help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2004-0023
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Elhaji, Y. A.
Right arrow Articles by Trifiro, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Elhaji, Y. A.
Right arrow Articles by Trifiro, M. A.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*(L)-ARGININE
*Genetics Home Reference
Molecular Endocrinology 18 (8): 1876-1886
Copyright © 2004 by The Endocrine Society

An Examination of How Different Mutations at Arginine 855 of the Androgen Receptor Result in Different Androgen Insensitivity Phenotypes

Youssef A. Elhaji, Jian Hui Wu, Bruce Gottlieb, Lenore K. Beitel, Carlos Alvarado, Gerald Batist and Mark A. Trifiro

Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital (Y.A.E., J.H.W., B.G., L.K.B., C.A., G.B., M.A.T.), Departments of Human Genetics (Y.A.E., M.A.T.), Medicine (M.A.T., L.K.B.) and Oncology (J.H.W., G.B.), McGill University, Montreal, Quebec, Canada H3T 1E2

Address all correspondence and requests for reprints to: M. A. Trifiro, M.D., Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote-Ste-Catherine Road, Montreal, Quebec, Canada H3T 1E2. E-mail: mark.trifiro{at}mcgill.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two substitutions at an identical location in the ligand-binding domain (LBD) of the human androgen receptor (AR), R855C and R855H, are associated with complete androgen insensitivity syndrome (AIS) and partial AIS, respectively. Kinetic analysis of the mutant receptors in genital skin fibroblasts and in transfected cells revealed very low total binding (Bmax) and increased rate constants of dissociation (k) for the R855C mutant; and normal Bmax and k, with slightly elevated equilibrium affinity constants (Kd), but decreased transactivational capacity for the R855H mutant. Further analysis of the R855H mutant revealed both thermolability and decreased N/C-terminal inter-actions in the presence and absence of the co-activator transcriptional intermediary factor 2. To establish the nature of these functional differences we have used molecular dynamic modeling to create four-dimensional models of each of the mutant receptors. Molecular dynamic modeling produced profoundly different models for each of the mutants: in modeling of R855C a surprisingly significant distant alteration in the position of helix 12 of the helix 12 positioning of the AR ligand binding domain (AR-LBD) occurs, which would predict severe ligand binding abnormalities and complete AIS; in modeling of R855H, no dramatic effect on the position of helix 12 was seen; thus, binding properties of the receptor are not compromised.

Molecular dynamics four-dimensional modeling clearly supports the biochemical and kinetic studies of both mutants. Such novel computational modeling may lead to a better understanding of the structure-function relationships and the molecular mechanics of ligand binding not only of the AR-LBD but also of other nuclear receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ANDROGEN RECEPTOR (AR) is a member of the nuclear receptor superfamily, a large family of ligand-activated, DNA-binding, transcription factors. The AR mediates the various age- and tissue-specific actions of androgens and is responsible for the development, maintenance, and regulation of the male reproductive system. It is encoded by an X-linked gene (AR), which consists of eight exons. The AR protein consists of approximately 919 amino acids (aa) and retains the well-characterized and conserved modular structure of the nuclear receptors: an N-terminal transactivational domain encoded by exon 1, a central DNA-binding domain encoded by exons 2 and 3, a hinge region that contains a bipartite nuclear localization signal encoded by exons 3 and 4, and a C-terminal ligand-binding domain (LBD) encoded by exons 4–8 (1, 2). It has two main transcription activation functions: a constitutive activation function (AF-1) in the N-terminal transactivational domain and a hormone-dependent activation function (AF-2) at the C terminus of the LBD.

AR mutations result in variable degrees of androgen insensitivity in XY individuals (3). Androgen insensitivity syndrome (AIS) ranges from complete AIS (CAIS) to mild AIS (MAIS), with a wide range of partial AIS (PAIS) in between. Phenotypically, it is a spectrum of abnormal sex differentiation ranging from XY female, in CAIS cases, to nearly normal male, in MAIS cases (3).

Most of the AR mutations reported so far are single-amino acid substitutions, and the vast majority are located in the LBD (4). Different substitutions at the same amino acid may result in receptors with different properties and thereby different degrees of AIS. Interestingly, identical substitutions at the same site may exhibit variable expressivity that results in different phenotypes not only in different families, but also within the same family (5, 6).

X-ray crystallographic studies of the AR LBD (7, 8) and other steroid receptor LBDs (9, 10, 11, 12) revealed that steroid receptors share a common LBD structure encompassing 12 {alpha}-helices and a ß-turn. These {alpha}-helices undergo similar conformational changes in response to ligand binding, resulting in the formation of a hydrophobic surface by helices 3, 4, 5, and 12 that constitutes the AF-2 domain, provides binding sites for coactivators and initiates N/C-terminal interactions (13, 14).

The ligand-dependent AR N/C interactions are essential for the transactivational properties of the receptor (15). The AR N/C-terminal interactions have been shown to be direct, mediated by conserved FXXLF and WXXLF motifs located in the AR N-terminal domain and the AF-2 domain in the LBD (16, 17), or indirect, wherein members of the family of p160 coactivators interact with the AR termini and mediate their communications (18, 19). The p160 coactivators interact with the AF-2 domain through conserved LXXLL motifs (18, 20, 21); however, their interactions with the N-terminal domain are LXXLL independent (16, 19). The major structural changes that occur upon ligand binding involve a change in conformation of the C-terminal helix 12 (H12) (22). This helix is completely repositioned upon ligand binding. Further studies have shown that when bound to activating ligands, H12 adopts a conformation that promotes the binding of coactivator proteins (23). These studies suggest that H12 is significantly more mobile than the main body of the protein but shows reduced mobility upon ligand binding. Thus, the dynamic properties of H12 may be a key to the regulation of transcriptional activity.

Recent studies of proteins and nucleic acids using molecular dynamics (MD) simulations, a key computational biology technique, have generated useful insights into the structural mechanisms underlying the biological function of a number of biomacromolecules (24, 25). Further, an implicit solvation method that is based on the generalized Born model (26) has been successfully implemented for use along with molecular mechanics force fields in MD simulations (27, 28, 29) [e.g. within the AMBER package (30)]. Recently, we have investigated a pathogenic AR LBD mutation (R744C), by examining details of its altered atomic structure using MD simulations extended over a 4-nsec time period (31); these simulations have strongly supported our analysis of the mutant phenotype including its kinetic properties as well as other biochemical data.

