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Molecular Endocrinology, doi:10.1210/me.2005-0134
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Molecular Endocrinology 19 (9): 2273-2282
Copyright © 2005 by The Endocrine Society

Androgen Receptor Mutations Identified in Prostate Cancer and Androgen Insensitivity Syndrome Display Aberrant ART-27 Coactivator Function

Wenhui Li, Claudio N. Cavasotto, Timothy Cardozo, Susan Ha, Thoa Dang, Samir S. Taneja, Susan K. Logan and Michael J. Garabedian

Departments of Microbiology (W.L., T.D., M.J.G.), Urology (W.L., S.H., T.D., S.S.T., S.K.L., M.J.G.), and Pharmacology (S.K.L., T.C.), New York University Cancer Institute, New York University School of Medicine, New York, New York 10016; and Molsoft (C.N.C.), La Jolla, California 92037

Address all correspondence and requests for reprints to: Susan K. Logan or Michael J. Garabedian, New York University Cancer Institute, New York University School of Medicine, 550 First Avenue, New York, New York 10016. E-mail for M.J.G.: garabm01{at}med.nyu.edu; E-mail for S.K.L.: logans02{at}med.nyu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The transcriptional activity of the androgen receptor (AR) is modulated by interactions with coregulatory molecules. It has been proposed that aberrant interactions between AR and its coregulators may contribute to diseases related to AR activity, such as prostate cancer and androgen insensitivity syndrome (AIS); however, evidence linking abnormal receptor-cofactor interactions to disease is scant. ART-27 is a recently identified AR N-terminal coactivator that is associated with AR-mediated growth inhibition. Here we analyze a number of naturally occurring AR mutations identified in prostate cancer and AIS for their ability to affect AR response to ART-27. Although the vast majority of AR mutations appeared capable of increased activation in response to ART-27, an AR mutation identified in prostate cancer (AR P340L) and AIS (AR E2K) show reduced transcriptional responses to ART-27, whereas their response to the p160 class of coactivators was not diminished. Relative to the wild-type receptor, less ART-27 protein associated with the AR E2K substitution, consistent with reduced transcriptional response. Surprisingly, more ART-27 associated with AR P340L, despite the fact that the mutation decreased transcriptional activation in response to ART-27. Our findings suggest that aberrant AR-coactivator association interferes with normal ART-27 coactivator function, resulting in suppression of AR activity, and may contribute to the pathogenesis of diseases related to alterations in AR activity, such as prostate cancer and AIS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ANDROGEN RECEPTOR (AR) is a transcriptional regulatory protein that transduces the signaling information conveyed by androgens (1). Upon androgen binding, the hormone-AR complex enters the nucleus, associates with specific DNA sequences, and modulates transcription initiation from nearby promoters (2). Activation of AR is essential for the maintenance of the prostate gland in adult males, and, in the absence of androgens, the prostate shrinks to a rudimentary form. For this reason, nonsteroidal antiandrogens are frequently used in conjunction with LHRH agonists to lower circulating androgen to treat advanced prostate cancer (3).

The transcriptional activation functions (AFs) of AR (4, 5) represent surfaces capable of interaction with general transcription factors and additional transcriptional regulatory factors termed coactivators. Coactivators have been identified that interact with the AR N-terminal AF-1 and the C-terminal AF-2 region to enhance AR-dependent gene transcription (6, 7). AR also interacts with the general transcription factor TFIIF (8) as well as the cyclin-dependent kinase-activating kinase of TFIIH (9).

AR trapped clone-27 (ART-27) was identified in our laboratory as an AR N-terminal coactivator (10). ART-27 binds to a region of AR encompassing AF-1a and AF-1b and activates AR-dependent transcription in a dose-dependent manner in cell-based assays. Endogenous ART-27 interacts with AR in nuclear extracts of LNCaP cells, and velocity gradient sedimentation of nuclear extracts suggests that native ART-27 is part of a multiprotein complex.

Indeed, the components of the ART-27 complex have recently been identified by mass spectrometric analysis of ART-27-associated proteins from HeLa whole-cell lysates (11). ART-27 associates with proteins that include RBP5, a subunit shared by RNA polymerases I, II, and III, an RBP5 binding protein called unconventional prefoldin RBP5 interactor and the ATPase/helicase TIP48 and TIP49, as well as other unidentified proteins. Thus, ART-27 appears to be part of a large multiprotein complex in human cells that includes proteins that function in transcriptional regulation.

