holo-LBD in AR in particular, and in the steroid/nuclear receptor superfamily in general.
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Discussion
In this study, we combined functional in-vitro assays and computational modelling to understand the structural basis for the strikingly different phenotypes caused by substitutions in AR residue M807. Our patient with the threonine substitution had partial AIS, whereas the valine and arginine substitutions have resulted in the more severe complete AIS (Adeyemo et al., 1993; Murono et al., 1995). The in-vitro reporter gene transactivation and ligand-binding assays correlated well, and demonstrated that disruption of AR transactivation function and ligand-binding capacity was most acute when methionine was mutated to arginine, followed by valine, and was least affected by the threonine substitution. In this regard it is relevant to note that although the valine mutant was partially active, it displayed only minimal AR activity at physiological doses (1–3 nmol/l) of DHT and testosterone, consistent with the observed complete AIS phenotype. It should be noted, however, that in some cases of AIS, the same amino acid substitution can result in differing phenotypes (Gottlieb et al., 1998), suggesting that overall genetic background may have a role in determining eventual phenotype.
The differences in AR binding capacity and transactivation function to some extent mirrored the differences in polarity and charges of the mutant residues, as methionine, threonine and arginine are non-polar, uncharged polar and basic respectively. Computer modelling indicated that M807 resides in H8 and is buried in the midst of a hydrophobic cluster of residues. The hydrophobic side-chain of M807 can form direct van der Waal contacts with nearby amino residues on H5 and H9 in the hydrophobic core, and with H7 and 10 from the other layer of the AR helical sandwich. For example, M807 interacts with at least six neighbouring side-chains, namely L859 and L863 in H10, L838 in H9, I799 and F794 in H7, and F747 in H5. A non-conservative mutation such as M807R would generate repulsive forces and destroy the hydrophobic, non-polar environment that surrounds the focal residue in position 807, thereby totally disrupting the LBD core resulting in complete AIS and the female phenotype. In contrast, the uncharged polar threonine substitution, while reducing side-chain interactions, still maintained two critical contacts, namely to L859 and L838 on H10 and H9 respectively, thereby weakening, but not totally disrupting, the forces binding the outer layer of the AR LBD to its inner hydrophobic core. This partial disruption to the scaffold is congruent with the partial AIS phenotype and the right shift of the dose–response curve for M807T, and explains why the function of the mutant AR, in vitro and in vivo, can still be rescued with ligands that have higher affinity for the ligand-binding pocket, such as DHT or MB.
However, it is puzzling to observe that the valine substitution, despite its conservative nature, resulted in complete AIS and almost total abrogation of AR activity at physiological doses of androgen. Valine, which is non-polar like WT methionine, surprisingly disrupted AR function to a greater degree than the polar substitution, threonine. Furthermore molecular modelling showed that V807 maintained more hydrophobic contacts than the threonine substitution (three contacts versus two contacts respectively), and yet the valine mutant was more defective in vivo and in vitro compared to threonine. One notable difference between these two substitutions was that the threonine, but not valine, substitution improved van der Waal contact with L859 in H10, suggesting the key importance of interactions between M807 on H8 in the hydrophobic core, to L859 on H10 in the other layer of the LBD helical sandwich. Since H10 and the contiguous H11 are linked to H12, the structure of the entire outer layer of the LBD sandwich could depend on this key interaction. Therefore, perturbations to the hydrophobic interactions between H8 and H10 in M807V may have a ripple effect on one layer of the AR LBD sandwich and in turn reducing the apo-LBD capacity to adopt a competent holo-LBD conformation and stable androgen binding. The end result would be defective androgen binding, transactivation dysfunction and complete AIS. Our approach of integrating biochemical data with molecular modelling therefore enabled us to define the key role of H8–H10 interactions for ligand-binding and transactivation function in the AR.
The possibility that H8–H10 interactions may be critical for AR function led us to examine other nuclear receptors for similar interactions. Strikingly, residues whose interactions were preserved in the threonine and valine substitutions proved to be highly conserved in the steroid/nuclear receptor superfamily. In particular, the residues corresponding to the critical residues L859 and L863 in H10 and L838 in H9 were almost invariably leucines. Furthermore the atomic distances from residues corresponding to L859 and L863 in H10 to M807 were highly conserved, suggesting that H8–H10 interactions might also be an important feature for other steroid receptors. In contrast, the molecular distances from M807 to residues in H5 and H7 were longer and tended to deviate more. Recently evidence was reported from mutations of the mineralocorticoid receptor, that interactions between H5 of the hydrophobic core and H3 of the outer layer may have an important general role in steroid receptor function (Geller et al., 2000). If so, M807 in H8 could be a focal residue as it can bind both outer layers to the hydrophobic core through its interactions with H10 in one other layer, and indirectly with H3, via H5, in the other.
The advantage of our combinatorial method of analyses (clinical profiles and molecular kinetic data coupled to algorithm modelling) is that it allows us to link function to tertiary structure. The various lines of evidence, such as patient phenotypes, transactivation data, ligand-binding studies and molecular models, suggest that residue 807 in H8 is a focal residue both structurally and functionally. This epicentric residue, M807, in the middle of the central helix in the hydrophobic core of the AR LBD, has van der Waal contacts, and could therefore bind both outer layers stably together to the hydrophobic core. Since every human AR mutation represents a natural `knock-out' experiment (Yong et al., 2000), combinatorial analyses have the potential to contribute to a comprehensive understanding of the structure and function relationships in the AR (Yong et al., 1998). Such knowledge may not only be applied to other steroid receptors, but could lead to rational pharmacological therapy.
Notes
3 To whom correspondence should be addressed. E-mail: obgyel{at}nus.edu.sg 
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Submitted on May 8, 2001; accepted on October 31, 2001.
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