In this study, we have identified and characterized two different substitutions at an identical position (R855H; R855C) in the LBD of the AR in two unrelated patients with MAIS and CAIS, respectively. Kinetic analysis has revealed that the alternate substitutions result in significantly different receptor abnormalities. The R855C mutant showed very low binding ability and had no transactivational activity. The R855H mutant retained normal or near-normal binding properties, but also exhibited a reduced transactivational capacity. An analysis of the R855H mutant revealed thermolability and decreased AR N/C-terminal interactions that may explain its pathogenicity. We have used MD modeling to correlate the structural alterations of these mutant receptors with the functional abnormalities that determine their very different phenotypes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutation Identification and PCR Mutagenesis
Two unrelated 46XY individuals (subjects 9025 and 82725) were diagnosed with MAIS and CAIS, respectively. Direct sequencing of the PCR products of exons 2–8 of the AR in each patient was performed and revealed a single-point mutation in exon 7 that resulted in the substitution of different aa for the conserved arginine at position R855. For subject 9025, a G-to-A transition at nucleotide 2926 resulted in the substitution of arginine with histidine (R855H), and for subject 82725, a C-to-T transition at nucleotide 2925 resulted in the substitution of arginine with cysteine (R855C). To further investigate these mutations, each substitution was recreated separately in the pSVhAR.BHEX and pcDNA3-hAR mammalian expression vectors. The HgaI restriction site, which is abolished by both mutations, was used to verify the incorporation of the mutations. Direct sequencing confirmed the presence of only the desired mutations.

The Effects of the Alternate Substitutions on Androgen Binding
Maximum androgen-binding capacity (Bmax), rate constant of dissociation (k), and apparent equilibrium dissociation constants (Kd) for wild-type and mutant ARs were measured in genital skin fibroblasts (GSFs) and transfected COS-1 cells with different ligands. The R855H mutant retained normal or near-normal Bmax and k but had an elevated Kd at physiological hormone concentrations both in COS-1 [for MT, k = 0.009 vs. 0.008 min–1 and Kd = 0.56 vs. 0.27 nM (Fig. 1Go)] and in GSFs (for MT, k = 0.014 vs. 0.012 min–1 and Kd = 0.8 vs. 0.21 nM) for R855H and wild-type ARs, respectively. Similar results were obtained when mibolerone (MB) and dihydrotestosterone (DHT) were used (data not shown). In contrast, the R855C mutant receptor had negligible binding activity at normal hormone concentrations. To examine whether this was due to an absolute lack of binding or an affinity problem, cells expressing R855C receptor were incubated with 100 nM 3H-labeled hormone. At these very high hormone concentrations, the mutant receptor was indeed able to bind up to 50% compared with the wild-type receptor. Western analysis revealed that the mutant ARs were of normal size (~110 kDa) and were stable in transfected COS-1 cells incubated for 24 and 48 h with and without ligand. MB binding in GSFs expressing the R855H mutant AR was normally up-regulated, from 87–198 fmol/mg protein after 2 h and overnight incubations, respectively.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1. MT-Binding Properties of the R855H Mutant ({bullet}) and Wild-Type ({square}) Receptors in Transfected COS-1 Cells

A, Rate constants of dissociation (k); and B, apparent equilibrium dissociation constants (Kd).

 
Thermolability of R855H Mutant Receptor
To further characterize the kinetic properties of the R855H mutant receptor, thermolability of R855H-androgen complexes was assessed. The AR is considered to be thermolabile if it loses more than 40% of its total binding when incubated at 42 C (32). R855H exhibited severe thermolability with both MB and methyltrienolone (MT). When exposed to 42 C, by 6 h R855H had lost approximately 70% and about 90% of its binding activity for MB and MT, respectively, compared with about 30% and approximately 27% for the wild-type AR (Fig. 2Go). Unlike the wild-type receptor, R855H was unable to recover from thermal stress when incubated at 42 C for 2 h and then shifted back to 37 C.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2. Thermolability of R855H-MB and R855H-MT Complexes Were Assayed in GSF Cultures from Patient (Subject 9025) ({bullet}) and a Wild-Type Strain ({square})

Total binding (100%) was measured after incubation at 37 C for 16 h with 3 nM 3H-labeled MB (A), or 3H-labeled MT (B). Cultures were then supplemented with cycloheximide and transferred to 42 C. The remaining binding activity was measured after 1, 2, 4, and 6 h of incubation at 42 C.

 
Transactivation Activity of Full-Length Receptors
The ability of the wild-type and mutant full-length receptors to transactivate an MMTV-GH reporter gene in the presence of both MB and MT was reduced by approximately 30% and about 40%, respectively, compared with that of wild-type AR (Fig. 3Go). The R855C receptor was unable to activate transcription of the reporter gene.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3. The Ability of the Full-Length Wild-Type and Mutant ARs to Transactivate the GH Reporter Gene in Cotransfected COS-1 Cells

Results are presented as the mean ± SD for wild-type (black), R855H (striped), and R855C (white) ARs. R855H transactivational activity was reduced in the presence of 3 nM MB or MT by 30% and 40%, respectively, compared with that of wild-type AR. The R855C mutant showed no transactivational induction in response to either ligand.

 
AF-2 Domain Transactivation Potential
The AF-2 domain is located in the LBD and is assembled and activated upon ligand binding. To examine the integrity of the AF-2 function, we investigated the transactivational ability of the wild-type and mutant LBDs in the presence of transcriptional intermediary factor 2 (TIF-2). Whereas the transactivational activities of R855H and wild-type LBDs were comparable, the R855C LBD showed no activity with or without TIF-2 (Fig. 4Go).



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4. The Transactivational Potential of the AF-2 Domain of the Mutant AR LBDs ± 3 nM MB Was Assessed in CV-1 Cells in the Presence or Absence of the Coactivator TIF-2

The mean ± SD of three different experiments is shown for wild-type (black), R855H (striped), and R855C (white).

 
AR N/C-Terminal Interactions
The ability of the mutant LBDs to interact with the AR N-terminal domain in vivo was reduced both in the presence and absence of the coactivator TIF-2 (Fig. 5Go). As expected for R855C, which was unable to bind ligand, no interaction was detected upon ligand addition. The N/C-terminal interaction for R855H LBD was only 32% (33.5 ± 9.5%) of that of the wild-type LBD in the absence of the coactivator TIF-2. However, the presence of TIF-2 appears to enhance the interaction of R855H LBD to 48% (47 ± 5%) of the wild-type LBD, reflecting an average fold induction of 9-fold for R855H compared with 6-fold for the wild-type LBD (Fig. 5Go).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 5. AR N/C-Terminal Interactions for the Wild-Type and mutant LBDs ± 3 nM MB Were Assayed in CV-1 Cells Using the Mammalian Two-Hybrid System

The mean ± SD of three independent experiments in the presence or absence of the coactivator TIF-2 is shown for wild-type (black), R855H (striped), and R855C (white) bars. The fold induction in the presence of TIF-2 is also indicated.