We have also shown that in normal adult human prostate, ART-27 protein is expressed in luminal epithelial cells, in contrast to the stroma, where ART-27 is not expressed (12). During prostate development in humans, ART-27 is expressed in differentiated luminal epithelial cells but is not detected in undifferentiated epithelial cell precursors, suggesting a role for ART-27 in AR-mediated growth suppression and differentiation. Consistent with a growth-suppressive function, ART-27 expression levels are negligible in human prostate cancer, and regulated expression of ART-27 in the androgen-sensitive LNCaP prostate cancer cell line inhibits androgen-mediated cellular proliferation (12). These findings suggest that ART-27 affects AR target genes important to prostate growth suppression and/or differentiation.

To examine the physiological contribution of ART-27 to AR-dependent processes, we examined a set of naturally occurring AR N-terminal mutations identified in prostate cancer and androgen insensitivity syndrome (AIS) for effects on the AR transcriptional response to ART-27. AR mutations have been identified in up to 50% of advanced, metastatic prostate cancers, suggesting that AR mutations may confer a growth advantage to the cells (13). In principle, AR somatic alterations associated with prostate cancer may represent gain-of-function mutations that enhance AR interaction with coactivators involved in cell proliferation. Alternatively, if coactivators such as ART-27 confer AR-dependent growth suppression and differentiation, the function of ART-27 may be compromised in certain receptor mutations isolated from prostate cancer patients. Interestingly, a growing list of mutations localizing to the N terminus is emerging, suggesting an important role for the AR N terminus and associated factors in prostate cancer.

Naturally occurring mutations are also found in individuals with AIS. There is a broad range of androgen insensitivity from complete AIS (CAIS) to partial AIS (PAIS). In CAIS, tissues are insensitive to androgen, whereas in PAIS tissues vary in their sensitivity to hormone. CAIS individuals are genetically male, yet phenotypically appear female as a result of a loss in AR activity, whereas a spectrum of phenotypic changes occur in PAIS patients depending on the severity of the defect in AR function (14, 15). The AR N-terminal mutations isolated from AIS individuals are presumed to produce alterations in AR function perhaps as a consequence of abnormal AR-coactivator interactions. Here we analyze naturally occurring AR mutations identified in prostate cancer and AIS for their ability to functionally interact with ART-27.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To examine the physiological contribution of ART-27 to AR-dependent processes, we tested a set of naturally occurring AR N-terminal mutations identified in prostate cancer and AIS for their effect on the AR transcriptional response to ART-27. We focused on AR N-terminal mutations because ART-27 has been shown to bind exclusively to this region of the receptor (10). Eleven AR mutations, spanning amino acids 2–491, were made in the N terminus. Of these mutations, five were mutations identified in human AIS (E2K, Q194R, N233K, L255P, and G491S), and six were mutations from individuals with prostate cancer (K180R, E198G, M266T, P269S, S334P, and P340L) (Fig. 1AGo).



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  		Fig. 1. AR Functional Domains and Predicted Structure

A, Schematic diagram of the functional domains of the human AR. Shown are a ploy-glutamine stretch (Q), AF-1a and AF-1b (black), the DNA binding domain (DBD) (hatched), and the ligand binding domain (LBD) and AF-2. The predicted secondary structure of the AR N terminus is also shown (bottom panel). The thick black line above the structure alignment depicts the ART-27 binding region. The AR N-terminal mutations identified in androgen insensitivity (AIS) (top) and prostate cancer (PCa) (bottom) are shown. A list of AR point mutations can be found at http://ww2.mcgill.ca/androgendb/. The secondary structure content for the AR N terminus from a multiple alignment comparing AR from eight different species to the human AR; these include AR from rat (P15207) (38 ), mouse (P19091) (39 ), dog (Q9TT90) (40 ), rabbit (P49699) (41 ), lemur (O97776) (42 ), chimpanzee (O97775) (42 ), macaque (O97952) (42 ), and baboon (O97960) (42 ). Gray cylinders represent {alpha}-helices; gray arrows are ß-sheets, and stippled gray lines are unstructured. AR mutants with a single asterisk denote an alteration that was found in conjunction with another mutation outside the AR N terminus. Predicted tertiary structures of wild-type AR (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 ) (panel B) and AR (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 ) E2K (panel C), wild-type AR (331–355) (panel D), and AR (331–355) P340L (panel E) are shown. The N-terminal regions are in red and C-terminal segments are shaded blue.

 
It is conceivable that these AR mutations induce changes in the receptor structure, such that unstructured regions would adopt structural features or, alternatively, that structured domains would be disrupted. Therefore, we analyzed the ability of the AR mutations to alter AR structure before embarking on the functional analysis. Initially, we analyzed the secondary structure of the wild-type AR N terminus from a multiple alignment among ARs from different species (Fig. 1AGo). This approach can achieve greater than 75% accuracy (16, 17) and revealed 16 regions with predicted structural features. Although the majority of the AR N terminus appears unstructured, this is in agreement with biophysical studies (18). Of the AR mutations analyzed, the majority had no apparent effect on the secondary structure of the AR N terminus (data not shown). The two mutations that did change the predicted structure of the AR N terminus were AR E2K, a germ line mutation identified in a patient with PAIS (19), and AR P340L, a somatic mutation identified in a localized prostate cancer (20). Analysis of the low-energy conformations (within 10 kcal/mol) accumulated during extensive peptide simulations of the AR residues 1–25 shows that the first 10 residues form an {alpha}-helical fold in the E2K mutant, whereas this region is largely unstructured in the wild-type AR (Fig. 1Go, B and C). The sequence-based prediction in Fig. 1AGo and the helical fold observed in residues 16–25 of the wild-type AR in Fig. 1BGo are in agreement with a recent report suggesting that AR amino acids 16–36 fold into a long amphipathic {alpha}-helix (21). Interestingly, the tertiary structure simulation for the AR 331–355 region shows a dramatic preference for wild-type peptide to adopt an {alpha}-helical fold at its C terminus between residues 341–355, whereas the P340L mutation folds into a {alpha}-helix near the N terminus flanked by residues 331–345 (Fig. 1Go, D and E). Therefore, the E2K and P340L mutations generate new structural elements, suggesting that they may represent functionally relevant alterations.

The AR mutants were initially analyzed for their ability to affect AR transcriptional activity as compared with wild-type AR in a cell-based assay using an AR-responsive mouse mammary tumor virus (MMTV)-luciferase reporter (Fig. 2AGo). The AR alterations E2K, Q194R, and P340L exhibited lower AR transcriptional activation, whereas the remaining mutations, K180R, E198G, N233K, L255P, M266T, P269S, S334P, and G491S, did not appear to significantly affect AR activity relative to the wild-type receptor. Immunoblot analysis revealed that both wild-type AR and all of the receptor variants are stabilized in the presence of R1881 and that some variability in AR protein expression is also observed among the mutant receptors (Fig. 2AGo). For example, the Q194R and P340L display elevated AR protein expression relative to wild-type receptor, but show lower receptor transcriptional activation, indicating that the decreased AR activity is not a result of reduced AR protein expression. In contrast, E2K, which also shows reduced AR-dependent activity, shows lower steady state levels of AR protein compared with the wild-type receptor, and this may contribute to the lower activity observed in the transcriptional activation assay. The AR E2K mutation, originally identified from a PAIS patient, has been previously shown to exhibit reduced receptor expression as a result of inefficient translation (19). Despite some variability of AR expression, a majority of the mutants exhibit activity comparable to wild-type AR. Thus, a subset of AR mutants affects AR transcriptional activity.



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  		Fig. 2. Transcriptional Response of AR Mutants to ART-27

A, Top panel: HeLa cells were transiently transfected as described in Materials and Methods with the indicated AR expression vector or vector only (VO), MMTV-luciferase reporter gene, and CMV-LacZ, together with pcDNA3:HA-ART-27 (+ART-27) or pcDNA3 (–ART-27). Cells were treated with 100 nM R1881 or an ethanol vehicle, and AR transcriptional activation was assayed for luciferase activity, normalized to ß-galactosidase activity and expressed as relative light units (RLU). Bottom panel: Whole-cell extracts were prepared from transfected cells, and the expression of AR variants was analyzed by Western blotting using an AR or an actin antibody, which serves as a control for loading. B, The relative fold-induction in response to 100 nM R1881 of each AR mutant in the absence (gray bars) and presence (black bars) of ART-27. Shown is a single experiment done in duplicate with the error bars representing the range of the mean. The experiment was repeated three times with similar results. C, Effect of the E2K and P340L mutations on the AR transcriptional response of ARR3 to ART-27. HeLa cells were transiently transfected with the rat probasin ARR reporter construct, ARR3-TK-luciferase, along with the wild-type AR (WT), AR E2K (E2K), AR P340L (P340L), or the empty expression vector (VO) in the absence and presence of ART-27. Luciferase activity was determined in the absence and presence and 100 nM R1881, as described previously. Shown is a representative experiment done in duplicate and repeated three times with similar results.

 
Next we examined the effect of ART-27 overexpression on wild-type and mutant AR transcriptional activation. The transcriptional activity of the wild-type AR was increased approximately 3-fold by ART-27 overexpression (Fig. 2AGo). Interestingly, the AR mutations E2K, Q194R, and P340L displayed reduced ART-27-dependent receptor transcriptional enhancement, whereas the majority of the other alterations appeared capable of increased activation in response to ART-27. Thus, of all the AR mutants tested, E2K, Q194R, and P340L appear to have a reduced capacity to utilize ART-27 as a coactivator.