 
MD Simulation of AR
The backbone root mean-square deviation (RMSD) of helix 10 (residues 849–886), relative to the crystal structure (pdb-1e3 g), was calculated for each structural snapshot during the 4-nsec simulations at 310 K and plotted against time for both the mutant and the wild-type receptors (Fig. 6AGo). There was clearly a large RMSD jump in the R855C mutant that occurred almost immediately and continued throughout the simulation. On the other hand, R855H showed only minor fluctuations from the wild type until almost the very end of the simulation at 4 nsec. To further explain the different local distortions created by each of the mutants we have analyzed the hydrogen bond network around residue 855. R855 in the wild-type AR forms four hydrogen bonds with residues L797 and E803 (Fig. 7Go). Residue C855 does not form any hydrogen bonds whereas residue H855 forms a hydrogen bond with residue S851 instead.



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 6. Backbone RMSD of A) Helix 10 (Residues 849–886) and B) Helix 12 (Residues 892–908) Relative to the Crystal Structure (1e3 g.pdb) for the Wild-Type AR LBD (—) and the R855C ({blacktriangleup}) and R855H ({triangleup}) Mutants

The data have been smoothed by performing a running average over 25 psec.

 


View larger version (79K):
[in this window]
[in a new window]
  		Fig. 7. Hydrogen-Bond Network around Residue 855 for the Wild-Type (Green) and R855H Mutant (Purple) ARs

Arginine 855 in the wild-type forms four hydrogen bonds: three with residue E803 and one with L797. Histidine at this position forms only one hydrogen bond with residue S851.

 
The critical replacement of H12 is paramount to stable ligand binding and promoting functionality of the stable ligand-receptor complex. To understand how such a local distortion at a remote distance from the binding pocket could affect ligand binding, the backbone RMSD of H12 (residues 892–908) was calculated for both the wild-type and mutant ARs (Fig. 6BGo). The backbone RMSD of H12 for the R855C differs significantly from that of the wild-type AR between 2.5–3.5 nsec, whereas R855H again had much less of a deviation throughout the simulation (Fig. 6BGo).

To examine the overall (H1–H12) structural differences, wild-type AR-LBD and mutant R855H and R855C models were constructed from 2.5–4 nsec MD simulations. Relative to the structure of the wild-type, the average backbone RMSD for R855C and R855H mutants are 3.1 Å and 2.8 Å, respectively. This indicates that overall structure of R855H is more similar to that of the wild type than the R855C mutant. To visually inspect these structural abnormalities, the averaged structures for both R855H and R855C mutants were superimposed on the backbone (Fig. 8Go). The average backbone RMSD between the structures of the two mutants themselves is as large as 3.7 Å. There is significantly more local structural distortion around residue 855 in the R855C mutant. Surprisingly, compared with the R855H mutant, H12 and its C-terminal extension (CTE) of the R855C mutant have adopted a significantly different position. Normally the CTE of H12 resides firmly in a pocket formed by helices 8 and 9 (33, 34). Therefore, the MD simulations have suggested that the R855C mutation, although 25 Å away from the bound R1881, repositions H12 and the CTE distinctly away from their normal position and therefore limits the receptor’s ability to bind ligand.



View larger version (73K):
[in this window]
[in a new window]
  		Fig. 8. Superimposed Average Structures Taken from 2.5–4 nsec MD Simulations at 310 K for Mutants AR-LDB R855C (Gray Ribbon) and R855H (White Ribbon)

Helices 10 and 11 and C terminus of R855C are orange, whereas H12 is green. Helices 10 and 11 and C terminus of R855H are Purple and its helix 12 is cyan. Residue 855 is a stick model. The bound ligand R1881 of R855C is an orange space-filled model.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have identified and characterized alternate mutations at a single position in the LBD of the AR, arginine 855, in two unrelated subjects with MAIS (subject 9025) and CAIS (subject 82725). A substitution of arginine 855 by histidine (R855H) and cysteine (R855C) was identified in subjects 9025 and 82725, respectively. Arginine 855 of the AR, which is conserved among the members of the steroid receptor subfamily, appears to be a hot spot for alterations, where substitutions have been reported in numerous unrelated AIS cases (4). In fact, arginine 855 substitutions, to histidine (nine cases) and cysteine (12 cases), including this report, have been associated with AIS cases more than any other AR amino acid substitutions (4). Whereas R855C is invariably associated with a female phenotype and complete AIS (35, 36, 37), R855H exhibits variable expressivity that results in significantly different phenotypes, including CAIS with normal female genitalia (35) and variable degrees of PAIS ranging from predominantly female external genitalia and female sex of rearing (38, 39, 40) to predominantly male external genitalia and male sex of rearing (40, 41, 42). The same set of substitutions has been also reported with high frequency for two other arginine residues in the AR LBD (arginine 774 and 840) (4). Similarly, both R774H and R840H substitutions have resulted in variable phenotypes ranging from PAIS to CAIS. However, whereas R774C results in CAIS, R840C has been associated only with PAIS. Interestingly, the R840C appears to be less disruptive and results in a milder phenotype than the R840H (43).

Androgen-binding analysis in the patients GSFs and transfected COS-1 cells revealed that a cysteine substitution for arginine 855 resulted in more severe binding abnormalities than the histidine substitution. Whereas the R855C mutant showed negligible binding, the R855H retained normal Bmax and k, and slightly elevated Kd with all ligands tested (DHT, MB, and MT). To ensure that the lack of androgen binding for R855C mutant was not due to protein instability and degradation, Western blot analysis was performed and showed that both mutant receptors were stable in the absence or presence of androgen.

The negligible ligand binding of the R855C mutant receptor suggested significant conformational abnormalities. A cysteine residue at this position may induce the formation and/or the replacement of a disulfide bridge, resulting in a conformation that is incompatible with ligand binding; however, no other cysteine residue appears nearby. Another possibility is that, whereas cysteine is uncharged, a positively charged residue such as arginine is required at position 855 of the AR for normal ligand binding. R855 is highly conserved in all AR-related members in the superfamily of steroid receptors (estrogen receptor, progesterone receptor, glucocorticoid receptor, mineralocorticoid receptor) as well as others not as closely related (human retinoid X receptor-{alpha}, chicken ovalbumin upstream promoter-transcription factor) (44). Thus, an arginine at position 855 seems to fulfill a strict functional requirement, most likely hydrogen bonding. This requirement may be fulfilled by the positively charged histidine, resulting in a normal ligand-binding capacity for the R855H mutant.