In addition to measuring total hormone-dependent AR activity, we also compared the "fold-induction" or the AR transcriptional response to ligand in the presence and absence of ART-27 (Fig. 2BGo). The relative fold-induction of AR in response to ART-27 is constant over a range of AR concentrations and, therefore, is a valid means of comparison among the receptor mutants that vary in expression (supplemental Fig. 1Go published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). The AR alterations E2K and P340L showed a decreased fold induction in response to overexpressed ART-27. In contrast, the Q194R mutation maintained a fold induction in response to ART-27 that was similar to wild-type AR. This suggests that the Q194R alteration has a more general effect on AR transcriptional activation, whereas the impact of E2K and P340L alterations appear specific to ART-27-mediated AR activation and will be the focus of our subsequent experiments (Fig. 2BGo).

We next examined the effect of the E2K and P340L substitutions on the receptor transcriptional response to ART-27 at other promoters and regulatory elements. Transcriptional activity of the mutations was compared with wild-type AR in a cell-based assay using the ARR3-luciferase reporter from the androgen responsive region (ARR) of the rat probasin promoter (22) and the synthetic TAT3-luciferase reporter. A similar reduction was also observed with the AR E2K and P340L mutations at the ARR3-luciferase reporter (Fig. 2CGo) and from the synthetic TAT3-luciferase reporter (data not shown). This indicates that the effect of the E2K and P340L substitutions on the receptor transcriptional response to ART-27 is evident at distinct androgen response elements and promoter elements.

To determine whether the decreased activity of AR E2K and AR P340L would be overcome by increasing the levels of ART-27 relative to receptor, we expressed wild-type receptor and the AR mutants E2K and P340L in the presence of increasing concentrations of ART-27 (Fig. 3AGo). Wild-type AR shows increased levels of AR transcriptional activation in response to ART-27, consistent with previous observation. On the other hand, AR E2K and AR P340L show a greatly diminished response in comparison to wild type at all the concentrations of ART-27 tested (Fig. 3AGo). Thus, increasing ART-27 protein levels cannot compensate for the receptor defects.



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  		Fig. 3. Diminished Response of AR E2K and AR P340L to ART-27 Is Not Restored by Increasing ART-27, AR, or Hormone Levels

A, Transcriptional response of WT AR, AR E2K, and AR P340L to increasing levels of ART-27. HeLa cells in six-well plates (1.5 x 105 cells per well) were transfected with an MMTV-luciferase reporter construct (100 ng), the AR derivatives (WT, E2K, or P340L; 200 ng), and increasing concentrations of ART-27 (0, 0.2, 0.5, 1.0, or 1.5 µg). Cells were treated with 100 nM R1881, and luciferase activity was assayed as described in Materials and Methods, normalized to ß-galactosidase activity, and expressed as relative light units (RLU). Western blot (bottom) shows the expression of ART-27. B, Transcriptional response of AR to ART-27 as a function of receptor concentration. HeLa cells in 24-well plates (3 x 104 cells per well) were transfected with ART-27 (100 ng) and increasing amounts of AR (20, 40, 80, 100, and 160 ng).

Cells were treated with 100 nM R1881 and AR transcriptional response from the MMTV-luciferase reporter gene was determined as above. Western blot (bottom) shows the expression of the AR variants. C, Increasing hormone levels do not compensate for the diminished transcriptional response of E2K and P340L to ART-27. HeLa cells were transfected with 40 ng of each AR variant and 100 ng of ART-27 and 100 ng MMTV-luciferase. Cells were then treated with the indicated concentration of R1881 and luciferase activity determined as above. WT, Wild type.

 
To determine the impact of AR expression levels on transcriptional activation, we titrated the amount of wild-type or mutant receptor and evaluated the ability to activate transcription (Fig. 3BGo). The AR E2K mutant cannot activate transcription to the level of wild-type receptor at any of the levels tested (Fig. 3BGo), consistent with its identification previously as a partial loss-of-function mutation from a PAIS patient (19). AR P340L also displays decreased responsiveness to ART-27 at all AR concentrations tested relative to the wild-type AR. Our findings indicate that AR E2K and AR P340L are defective in response to ART-27 at levels of receptor comparable to the wild-type AR.

To evaluate the effect of hormone concentration on the activity of the AR E2K and AR P340L mutants relative to wild-type AR, cells were cotransfected with the AR or AR mutants and ART-27, and treated with hormone ranging from 10–7 to 10–11 M (Fig. 3CGo). The results indicate that both AR E2K and AR P340L show reduced receptor transcriptional activity at all hormone concentrations tested. Interestingly, AR E2K showed a more dramatic reduction in AR activity at lower hormone concentrations. For example, whereas the wild-type AR and AR P340L achieved 80% of maximal activation in response to 10–10 M R1881, AR E2K achieved only 30% of its maximal activity at this same hormone concentration (Fig. 3CGo). Therefore, increased hormone concentration does not compensate for the decreased AR transcriptional activity of AR E2K and AR P340L to ART-27. Thus, increasing ART-27, AR, or hormone concentration does not compensate for the defect in the AR E2K and P340L.