Recently, the availability of a three-dimensional model of the AR LBD (7) has permitted researchers to examine the possible effects that AR LBD mutations may have on the conformation and actual functioning of the ligand-binding pocket. In analyzing the x-ray crystal structure of the human AR LBD, only 18 specific residues were predicted to be in sufficiently close contact with the bound ligand (R1881) to affect ligand binding (7). Mutations at the R855 residue are particularly puzzling because they are almost 25 Å from the ligand-binding pocket, and positioned on the outside of the crystal structure, as are a significant number of the reported AR LBD mutations, again located far from the ligand-binding pocket (4).

Therefore, to elucidate how R855 mutations could possibly affect the structure of the ligand-binding pocket, and thereby explain their different effect on ligand binding, we have used MD simulations over extended periods of time (≤4 nsec) to create, in effect, four-dimensional structures of the AR LBDs. Our results clearly show a significant local distortion around residue 855 at the N terminus of H10 (Figs. 7Go and 8Go). In the wild-type AR, residue R855 forms multiple hydrogen bonds with E803 (helix 8) and L797 (helix 7). Residue E803 is one of very few superconserved aa in the LBD present in both related and unrelated members of the steroid receptor family (44). Residue L797 is also highly conserved in the AR-related family members. In the R855H mutant, these hydrogen bonds do not exist, but a hydrogen bond between H855 and S851 is formed that could compensate for the loss of normal hydrogen bonding around residue 855. C855 does not participate in hydrogen bonding; the lack of such bonding can introduce more local flexibility, which may lead to further structural alterations.

These observations alone, however, cannot by themselves explain the kinetic abnormalities associated with both R855 mutants. Helix 12 has been widely implicated as being crucial to the ligand-binding process in the nuclear receptor family (22, 45, 46). The MD simulations clearly illustrate that whereas a significant structural distortion of H12 was caused by the R855C mutation, the R855H mutation resulted in much less H12 alteration, which would explain its near-normal ligand binding. Helix 12 appears to be the most singular flexible part of the steroid receptor LBDs and probably, because of its inherent flexibility, is most apt to adopt a favorable position such that the whole LBD can be made to accommodate specific missense mutations (23, 47) albeit at the potential risk of losing ligand-binding capability. Helix 12 appears to be fixed in its ultimate position by antiparallel ß-sheet interactions between its CTE and residues 815–817 (loop 8–9) (7). This is obviously lacking in R855C (Fig. 8Go).

Although modeling offers a possible explanation for the phenotypic effects of R855C, it does not fully explain the phenotypic abnormalities associated with the R855H mutation. The main immediate difference was that R855H-androgen complexes in GSFs exhibited severe thermolability. In addition, we investigated the transactivational properties of the mutant receptors, by first testing the activation ability of the ligand-dependent AF-2 domain. The AR AF-2, unlike other steroid receptors, has very weak transcriptional activity in mammalian cells in the absence of the N-terminal domain (48); however, it is highly inducible by the coactivator TIF-2 (16). Interestingly, the R885H-AR-LBD was able to transactivate the reporter gene in a similar manner to the wild type. This indicated an intact AF-2 function and also suggested that the R855H mutation did not reduce the ability of the LBD to recruit and interact with TIF-2. However, when we tested the transactivational properties of the full-length mutant receptors, activation of the reporter gene by the R855H was 30% and 40% less than the wild type in response to MB and MT, respectively. Again, as expected for the R855C, there was no induction of the reporter gene upon ligand addition.

AR N/C-terminal interactions are essential for the transactivational activity of the receptor (49, 50). The AF-2 domain initiates AR N/C-terminal interactions and provides binding sites for the recruitment of coactivators (e.g. steroid receptor coactivator 1, TIF-2, p/CIP), which may further enhance the N/C-terminal interactions (51, 52). As we have shown previously, diminished N/C interactions may correlate with and explain the clinical phenotypes of some LBD mutants, even if their ligand-binding activities are normal (52). To characterize the N/C-terminal interactions of the R855 substitutions, we used the mammalian two-hybrid system. Our results showed no interaction for the R855C mutant, which was consistent with its lack of ligand-binding activity. The R855H mutant resulted in significantly reduced interaction compared with that of wild type, both in the absence and presence of TIF-2. The presence of TIF-2 partially rescued the defective N/C interaction of the R855H LBD, confirming the ability of the AF-2 domain of the R855H mutant to recruit and interact with TIF-2. In fact, the presence of TIF-2 resulted in a greater fold induction for the R855H N/C-terminal interactions than the wild type: 9-fold increase for R855H compared with 6-fold for the wild-type receptor. It has been suggested that the TIF2-LXXLL motif competes with the more favorable AR N-terminal-FXXLF motif for an overlapping binding site in the AF-2 domain (53). Thus, the higher fold induction for the R855H mutant may reflect increased interactions with TIF-2 due to the decreased competition of the FXXLF motifs in the N-terminal domain (i.e. decreased N/C-terminal interactions). Enhanced coactivator/AF-2 interactions may amplify the role of coactivators in the transactivational ability of the receptor and thereby their role in the variable expressivity exhibited by the R855H mutation. Enhanced TIF-2/AF-2 interaction due to reduced AR N/C-terminal interactions has been suggested as a possible mechanism for AR activation after androgen blockade in prostate cancer patients. It was proposed that, in the absence of testicular androgens, increased binding to adrenal androgens and other steroids that induce less N/C-terminal interaction result in enhanced TIF-2/AF-2 interactions and, thereby, AR transactivational ability (54).

Our results show that different phenotypes associated with alternate substitutions at the same amino acid may reflect different receptor abnormalities. Whereas the cysteine substitution that resulted in CAIS correlated with an abolished AR binding ability, the histidine substitution that resulted in PAIS reduced AR N/C-terminal interaction and the transactivational activity of the receptor without compromising its binding properties. In addition, the ability of the TIF-2 coactivator to partially rescue N/C interaction for the R855H mutation suggests an important role for coactivators in the variable expressivity exhibited by certain mutations.

The use of MD simulations strongly suggested that the lack of ligand binding of the R855C mutant is due to a local distortion that leads to the critical misplacement of H12, which is essential for ligand binding. Furthermore, it suggests how mutations in the AR that are located far away from the ligand-binding pocket can still affect ligand binding. Much more extensive MD simulations may be used to document, in a time-dependent manner, the exact series of physical changes that H12 may undergo in response to ligand binding and abnormal order of events in respect to specific missense mutations. This will be particularly important not only because of the many phenotypically significant mutations that are located at a considerable distance from the ligand-binding pocket of the AR, but also in shedding light on the role of H12 position in ligand binding.

Combined studies employing MD simulation of the mutant ARs in conjunction with the classical molecular and kinetic analysis will certainly help in establishing the widespread application of MD simulation that would be pertinent to other proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Subject and Mutation Identification
Subject 82725 was a 19-yr-old 46XY individual diagnosed with CAIS, with primary amenorrhea, sparse pubic hair, normal female external genitalia, and a 4-cm blind-ending vagina.

Subject 9025 was a 10-wk-old 46XY infant with ambiguous genitalia, a micropenis, and hypospadia.

To identify AR mutations, DNA was extracted from the GSFs from each patient. PCR amplification and direct sequencing of exons 2–8 of the AR from each patient were performed as previously described (55).

Full-Length Mutant AR Plasmid Constructs
The mutations detected (R855H and R855C) were recreated in the pSVhAR.BHEX and pcDNA3-hAR expression vectors, which encode the full-length AR, by the overlap extension method. For each mutation, two overlapping fragments were amplified using the pSVhAR.BHEX as a template and the appropriate primers to incorporate each mutation into the PCR product. The internal primers used for the R855H mutation were:

9025A-(5'-TGCTCAAGACACTTCTACCAGC-3') and

9025B-(5'-TGGTAGAAGTGTCTTGAGCAGG-3'); and for the R855C mutation were

82725A-(5'-TGCTCAAGATGCTTCTACCAGC-3') and

82725B-(5'-TGGTAGAAGCATCTTGAGCAGG-3')

The substituted nucleotides are underlined. The same external primers were used for both mutations; TCFA (5'-GTCCCGGATATACAGCCAG-3') and P3' (5'-CACCAA-CCTTCTGATAGGCAGC-3'). One microliter of each of the corresponding overlapping fragments was used for the second PCR: an extension step for five cycles followed by 25 cycles with the external primers. The products of the second PCR were digested with EcoRI and BamHI and ligated into similarly digested pSVhAR.BHEX. The full-length AR mutants in pSVhAR.BHEX were then transferred into pcDNA3-hAR by NheI/BamHI double digestion and ligations. The insertion of the desired sequences was confirmed by restriction enzyme digestions and direct sequencing using the Thermo Sequenase Radiolabeled Terminator Cycle sequencing kit (United States Biochemical Corp., Cleveland, OH).

Mammalian Two-Hybrid Constructs
The wild type pM-AR(LBD) expression vector, encoding the GAL4-DNA-binding domain fused in frame with aa 628–919 of the AR, the pVP16-AR(TAD20Q) plasmid, coding for the VP16 activation domain fused in frame with aa 1–565 of the AR, and the pGAL4-Luc reporter vector containing GAL4-DNA-binding sites and a luciferase reporter gene were kindly provided by Dr. E. L. Yong, National University of Singapore (51). The pM-mutant AR(LBD) expression constructs were created for each mutant by a double digest of the corresponding mutant pcDNA3-hAR constructs with BstBI/XbaI and religating in frame with the similarly digested wild-type pM-AR(LBD). The pSG5-TIF-2 construct codes for the full-length transcriptional coactivator TIF-2 (51).

Cell Culture and Transfections
GSFs and COS-1 and CV-1 monkey kidney cells were maintained in Opti-MEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5%–10% fetal bovine serum, 50 U/ml of penicillin, and 50 µg/ml of streptomycin [penicillin-streptomycin solution (Life Technologies, Inc.)]. Cell transfections were performed in six and 12-well plates using LipofectAMINE 2000 (LF2000; Life Technologies, Inc.) as recommended by the manufacturer. All transfections included 1–2 µg pCMV-ß-galactosidase (ß-gal) as a control for transfection efficiency.

Androgen-Binding Assays
Androgen-binding studies in both GSFs and transfected COS-1 cells were performed as previously described (43). Androgens used were DHT, and the synthetic nonmetabolizable androgens MB and MT (R1881).

Thermolability of R855H-Androgen Complexes
Thermolability of R855H-MB and R855H-MT complexes was assayed in patient GSFs and a wild-type strain GSF culture plated in six-well plates. Four wells of each plate were incubated with 3 nM [3H]androgen and two with 3 nM [3H]androgen plus a 200-fold excess of androgen at 37 C. After 16 h, one plate was used to measure androgen-binding activity. The medium in the remaining plates was replaced by fresh medium supplemented with 10 µM cycloheximide before incubation at 42 C. Androgen binding was measured after 1, 2, 4, and 6 h of incubation at 42 C.

Ability to Up-Regulate and Stability of the Mutant ARs
To determine the ability of the mutant ARs to up-regulate, GSFs were plated in two six-well plates, labeled, and incubated as described above at 37 C. Androgen-binding capacity was measured after 2-h and overnight incubations. To assess the stability of mutant ARs in transfected COS-1 cells, cells were incubated with and without 10 nM MB at 37 C in the presence of 100 µM cycloheximide for 24 and 48 h. Cells were harvested and lysed in 1x Reporter Lysis buffer (Promega Corp., Madison, WI). Total protein (100 µg) was used for SDS-PAGE and Western blotting using AR 441, a mouse monoclonal antibody directed to aa 299–315 of human AR (NeoMarkers, Fremont, CA) and anti-heat shock protein 70 monoclonal antibody (Stress Gen Biotechnologies Corp., Victoria, British Columbia, Canada) as a control for protein loading. Detection was performed using ECL Plus kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

Transactivation Assay
COS-1 cells were plated in 12-well plates at a density of 3 x 105 cells per well in 1 ml Opti-MEM per well, supplemented with 5% fetal bovine serum and without antibiotics. The following day, cells were cotransfected with 300 ng wild-type or mutant full-length AR, 1 µg of the reporter construct pMMTV-GH, and 300 ng pCMV-ß-gal complexed with 4 µl LF2000 reagent per well. Cells were incubated with 3 nM MB or MT. After 72 h, media were collected and secreted hGH was measured using an hGH-ELISA Kit (MEDICORP Inc., Montreal, Quebec, Canada). Lowry and ß-gal assays were used to determine cellular protein and transfection efficiency.