To determine whether these AR mutants show decreased responsiveness to coactivators other than ART-27, cells were transfected with wild-type AR, AR E2K, and AR P340L along with ART-27 or the p160 coactivators, glucocorticoid receptor-interacting protein 1 (GRIP-1) or steroid receptor coactivator 1 (SRC-1) (23, 24). As before, the AR P340L substitution showed a diminished capacity to respond to ART-27 relative to the wild-type AR (Fig. 4Go). In contrast, the AR transcriptional response to SRC-1or GRIP-1 was not affected by the AR P340L substitution relative to the wild-type receptor. Analysis of the AR E2K mutation again showed a reduced ability to respond to ART-27 (Fig. 4Go). Surprisingly, AR E2K shows an increase in transcriptional activity in response to SRC-1 (Fig. 4Go). Overall, our findings indicate that the E2K and P340L mutations selectively affect AR functional interactions with ART-27.



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  		Fig. 4. Specificity of the Mutant AR Transcriptional Response to ART-27

HeLa cells were transiently transfected with the indicated AR derivatives, the MMTV-luciferase reporter gene, and ART-27, GRIP-1, or SRC-1 expression constructs. Cells were treated with 100 nM R1881or an ethanol vehicle, and luciferase activity was determined as described in Fig. 2Go. Shown is a representative of three independent experiments done in triplicate with the error bars representing the SE. Western blot (bottom) shows the expression of the AR derivatives. RLU, Relative light units.

 
We next tested the AR mutants for physical interaction with ART-27 by coimmunoprecipitation. HeLa cells were transfected with a hemagglutinin (HA)-tagged version of ART-27 along with expression vectors for either the wild-type AR, AR E2K, or AR P340L. Cells were treated with hormone and lysed under conditions that preserve the interaction between AR and ART-27. AR immunoprecipitates were analyzed by immunoblotting with an HA antibody specific for the HA-tagged ART-27. As seen in Fig. 5AGo, ART-27 was coimmunoprecipitated with both wild-type AR, AR E2K, and AR P340L. About half the level of ART-27 was detected in association with AR E2K as compared with the wild-type receptor. Thus, the AR E2K alteration reduces interaction with ART-27. In contrast, AR P340L showed an increase in its association with ART-27, despite its inability to enhance AR transcription. To investigate whether the increased ART-27 associated with AR P340L mutant might reflect a tighter binding, reciprocal immunoprecipitation experiments between AR and ART-27 were performed under more stringent conditions. Under these circumstances, little ART-27 is detected in association with the wild-type AR (Fig. 5BGo). In contrast, ART-27 is readily detected in association with the AR P340L substitution, indicating that the interaction of AR P340L with ART-27 is more stable than that of the wild-type receptor. This finding suggests that the AR P340L mutant is binding inappropriately to ART-27 and thus fails to facilitate AR-dependent transcriptional activation.



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  		Fig. 5. Aberrant Binding of ART-27 to AR P340L and AR E2K Mutations

A, HeLa cells were transfected with HA-ART-27 and either the wild-type AR, the AR E2K, or AR P340L, treated with 100 nM R1881 for 2 h, and were immunoprecipitated with an AR antibody from nuclear extracts under low-stringency conditions as described in Materials and Methods. Associated proteins were resolved by SDS-PAGE. HA-ART-27 associated with AR was detected by immunoblotting with an antibody against HA. The left panel shows the expression of AR and ART-27 before immunoprecipitation (input) and the right panel reveals the ART-27 that was immunoprecipitated with AR (IP). The total amount of AR immunoprecipitated (bottom panel) was used to standardize the amount of associated ART-27 by densitometry, with the wild-type AR:ART-27 ratio arbitrarily set as 1. B and C, HeLa cells were transfected as above, and whole cell lysates were prepared in RIPA buffer (high-stringency conditions), and reciprocal immunoprecipitations were performed using antibodies against AR (panel B) or HA (ART-27) (panel C). The left panel shows the expression of AR and ART-27 before immunoprecipitation (input). Associated proteins were resolved by SDS-PAGE and revealed by immunoblotting with the corresponding AR or ART-27 (HA) antibody. Shown are representative experiments that were repeated three times with similar results. WT, Wild type.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Although AR plays a role in normal and malignant prostate cell function, the impact of AR N-terminal mutations in the etiology of prostate cancer and AIS is not clear (25). One possibility is that the AR N-terminal mutations increase or decrease interaction with AR cofactors. AR mutations in prostate cancer could enhance the function of a coactivator involved in AR-dependent proliferation or inhibit the activity of a coactivator mediating AR-dependent differentiation. The cofactors themselves could also be altered and collaborate with AR mutations to promote cellular proliferation. A recent report indicates that recurrent prostate cancers express higher than normal levels of the p160 coactivators, transcriptional intermediary factor-2/GRIP-1 and SRC-1 (26). In addition, the expression levels of multiple AR coactivators varied between normal and malignant prostate tissue samples (27, 28, 29). Further, our group has found that ART-27 protein levels are decreased in prostate cancer, suggesting a role for ART-27 in growth inhibition (12). The AR N-terminal mutations in AIS, which presumably reduce AR activity in vivo, could also result from an alteration in cofactor binding. Recently, a patient with AIS was described whose cells lacked AR transcriptional activity, probably through the loss of an as-yet-unidentified AR N-terminal cofactor (30). This underscores the importance of the AR N terminus and associated factors in AIS.