The Mammalian Two-Hybrid Assay
To assess AR N/C-terminal interactions in vivo, mammalian two-hybrid assays were performed. CV-1 cells were used for the AR N/C interaction studies due to their low background compared with COS-1 cells in our experimental settings. CV-1 cells were plated in six-well plates at a density of 5 x 105 cells per well. Transfections were performed, as described above, using 6 µl LF2000 reagent, 0.5 µg wild-type or mutant pM-AR(LBD) construct, 0.5 µg pVP16-AR(TAD20Q), 2 µg pGAL4-Luc reporter vector, and 0.5 µg pCMV-ß-gal with or without 0.5 µg pSG5-TIF-2 per well. Empty pVP16 or pM vectors were used as controls. Twenty-four hours after transfection, four wells were treated with 3 nM MB. Seventy-two hours after transfection, the cells were harvested and lysed in 1x Reporter Lysis Buffer and assayed for luciferase and ß-gal activity according to Promega protocols. Total protein content was determined using BCA Protein Assay Kit (Pierce Chemical Co., Rockford, IL).

AF-2 Domain Transactivation Potential
To assess the transactivational potential of the AF-2 domain of the mutant receptors, CV-1 cells were cotransfected as described for the two-hybrid assay, except for the pVP16-AR(TAD20Q) vector, with 0.5 µg wild-type or mutant pM-AR(LBD) construct, 2 µg pGAL4-Luc reporter vector, 0.5 µg pCMV-ß-gal, and 0.5 µg pSG5-TIF-2 per well. Empty pM vector was used as a control. Cells were incubated with 3 nM MB, harvested, and assayed for luciferase induction as described above.

Protein Modeling
The crystal structure of AR-LBD complexed with R1881 was obtained from the Protein Data Bank (PDB entry: 1e3 g), and the crystallographic waters were removed. Hydrogen atoms were added using the BIOPOLYMER module of Insight II package (Accelrys, Inc., San Diego, CA). The starting structure of the AR-LBD R855 mutants were obtained by mutating residue 855 from R to C and H in the AR-LBD crystal structure. The bound ligand, R1881, was retained in both wild-type and mutant AR-LBD.

MD Simulations
All MD simulations presented in this work were performed using the AMBER 7.0 simulation package (30) and ff99 force field (56). The detailed procedure has been described elsewhere (31). Briefly, the generalized Born solvation model instead of explicit water was used (57). Before MD simulation was initiated, the possible bad contacts resulting from amino acid substitutions were relaxed by 300 cycles of the steepest descent energy minimizations whereas the heavy atoms were restrained with a force constant of 5 kcal/mol·Å2. The MD simulations were performed at 310 K without any restraints. The average structures from a specific period of MD simulations were calculated using the PTRAJ module of AMBER package. All RMSDs were mass weighted and calculated using the CARNAL module in AMBER.


   FOOTNOTES
 
This work was supported by the Canadian Institutes for Health Research, the Canadian Breast Cancer Research Initiative, and the Montreal Breast Cancer Foundation. Y.E. was the recipient of a full scholarship from the Secretariat of Education and Scientific Research of Libya (SESR).

Abbreviations: aa, Amino acids; AF-2, activation function 2; AIS, androgen insensitivity syndrome; AR, androgen receptor; CAIS, complete AIS; CTE, C-terminal extension; DHT, dihydrotestosterone; ß-gal, ß-galactosidase; GSFs, genital skin fibroblasts; H12, C-terminal helix 12; LBD, ligand-binding domain; MAIS, mild AIS; MB, mibolerone; MD, molecular dynamics; MT, methyltrienolone; PAIS, partial AIS; RMSD, root mean square deviation; TIF-2, transcriptional intermediary factor 2.

Received for publication January 20, 2004. Accepted for publication April 21, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Zhou ZX, Sar M, Simenthal JA, Lane MV, Wilson EM 1994 A ligand-dependent bipartite nuclear targeting signal in the human androgen receptor. Requirement for the DNA-binding domain and modulation by NH2-terminal and carboxyl-terminal sequences. J Biol Chem 269:13115–13123[Abstract/Free Full Text]
  2. Quigley CA, De Bellis A, Marschke KB, El-Awady MK, Wilson EM, French FS 1995 Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr Rev 16:271–321[Abstract/Free Full Text]
  3. Gottlieb B, Pinsky L, Beitel LK, Trifiro M 1999 Androgen insensitivity. Am J Med Genet 89:210–217[CrossRef][Medline]
  4. Gottlieb B, Beitel LK, Lumbroso R, Pinsky L, Trifiro M 1999 Update of the androgen receptor gene mutations database. Hum Mutat 14:103–114[CrossRef][Medline]
  5. Boehmer AL, Brinkmann O, Brüggenwirth H, van Assendelft C, Otten BJ, Verleun-Mooijman MCT, Niermeijer MF, Brunner HG, Rouwé CW, Waelkens JJ, Oostdijk W, Kleijer WJ, van der Kwast TH, de Vroede MA, Drop SLS 2001 Genotype versus phenotype in families with androgen insensitivity syndrome. J Clin Endocrinol Metab [Erratum (2002) 87:3109] 86:4151–4160
  6. Gottlieb B, Beitel LK, Trifiro MA 2001 Variable expressivity and mutation databases: the androgen receptor gene mutations database. Hum Mutat 17:382–388[CrossRef][Medline]
  7. Matias PM, Donner P, Coelho R, Thomas M, Peixoto C, Macedo S, Otto N, Joscho S, Scholtz P, Wegg A, Abasler S, Schafer M, Engers U, Carrondo MA 2000 Structural evidence for ligand specificity in the binding domain of the human androgen receptor. Implications for pathogenic gene mutations. J Biol Chem 275:26164–26171[Abstract/Free Full Text]
  8. Sack JS, Kish KF, Wang C, Attar RM, Kiefer SE, Yongmi A, Wu GY, Scheffler JE, Salvati ME, Krystek Jr SR, Roberto W, Einspahr HM 2001 Crystallographic structures of the ligand-binding domains of the androgen receptor and its T877A mutant complexed with the natural agonist dihydrotestosterone. Proc Natl Acad Sci USA 98:4904–4909[Abstract/Free Full Text]
  9. Wagner RL, Apriletti, JW, McGrath ME, West BL, Baxter JD, Fletterick RJ 1995 A structural role for hormone in the thyroid hormone receptor. Nature 378:690–697[CrossRef][Medline]
  10. Parker MG, White R 1996 Nuclear receptors spring into action. Nat Struct Biol 3:113–115[CrossRef][Medline]
  11. Brzozowski, AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engström O, Öhman L, Greene GL, Gustafsson J-Å, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  12. Williams SP, Sigler PB 1998 Atomic structure of progesterone complexed with its receptor. Nature 393:392–396[CrossRef][Medline]
  13. Feng W, Ribeiro RC, Wagner RL, Nguyen H, Apriletti JW, Fletterick RJ, Baxter JD, Kushner PJ, West BL 1998 Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors. Science 280:1747–1749[Abstract/Free Full Text]
  14. Weatherman RV, Fletterick RJ, Scanlan TS 1999 Nuclear-receptor ligands and ligand-binding domains. Annu Rev Biochem 68:559–581[CrossRef][Medline]
  15. Whitfield GK, Jurutka PW, Haussler CA, Haussler MR 1999 Steroid hormone receptors: evolution, ligands, and molecular basis of biologic function. J Cell Biochem(Suppl 32–33):110–122
  16. He B, Kemppainen, JA, Voegel JJ, Gronemeyer H, Wilson EM 1999 Activation function 2 in the human androgen receptor ligand binding domain mediates interdomain communication with the NH2-terminal domain. J Biol Chem 274:37219–37225[Abstract/Free Full Text]
  17. He B, Kemppainen JA, Wilson EM 2000 FXXLF and WXXLF sequences mediate the NH2-terminal interaction with the ligand binding domain of the androgen receptor. J Biol Chem 275:22986–22994[Abstract/Free Full Text]
  18. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736[CrossRef][Medline]
  19. Ma H, Hong H, Huang SM, Irvine RA, Webb P, Kushner PJ, Coetzee GA, Stallcup MR 1999 Multiple signal input and output domains of the 160-kilodalton nuclear receptor coactivator proteins. Mol Cell Biol 19:6164–6173[Abstract/Free Full Text]
  20. Hong H, Darimont BD, Ma H, Yang L, Yamamoto KR, Stallcup MR 1999 An additional region of coactivator GRIP1 required for interaction with the hormone-binding domains of a subset of nuclear receptors. J Biol Chem 274:3496–3502[Abstract/Free Full Text]
  21. Lim J, Ghadessy FJ, Abdullah AAR, Pinsky L, Trifiro M, Yong EL 2000 Human androgen receptor mutation disrupts ternary interactions between ligand, receptor domains, and the coactivator TIF2 (transcription intermediary factor 2). Mol Endocrinol 14:1187–1197[Abstract/Free Full Text]
  22. Egea PF, Klaholz BP, Moras D 2000 Ligand-protein interactions in nuclear receptors of hormones. FEBS Lett 476:62–67[CrossRef][Medline]
  23. Kallenberger BC, Love JD, Chatterjee KK, Schwabe JWR 2003 A dynamic mechanism of nuclear receptor activation and its perturbation in a human disease. Nat Struct Biol 10:136–140[CrossRef][Medline]
  24. Vitkup D, Ringe D, Petsko GA, Karplus M 2000 Solvent mobility and the protein glass transition. Nat Struct Biol 7:34–38[CrossRef][Medline]
  25. Young MA, Gonfloni S, Superti-Furga G, Roux B, Kuriyan J 2001 Dynamic coupling between the SH2 and SH3 domains of c-Src and hck underlies their inaction by C-terminal tyrosine phosphorylation. Cell 105:115–126[CrossRef][Medline]
  26. Qiu D, Shenkin PS, Hollinger FP, Still WC 1997 The GB/SA continuum model for solvation. A fast analytical method for the calculation of approximate Born radii. J Phys Chem A 101:3005–3014[CrossRef]
  27. Tsui V, Case DA 2001 Theory and applications of the generalized Born solvation model in macromolecular simulations. Biopolymers 56:275–291
  28. Cornell W, Abseher R, Nilges M, Case DA 2001 Continuum solvent molecular dynamics study of flexibility in interleukin-8. J Mol Graph Model 19:136–145[CrossRef][Medline]
  29. Bursulaya BD, Brooks III CL 2000 Comparative study of the folding free energy landscape of a three-stranded ß-sheet protein with explicit and implicit solvent models. J Phys Chem B 104:12378–12383[CrossRef]
  30. Case DA, Pearlman DA, Caldwell JW, Cheatam III TE, Wang J, Ross WS, Simmerling CL, Darden TA, Merz KM, Stanton RV 2002. AMBER 7 user manual. San Francisco: University of California, San Francisco
  31. Wu JH, Gottlieb B, Batist G, Sulea T, Purisima EO, Beitel LK, Trifiro M 2003 Bridging structural biology and genetics by computational methods: an investigation into how the R774C mutation in the AR gene can result in complete androgen insensitivity syndrome. Hum Mutat 22:465–475[CrossRef][Medline]
  32. Griffin JE, Durrant JL 1982 Qualitative receptor defects in families with androgen resistance: failure of stabilization of the fibroblast cytosol androgen receptor. J Clin Endocrinol Metab 55:465–474[Abstract/Free Full Text]