Previous studies have shown that the AR E2K mutation decreases receptor translation, resulting in lower steady state AR levels (19). Our results also indicate that this mutant shows a diminished interaction and transcriptional response to ART-27. Because the AR E2K mutation is located outside of the ART-27 binding region (Fig. 1AGo) and induces a local conformational change (Fig. 1CGo), we suggest that the change in conformation affects the global architecture of the receptor, which reduces ART-27 binding (Fig. 5Go). The unexpected finding that the AR E2K displays an enhanced transcriptional response to SRC-1 is consistent with the notion that the E2K mutation affects global AR conformation. Such changes in the AR response to coactivators may be an important determinant in the AIS phenotype.

Our results also indicate that the ability of ART-27 to function as an AR coactivator is greatly decreased by the P340L substitution. Although the expectation was that this reduced activity is a result of diminished ART-27 binding to the receptor, this is not the case (Fig. 4Go). Instead, our findings demonstrate that the AR P340L mutant associates more avidly with ART-27 (Fig. 5Go). In principle, increased ART-27 binding to AR could affect the association of a different regulatory cofactor. In support of this idea, AR P340L lies near a stretch of amino acids that has been shown to interact with TFIIF, a component of the basal transcription machinery consisting of two subunits, RAP74 and RAP30 (8, 31). Elegant work from the McEwan laboratory (8) has revealed that RAP74 interacts with AR at multiple sites including two motifs (PSTLSL) located between residues 159–164 and 340–345 in the AR N terminus. It is possible that the AR P340L mutation creates a new surface for ART-27 binding and eliminates a motif existing in the wild-type AR for cofactor binding, which is consistent with the structure prediction (Fig. 1EGo). This could explain the tendency of AR P340L mutant to exhibit increased ART-27 binding, but decreased AR activity.

Another possibility is that ART-27 functions as a chaperone to help "load" TFIIF (or another factor) onto the receptor or maintain AR in a conformation competent for cofactor binding. Once this is accomplished, ART-27 would then dissociate from the receptor. In the AR P340L mutant, however, ART-27 would be unable to correctly place the cofactor onto the receptor or promote a receptor conformation compatible with cofactor binding and would neither dissociate nor coactivate. This idea is not inconceivable because ART-27 shows homology to prefoldins, which are small molecular weight proteins that assemble into molecular chaperone complexes to affect protein folding. Recently, ART-27 has been shown to be part of a transcriptional regulatory complex that contains an unconventional prefoldin that controls a transcription program in response to nutrient deprivation (11). This highlights the biological relevance of prefoldin-type proteins in the regulation of gene expression.

Our recent studies indicate that ART-27 is present in normal adult prostate but is absent in prostate cancer (12). Further, examination of ART-27 protein expression in prostate development demonstrates that ART-27 is detected only when the prostate gland has proceeded from a solid mass of undifferentiated cells to a stage at which differentiated luminal epithelial cells are evident (12). In light of these findings, we suggest that ART-27 plays a role in suppressing prostate cancer development by contributing to the maintenance of a program of AR-mediated differentiation. Thus, the AR P340L mutant would facilitate prostate cancer progression by preventing the normal "growth-suppressive" function of ART-27. This may represent a novel mechanism of pathogenesis, whereby an AR mutation acts in a dominant negative fashion to reduce the action of ART-27 in maintaining differentiation and further highlights the importance of loss of ART-27 function in oncogenic transformation of prostate epithelial cells.

Unexpectedly, our findings indicate that a defect in ART-27 coactivator function may play a role in AIS as well as prostate cancer. This may seem surprising in that AIS results from a loss of AR function, whereas prostate cancer is generally thought to stem from enhanced AR activity, through, for example, increased receptor expression (32). However, both the E2K and P340L AR mutations may reflect defects in the induction and maintenance of differentiation by AR and ART-27. It is well recognized that AR exerts a range of effects during development and in fully differentiated adult tissue. The AR E2K mutant results in a developmental defect because it is a germline mutation causing PAIS. In development ART-27 is expressed in differentiated epithelial cells of the urogenital sinus. During prostate development, ART-27 is not expressed until a solid mass of cells in the prostatic bud begins to differentiate into luminal epithelial cells. The fact that AR E2K exhibits decreased protein levels due to inefficient translation explains, in part, the androgen insensitivity phenotype. However, we suggest that this phenotype is exacerbated by reduced receptor binding ART-27 and decreased transcription from the ART-27/AR complex. Together, this would have a negative effect on prostate epithelial cell differentiation.

The AR P340L mutation is a somatic mutation found in prostate cancer. Therefore, a phenotype caused by this mutation would not be revealed in development. Our previous findings suggest that ART-27, in conjunction with AR, represses cell growth. We hypothesize that the aberrant interaction of ART-27 with AR results in derepression of growth-stimulatory genes, thus contributing to cellular hyperplasia and cancer.

Thus, the AIS mutation AR E2K is unable to induce AR-mediated differentiation during development, whereas the prostate cancer AR mutation P340L is unable to maintain growth arrest and differentiation in the adult prostate. Therefore, it is conceivable that ART-27 is an important element in the pathogenesis of both AIS and prostate cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Construction
The AR mutants were generated by QuikChange Site-Directed Mutagenesis system (Stratagene, La Jolla, CA) using the oligonucleotides described in supplemental Table 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org and the wild-type pcDNA3:hAR expression plasmid as the template. All mutations were confirmed by DNA sequencing. The p160 expression vectors, pcDNA3-GRIP-1 and pCR3-hSRC-1A, have been described previously (33).

Cell Culture and Transient Transfection
HeLa cells were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT) and 2 mM L-glutamine (Invitrogen, San Diego, CA). Cells were seeded in a 24-well plate at a density of 3 x 104 or a six-well plate at a density of 1.5 x 105 in phenol red-free DMEM supplemented with 10% charcoal-stripped fetal bovine serum. Transfection was performed using Lipofectamine Plus (Invitrogen) according to manufacturer’s instructions. For transfection of cells in 24-well plates, each well received 40 ng of the pcDNA3:hAR expression vector, 100 ng of androgen-responsive reporter plasmid MMTV-luciferase, and 10 ng of cytomegalovirus-LacZ, together with 100 ng of pcDNA3:HA-ART-27. The total amount of DNA transfected was held constant using the corresponding empty vector. For SRC-1 and GRIP-1, 100 ng of each plasmid was used. For six-well plates, all plasmid amounts were increased 5-fold. After a 3-h incubation, the transfection mixtures were removed and the cells were refed with phenol red-free medium. The next day, the indicated amount of R1881 (PerkinElmer, Norwalk, CT) or an equal volume of an ethanol vehicle was added. After 24 h, the cells were washed with PBS and lysed in 1x luciferase cell culture lysis reagent (Promega Corp., Madison, WI; catalog no. E1500). The cell extracts were analyzed for luciferase activity, and the values were normalized to ß-galactosidase activity. Luciferase activity was quantified in a reaction mixture containing 15 µl of lysate and 100 µl of luciferase assay reagent [25 mM glycylglycine (pH 7.8), 10 mM MgSO4, 1 mM ATP, 0.1 mg/ml BSA, 1 mM dithiothreitol (DTT)], using an LMax microplate reader luminometer and 1 mM D-luciferin as substrate. Parallel sets of cells were analyzed for AR protein expression.

Coimmunoprecipitation
For each 10-cm dish of HeLa cells, 10 µg of the wild-type or mutant AR was cotransfected with 10 µg of HA-ART-27. The cells were treated 3 h posttransfection with 100 nM R1881 or ethanol vehicle for 16 h, washed with cold PBS, scraped, and collected into a 15-ml conical tube by low-speed centrifugation. For interactions under low-stringency conditions, nuclear extracts were prepared from cell pellets resuspended in 1.5-fold of the packed cell volume (typically 300 µl) of buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 2 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail). The cells were then lysed by five passes through a 24-gauge needle and centrifuged at 14,000 rpm for 5 min at 4 C. The nuclear pellet was resuspended in two thirds of the original volume (typically 200 µl) of buffer C [20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 2 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, and the protease inhibitor cocktail] and incubated on ice with stirring for 30 min. The nuclear extracts were obtained by centrifugation at 14,000 rpm for 5 min at 4 C.

The coimmunoprecipitation under high-stringency conditions was performed by lysing the cells directly on the plate in 300 µl of RIPA buffer [150 mM NaCl, 0.2% sodium dodecyl sulfate (SDS), 50 mM Tris (pH 7.4), 1% Nonidet P-40, 1% deoxycholate) on ice and centrifugation at 14,000 rpm for 5 min at 4 C.

The total protein concentration was normalized and 10 µg of a mouse monoclonal antibody to HA (Covance Laboratories, Inc., Berkeley, CA) or AR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; catalog no. sc-7305) was added and incubated overnight at 4 C. After the incubation, 60 µl of a 50% slurry of Protein G Sepharose beads (Amersham Pharmacia Biotech, Inc., Arlington Heights, IL) was added and incubated for an additional 2 h at 4 C with rocking. The beads were collected by centrifugation, and the immune complexes were washed three times with HEMG buffer (20 mM HEPES, pH 7.9; 12.5 mM MgCl2; 0.2 mM EDTA) for the low-stringency precipitation. For the high-stringency conditions, the immune complexes were washed three times in low-salt buffer (150 mM NaCl; 20 mM Tris, pH 8.1; 2 mM EDTA; 0.1% SDS; 1% Triton X-100), twice with high-salt buffer (500 mM NaCl; 20 mM Tris, pH 8.1; 2 mM EDTA; 0.1% SDS; 1% Triton X-100), and twice with a nonionic detergent containing wash buffer (250 mM LiCl; 0.5% Nonidet P-40; 0.5% deoxycholate; 1 mM EDTA; 10 mM Tris, pH 8.1). The beads were resuspended in 2x SDS sample buffer, boiled for 5 min, placed on ice, and stored at –20 C.

Immunoblotting
Proteins were separated by 12% SDS-PAGE and transferred to Immobilon paper (Millipore Corp., Bedford, MA). The membranes were blocked in 5% BSA in Tris-buffered saline, pH 7.4 (TBS) at 4 C overnight. The membranes were incubated in the blocking buffer with primary antibody at room temperature for 2–4 h [1:500 of a mouse monoclonal antibody to AR; 1:1000 of a goat polyclonal antibody against actin (Santa Cruz Biotechnology catalog no. sc-1616); and 1:1000 of a mouse monoclonal antibody to HA]. The membranes were washed three times for 10 min in TBS/0.1% Triton X-100 and were incubated for 1 h at room temperature with antirabbit, antimouse, or antigoat-IgG conjugated to horseradish peroxidase, washed five times for 10 min in TBS/0.1% Triton X-100, followed by TBS, and developed with enhanced chemiluminescence (Amersham Pharmacia Biotech, Inc.).

Structure Analysis
Secondary structure predictions were based on the Frishman and Argos (17) method as implemented in the Internal Coordinates Mechanics program.

Peptide Simulations
The 25-residue peptides M1-EVQLGLGRVYPRPPSKTYRGAFQ-N25 and A331-GSSGTLELPSTLSLYKSGALDEA-A355 (together with E2K and P340L mutations) were built into the Internal Coordinates Mechanics program using an all-atom representation according to the ECEPP/3 force field (34). The total energy also included an entropy term (35) plus a generalized Born-based electrostatic term (36). For each peptide, 10 parallel independent Biased Probability Monte Carlo (35) global energy optimizations of all backbone and side chain torsion angles were performed starting from different randomized conformations, and the low-energy conformations were collected in a conformational stack (37). Simulations were terminated after 50 million energy evaluations (which corresponds to ~100,000 random steps followed by ~500 local minimization steps), and redundant conformations were eliminated from the stack.


   ACKNOWLEDGMENTS
 
We thank R. Miesfeld for the human AR expression vector, R. Matusik for the ARR3 reporter construct, and I. Pineda Torra, I. Rogatsky, and N. Tanese for critically reading the manuscript.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK58024 (to M.J.G.); Edwin Beer Foundation (to S.K.L.) the St. Lawrence Seaway Corporation (to M.J.G. and S.S.T.) and a Department of Defense Prostate Cancer postdoctoral fellowship (to W.L.).

First Published Online May 26, 2005

Abbreviations: AF, Activation function; AIS, androgen insufficiency syndrome; AR, androgen receptor; ARR, androgen responsive region; ART-27, AR trapped clone-27; CAIS, complete AIS; DTT, dithiothreitol; GRIP, glucocorticoid receptor interacting protein; HA, hemagglutinin; MMTV, mouse mammary tumor virus; PAIS, partial AIS; SDS, sodium dodecyl sulfate; SRC, steroid receptor coactivator; TBS, Tris-buffered saline.

Received for publication March 22, 2005. Accepted for publication May 11, 2005.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   AR
Coregulators:   ART-27  |  SRC-1  |  GRIP1
Ligands:   R1881



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