  33. Bledsoe RK, Montana VG, Stanley TB, Delves CJ, Apolito CJ, McKee DD, Consler TG, Parks DJ, Stewart EL, Willson TM, Lambert MH, Moore JT, Pearce KH, Xu HE 2002 Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition. Cell 110:93–105[CrossRef][Medline]
  34. Tahiri B, Auzou G, Nicolas JC, Sultan C, Lupo B 2001 Participation of critical residues from the extreme C-terminal end of the human androgen receptor in the ligand binding function. Biochemistry 40:8431–8437[CrossRef][Medline]
  35. McPhaul MJ, Marcelli M, Zoppi S, Wilson CM, Griffin JE, Wilson JD 1992 Mutations in the ligand-binding domain of the androgen receptor gene cluster in two regions of the gene. J Clin Invest 90:2097–2101
  36. Brinkmann AO, Jenster G, Ris-Stalpers C, van der Korput JA, Bruggenwirth HT, Boehmer AL, Trapman J 1995 Androgen receptor mutations. J Steroid Biochem Mol Biol 53:443–448[CrossRef][Medline]
  37. Ahmed SF, Cheng A, Dovey L, Hawkins JR, Martin H, Rowland J, Shimura N, Tait AD, Hughes IA 2000 Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome. J Clin Endocrinol Metab 85:658–665[Abstract/Free Full Text]
  38. Marcelli M, Tilley WD, Zoppi S, Griffin JE, Wilson JD, McPhaul MJ 1992 Molecular basis of androgen resistance. J Endocrinol Invest 15:149–159
  39. Weidemann W, Linck B, Haupt H, Mentrup B, Romalo G, Stockklauser K, Brinkmann AO, Schweikert HU, Spindler KD 1996 Clinical and biochemical investigations and molecular analysis of subjects with mutations in the androgen receptor gene. Clin Endocrinol (Oxf) 45:733–739[CrossRef][Medline]
  40. Boehmer AL, Brinkmann AO, Niermeijer MF, Bakker L, Halley DJ, Drop SL 1997 Germ-line and somatic mosaicism in the androgen insensitivity syndrome: implications for genetic counseling. Am J Hum Genet 60:1003–1006[Medline]
  41. Batch JA, Williams DM, Davies HR, Brown BD, Evans BA, Hughes IA, Patterson MN 1992 Androgen receptor gene mutations identified by SSCP in fourteen subjects with androgen insensitivity syndrome. Hum Mol Genet 1:497–503[Abstract/Free Full Text]
  42. Hiort O, Sinnecker GH, Holterhus PM, Nitsche EM, Kruse K 1996 The clinical and molecular spectrum of androgen insensitivity syndromes. Am J Med Genet 63:218–222[CrossRef][Medline]
  43. Beitel LK, Kazemi-Esfarjani P, Kaufman M, Lumbroso R, DiGeorge AM, Killinger DW, Trifiro MA, Pinsky L 1994 Substitution of arginine-839 by cysteine or histidine in the androgen receptor causes different receptor phenotypes in cultured cells and coordinate degrees of clinical androgen resistance. J Clin Invest 94:546–554
  44. Wurtz JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D, Gronemeyer H 1996 A canonical structure for the ligand-binding domain of nuclear receptors. Nat Struct Biol [Erratum (1996) 3:206] 3:87–94
  45. Vaisanen S, Perakyla, M, Karkkainen JI, Steinmeyer A, Carlberg C 2002 Critical role of helix 12 of the vitamin D3 receptor for the partial agonism of carboxylic ester antagonists. J Mol Biol 315:229–238[CrossRef][Medline]
  46. Pearce ST, Liu H, Jordan VC 2003 Modulation of estrogen receptor {alpha} function and stability by tamoxifen and a critical amino acid (Asp-538) in helix 12. J Biol Chem 278:7630–7638[Abstract/Free Full Text]
  47. Kauppi B, Jakob C, Farnegardh M, Yang J, Ahola H, Alarcon M, Calles K, Engstrom O, Harlan J, Muchmore S, Ramqvist A-K, Thorell S, Ohman L, Greer J, Gustafsson J-A, Carlstedt-Duke J, Carlsquist M 2003 The three-dimensional structures of antagonistic and agonistic forms of the glucocorticoid receptor ligand-binding domain. J Biol Chem 278:22748–22754[Abstract/Free Full Text]
  48. Berrevoets CA, Doesburg P, Steketee K, Trapman J, Brinkmann AO 1998 Functional interactions of the AF-2 activation domain core region of the human androgen receptor with the amino-terminal domain and with the transcriptional coactivator TIF2 (transcriptional intermediary factor 2). Mol Endocrinol 12:1172–1183[Abstract/Free Full Text]
  49. McKenna NJ, O’Malley 2002 Minireview: nuclear re-ceptor coactivators—an update. Endocrinology 143:2461–2465[Abstract/Free Full Text]
  50. Heinlein CA, Chang C 2002 Androgen receptor (AR) coregulators: an overview. Endocr Rev 23:175–200[Abstract/Free Full Text]
  51. Ghadessy FJ, Lim J, Abdullah AA, Panet-Raymond V, Choo CK, Lumbroso R, Tut TG, Gottlieb B, Pinsky L, Trifiro MA, Yong EL 1999 Oligospermic infertility associated with an androgen receptor mutation that disrupts interdomain and coactivator (TIF2) interactions. J Clin Invest 103:1517–1525[Medline]
  52. Ghali SA, Gottlieb B, Lumbroso R, Beitel LK, Elhaji Y, Wu JH, Pinsky L, Trifiro MA 2003 The use of androgen receptor amino/carboxyl-terminal interaction assays to investigate androgen receptor gene mutations in subjects with varying degrees of androgen insensitivity. J Clin Endocrinol Metab 88:2185–2193[Abstract/Free Full Text]
  53. He B, Wilson EM 2003 Electrostatic modulation in steroid receptor recruitment of LXXLL and FXXLF motifs. Mol Cell Biol 23:2135–2150[Abstract/Free Full Text]
  54. Gregory CW, He B, Johnson RT, Ford OH, Mohler JL, French FS, Wilson EM 2001 A mechanism for androgen receptor-mediated prostate cancer recurrence after androgen deprivation therapy. Cancer Res 61:4315–4319[Abstract/Free Full Text]
  55. Trifiro M, Prior RL, Sabbaghian N, Pinsky L, Kaufman M, Nylen EG, Belsham DD, Greenberg CR, Wrogemann K 1991 Amber mutation creates a diagnostic MaeI site in the androgen receptor gene of a family with complete androgen insensitivity. Am J Med Genet 40:493–499[CrossRef][Medline]
  56. Wang J, Cieplak P, Kollman PA 2000 How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J Comput Chem 21:1049–1074[CrossRef]
  57. Onufriev A, Bashford D, Case DA 2000 Modification of the generalized Born model suitable for macromolecules. J Phys Chem B 104:3712–3720[CrossRef]

NURSA Molecule Pages Link:

Nuclear Receptors:   AR
Coregulators:   GRIP1
Ligands:   Dihydrotestosterone  |  R1881



This article has been cited by other articles:


Home page
 J Mol EndocrinolHome page
J Jaaskelainen, A Deeb, J W Schwabe, N P Mongan, H Martin, and I A Hughes
Human androgen receptor gene ligand-binding-domain mutations leading to disrupted interaction between the N- and C-terminal domains.
J. Mol. Endocrinol., April 1, 2006; 36(2): 361 - 368.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
Y. A. Elhaji, I. Stoica, S. Dennis, E. O. Purisima, and M. A. Trifiro
Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity
Hum. Mol. Genet., March 15, 2006; 15(6): 921 - 931.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Elhaji, Y. A.
Right arrow Articles by Trifiro, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Elhaji, Y. A.
Right arrow Articles by Trifiro, M. A.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*(L)-ARGININE
*Genetics Home Reference

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals