Cancer Research Annual Meeting 2010 Sign up for Cancer Research eTOC's
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 10087, October 15, 2007. doi: 10.1158/0008-5472.CAN-07-1646
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Buchanan, G.
Right arrow Articles by Tilley, W. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Buchanan, G.
Right arrow Articles by Tilley, W. D.

Endocrinology

Control of Androgen Receptor Signaling in Prostate Cancer by the Cochaperone Small Glutamine–Rich Tetratricopeptide Repeat Containing Protein {alpha}

Grant Buchanan1, Carmela Ricciardelli1, Jonathan M. Harris2, Jennifer Prescott3, Zoe Chiao-Li Yu2, Li Jia3, Lisa M. Butler1, Villis R. Marshall1, Howard I. Scher4, William L. Gerald4, Gerhard A. Coetzee3 and Wayne D. Tilley1

1 Dame Roma Mitchell Cancer Research Laboratories, School of Medicine, The University of Adelaide/Hanson Institute, Adelaide, South Australia, Australia; 2 Centre for Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, Brisbane, Queensland, Australia; 3 Department of Preventive Medicine, USC/Norris Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California; and 4 Genitourinary Oncology Service, Division of Solid Tumor Oncology, Memorial Sloan-Kettering Cancer Center, Department of Medicine, Joan and Sanford I. Weill College of Medicine, New York, New York

Requests for reprints: Grant Buchanan and Wayne D. Tilley, Department of Medicine, Dame Roma Mitchell Cancer Research Laboratories, School of Medicine, The University of Adelaide/Hanson Institute, P.O. Box 14, Rundle Mall, Adelaide, South Australia 5000, Australia. Phone: 61-88222-3261; Fax: 61-88222-3217; E-mail: grant.buchanan{at}imvs.sa.gov.au and wayne.tilley{at}imvs.sa.gov.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the androgen receptor (AR) is accepted as the major determinant of prostate cancer cell survival throughout disease progression, it is currently unclear how the receptor sustains genomic signaling under conditions of systemic androgen ablation. Here, we show that the evolutionarily conserved Hsp70/Hsp90 cochaperone, small glutamine–rich tetratricopeptide repeat containing protein {alpha} ({alpha}SGT), interacts with the hinge region of the human AR in yeast and mammalian cells. Overexpression and RNA interference revealed that {alpha}SGT acts to (a) promote cytoplasmic compartmentalization of the AR, thereby silencing the receptors basal/ligand-independent transcriptional activity, (b) regulate the sensitivity of receptor signaling by androgens, and (c) limit the capacity of noncanonical ligands to induce AR agonist activity. Immunofluorescence, coactivator, and chromatin immunoprecipitation analyses strongly suggest that these effects of {alpha}SGT on AR function are mediated by interaction in the cytoplasm and are distinct from the receptors response to classic coregulators. Quantitative immunohistochemical analysis of {alpha}SGT and AR levels in a cohort of 32 primary and 64 metastatic human prostate cancers revealed dysregulation in the level of both proteins during disease progression. The significantly higher AR/{alpha}SGT ratio in metastatic samples is consistent with the sensitization of prostate tumor cells to androgen signaling with disease progression, particularly in a low-hormone environment. These findings implicate {alpha}SGT as a molecular rheostat of in vivo signaling competence by the AR, and provide new insight into the determinants of androgen sensitivity during prostate cancer progression. [Cancer Res 2007;67(20):10087–96]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The requirement for androgen signaling in the development and progression of prostate cancer (PCa) underpins hormonal strategies [e.g., androgen ablation therapy and/or androgen receptor (AR) antagonists] in disease treatment. Although virtually all prostate tumors initially respond to androgen ablation, the majority relapse, and the disease progresses. It is now recognized that the failure of these hormonal strategies can be explained in many cases by the selection of a hyperactive AR in a low-androgen environment (1). In particular, we and others have reported increased AR levels in both animal models and clinical PCa, the selection of permissive AR mutations during progression, and the potential for crosstalk between AR and other signaling pathways (2). Whereas these findings suggest that deregulation of AR signaling is a common phenomenon in PCa progression, they can only partially explain how increased AR signaling is achieved, and particularly, how tumors with an apparently intact receptor expressed at levels comparable to that of the normal prostate sustain signaling during androgen ablation therapy. It has recently been proposed that nucleocytoplasmic shuttling of the AR integrates the signaling environment in the cytoplasm with AR activity in the nucleus (3). Factors such as chaperones that modulate this balance are likely to have profound consequences for AR signaling.

Molecular chaperones are essential for the activity of diverse signaling molecules, including steroid and tyrosine kinase receptors, p53 and telomerase, and are emerging as critical players in the pathogenesis of human diseases (4–8). For example, cancer cells have a highly primed and sensitized Hsp90 chaperone system that allows more rapid responses to insult, buffers cellular signaling pathways against genetic instability, and enhances sensitivity to extracellular signaling (6). The same molecular adaptations also make cancer cells particularly susceptible to Hsp90 inhibitors, such as geldanamycin and its derivatives (9).

Whereas the Hsp70/Hsp90 chaperone system has traditionally been ascribed to folding and stabilization of signaling-competent client proteins, there is emerging evidence for additional roles in client movement in the cytoplasm and nucleus, signal and/or transcriptional competence following activation, and for the biologically diverse actions mediated by structurally related proteins (4, 10, 11). Consequently, they are now considered key contributors to the diversity of hormone signaling, acting in unique combinations in a cell-, receptor-, and ligand-specific manner at multiple stages of steroid receptor activation (11). Beginning with Hsp70 and DnaJ (Hsp40), the ordered and stepwise association of nascent steroid receptors with heat shock proteins and chaperones in the cytoplasm mediates folding and the acquisition and maintenance of ligand-binding competence (11). In the final stages of maturation, the receptor becomes dynamically associated with a dimer of Hsp90, p23, and one of a small group of tetratricopeptide repeat (TPR)–containing proteins that exhibit preference for particular steroid receptor complexes (11). The Hsp90 heterocomplex contributes to four stages of steroid receptor movement; through the cytoplasm, across the nuclear pore complex, in the nucleus, and cycling between active sites of transcription and nucleoplasmic stores of cofactors (10). Indeed, the earliest event in steroid receptor activation and cellular redistribution is thought to be ligand-induced exchange of TPR proteins in the mature receptor/Hsp90 heterocomplex (12). However, the precise manner in which these events are orchestrated, how specificity is conferred for different receptors, and how equilibrium between nuclear and cytoplasmic compartments is controlled in a given cell type have not been elucidated.

We report here that the microtubule-associated and evolutionarily conserved small glutamine–rich tetratricopeptide repeat containing protein {alpha} ({alpha}SGT) is a candidate AR-specific Hsp70/Hsp90 cochaperone TPR partner. Interacting with the AR hinge, {alpha}SGT promotes cytoplasmic retention of the receptor in vivo and as a determinant of the sensitivity and specificity of AR activation, thereby buffering ligand-dependent and ligand-independent transactivation responses. We show an increase in the AR/{alpha}SGT ratio in metastatic human prostate tumor samples compared with primary lesions, which is predicted to result in decreased control of AR function and to exacerbate AR-mediated disease progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and tissues. Cell lines were maintained in RPMI 1640 with 5% fetal bovine serum and treated with steroids in phenol red–free RPMI 1640 with 5% to 10% dextran-coated charcoal-treated fetal bovine serum. Multitissue blocks of formalin-fixed, paraffin-embedded tissue, consisting of three representative 0.6-mm cores from diagnostic areas of each of 36 primary PCa samples, 79 metastatic PCa lesions, and nonneoplastic prostate tissue samples, have been described previously (13).

Microarray analysis. Affymetrix U95 microarray data collected as part of our previous studies from 23 PCa samples (manually microdissected for PCa epithelial cells) was analyzed for gene expression as previously described (13).

Immunohistochemistry. Immunohistochemistry/video image analysis was done as described previously (14) on 20 contiguous fields per sample (40x magnification) for serial 5 µm formalin-fixed paraffin tissue sections stained with rabbit AR (1:300; AR-U402, AR-U407) or {alpha}SGT (1:5,000; {alpha}SGT-C18; Zymed Laboratories, Inc.). Samples were discounted if they did not contain sufficient informative stained area. Statistical significance was assessed using the Mann-Whitney U test.

Yeast assays. A yeast two-hybrid screen was done with pAS2-AR(618–754) and a pACT2-pooled prostate cDNA library (BD Biosciences Clontech). Specific interactions were shown by re-transforming yeast with either empty pAS2, pAS2-AR(618–754), or pAS2-AR(618–917) and positive pACT2 clones by luminescent liquid ß-galactosidase assays on triplicate colonies.

Transactivation assays. Steroid receptor–negative PC-3 or COS-1 cells (10,000–20,000/well of a 96-well plate) were transfected with 0.1 to 10 ng of full-length AR (pCMV-AR; pcDNA3.1AR), AR deleted for amino acids 636 to 646 (pCMV-AR{Delta}638–646), or ER (pHEGO) vectors, and 100 ng of androgen (probasin ARR3-tk-Luc) or estrogen (ERE-tk-luc) reporter constructs, treated for 24 h with vehicle control (ethanol) or steroids, and assayed for luciferase activity as previously described (15). Five to 50 ng of pSG5/HA-{alpha}SGT, pSG5/HA-Hic5, or pSG5/HA-glucocorticoid receptor–interacting protein 1 (GRIP1) expression vectors were included as appropriate. All transfection mixes were balanced with respect to the molar ratio of expression vectors (with appropriate empty vector) and total plasmid [with pCAT-basic or pBS-sk(–)]. Mammalian two-hybrid assays were done similarly in COS-1 cells with equal molar amounts of vectors expressing GAL4-DBD and VP16-AD fusions of {alpha}SGT (maximum 5 ng/well), and 25 ng of the GAL4-responsive luciferase reporter, pGK1.

Immunoblot and coimmunoprecipitation. Immunoblot analysis was done using rabbit AR U407 (1:1,000), N20 (1:1,000; Santa Cruz Biotechnology), or C-19 (1:1,000; Santa Cruz Biotechnology), rabbit {alpha}SGT (1:3,000; Zymed Laboratories, Inc.), and/or goat ß-actin (I19; Santa Cruz Biotechnology) antisera (14). For immunoprecipitation, lysates of COS-1 cells (untransfected or transfected with control, AR, and/or {alpha}SGT expression vectors) were incubated with 5 µg of the appropriate antisera. Antibody-bound proteins were collected using Dynal beads (Invitrogen).

GST pulldown assays. {alpha}SGT and AR (amino acids 534–917) were expressed from GST pGEX-4T (Amersham Pharmacia) in the BL21 strain. Purified proteins (5 µg) were immobilized with 50% glutathione 4B beads and incubated with 200 µg of precleared lysates from COS-7 cells transfected with AR or {alpha}SGT expression plasmids with or without 10 nmol/L of 5{alpha}-dihydrotestosterone (DHT).

Confocal microscopy. Confocal microscopy was done on PC-3 cells (50,000 cells/well) transfected for 40 h with AR (50 ng/well) and {alpha}SGT (500 ng/well) expression vectors, or equivalent molar amounts of control, and treated with or without 0.1 to 10 nmol/L of DHT (16). AR cellular localization was manually scored for each treatment in 25 to 50 stained cells in three independent experiments.

{alpha}SGT knockdown by small interfering RNA. C4-2B cells (100,000 cells/well) were transfected using OligofectAMINE (Invitrogen) for 72 h with 2.86 µg of one of two chemically synthesized 21-bp small interfering RNA (siRNA) duplexes (sense, AGCUCGGUCACUUGAGUGUTT; antisense, ACACUCAAGUGACCGAGCUTT; or sense, ACUUUGAAGCUGCCGUGCATT; antisense, UGCACGGCAGCUUCAAAGUTT) or a nonspecific negative control (sense, AGAUCUGGCUAUCGCGGUATT; antisense, UACCGCGAUAGCCAGAUCUTT), and treated with or without ligand for 24 h. RNA was reverse-transcribed and assessed for prostate-specific antigen (PSA) and glyceraldehyde-3-phosphate dehydrogenase expression by quantitative real-time PCR in triplicate reactions (16).

Molecular modeling. Models of {alpha}SGT were created using SwissModel5 based on crystallographic structures of protein phosphatase 5 (PP5) and cyclophilin-40 (Cyp40), and confirmed by WhatIf.6 The AR hinge peptide (amino acids 638–675) was constructed in silico using Chemsite Pro (Pyramid), folded by homology, and energy-minimized using AMBER.7 Docking simulations were done with BiGGER,8 with clustering/scoring according to geometric fit and electrostatic complementarity. Models were rendered with PovRay.9


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Interaction of the AR hinge with a novel molecular chaperone and with Hsp90ß. The hinge region of steroid receptors is a short, poorly conserved structure between the canonical domains for ligand and DNA binding. Whereas little is known about the role of the hinge in steroid signaling, recent data implicates it as a major site for chaperone interaction, posttranslational modification, proteasome targeting, and phosphorylation (12, 17–20). In order to identify the determinants of AR hinge function and how it relates to PCa, we undertook a yeast two-hybrid screen using a human prostate cDNA library and a fragment of the human AR (amino acids 618–754) encompassing the hinge (amino acids 625–669). The screen yielded 23 independent clones, with the most highly represented encoding full-length {alpha}SGT (four clones) and the COOH-terminal region of Hsp90ß (nine clones), the latter being the classic AR chaperone suspected of interacting with at least amino acids 704 to 758 of the receptors ligand-binding domain (11). A specific interaction of AR with {alpha}SGT and Hsp90ß was confirmed in yeast cells using luminescent ß-galactosidase assays (Fig. 1A ). Yeast growth on highly selective media determined that the complete AR hinge and ligand-binding domain (amino acids 618–919) retained interaction with {alpha}SGT, whereas interaction with Hsp90ß was markedly diminished (data not shown). In yeast, neither interaction was affected by the addition of DHT. To analyze the AR/{alpha}SGT interaction further, we raised a rabbit polyclonal antibody to the COOH terminal 18 amino acids of {alpha}SGT. This antisera shows immunoreactive bands in mammalian cells corresponding to native {alpha}SGT and transfected HA-tagged {alpha}SGT proteins at molecular weights of ~43 and 45 kDa, respectively, which could be abrogated by a 5-fold excess by weight of specific peptide (Supplementary Data 1A). The AR/{alpha}SGT interaction was confirmed in mammalian cells by coimmunoprecipitation of full-length proteins, and by GST pulldown assays (Fig. 1B and C). In the native cellular context, the AR/{alpha}SGT interaction is dissociated by DHT (Fig. 1B).


Figure 1
View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. AR interacts with the molecular cochaperone, {alpha}SGT. A, interaction of AR (amino acids 618–754) and full-length {alpha}SGT or the COOH-terminal region of Hsp90ß (amino acids 609–724) in yeast cells. Columns, luminescent ß-galactosidase signal and mean fold activity for three individual transformants over AR alone; bars, SE. B, coimmunoprecipitation of AR and HA-tagged {alpha}SGT in transfected COS-1 cells in the absence (left) or absence and presence (right) of 10 nmol/L of DHT. Immunoprecipitation was done with AR (N20), {alpha}SGT, HA, or rabbit IgG (rIgG) antisera, and immunoblotted with AR-N20 antisera. C, GST pulldown assays with AR and {alpha}SGT from transfected COS-7 cells (immunoblot with {alpha}SGT antisera) and vice versa (immunoblot with AR C-19 antisera). D, expression of the steroid receptor–interacting Hsp70/Hsp90 TPR cochaperones relative to AR in prostate cancer samples (columns, mean; bars, SE) from Affymetrix microarray data. Left, microdissected epithelial cells of 23 primary prostate cancers (ranked by decreasing relative expression). Right, the chaperone/AR ratio in seven metastatic prostate tumors (black columns) relative to primary samples (gray columns; ranked by decreasing significance for a decline).

 
There are only two recognizable domains in the {alpha}SGT protein, a centrally located TPR protein-protein interaction domain and a small glutamine–rich COOH terminus of unknown function (Supplementary Data 1B). Consistent with a potential role in AR function, the TPR repeat of {alpha}SGT exhibits the highest similarity with the analogous regions of the small group of TPR-containing proteins previously implicated in chaperoning and/or maturation of steroid hormone receptors (refs. 11, 21, 22; Fig. 1E). Importantly, homology modeling of the {alpha}SGT TPR suggests remarkable structural conservation with the solved crystal TPR domain structures of PP5 and Cyp40 (Supplementary Data 1C). The importance of this class of TPR proteins to AR signaling is highlighted by the phenotype of FKBP52 knockout mice, which exhibit defects in male reproductive tissues (23, 24). Expression of {alpha}SGT in a cohort of microdissected human PCa epithelial cells is second only to that of FKPB52 among this steroid receptor–targeting TPR proteins (Fig. 1D). Moreover, the level of {alpha}SGT is the most significantly altered relative to AR in the progression to metastatic disease (Fig. 1D). Along with the known interaction of {alpha}SGT with Hsp90 and Hsp70 (25), these findings implicate {alpha}SGT as a potentially important cochaperone of the AR.

The relative cellular levels of AR and {alpha}SGT determine AR transcriptional capacity. Overexpression of {alpha}SGT relative to AR in PC-3 PCa cells resulted in a 2-fold decrease in DHT-mediated AR transactivation activity (Fig. 2A ), and reduced basal receptor activity (i.e., activity in the absence of exogenous ligand) by 92.9 ± 0.4% (Fig. 2A, inset). As a consequence of reduced basal activity, the fold induction in receptor activity caused by 10 nmol/L of DHT was dramatically increased from 47.6 ± 3.6-fold for AR alone to 365.5 ± 38.5-fold in the presence of {alpha}SGT. The basal activity is AR-dependent as (a) there is negligible promoter activity in the absence of transfected AR, and (b) deletion of activation function 1 (AF1), which is essential for AR transcriptional capacity, decreased basal activity by 74.8 ± 5.4% (Supplementary Data 2A). In human C4-2B PCa cells that express both {alpha}SGT and AR, reducing {alpha}SGT levels with a specific siRNA resulted in a marked increase in basal and DHT-induced expression from (a) the endogenous androgen-responsive PSA gene as determined by quantitative real-time PCR (Fig. 2C), and (b) from a transfected reporter gene (data not shown). Similar results were obtained with LNCaP PCa cells, in both C4-2B and LNCaP cell lines treated with an independent siRNA targeting a different sequence in the {alpha}SGT mRNA, and for ectopic AR in transfected PC-3 cells (data not shown). Consistent with the cellular level of {alpha}SGT being a determinant of AR function, increasing the level of AR over the endogenous level of {alpha}SGT in PC-3 cells resulted in the same outcome as for the siRNA experiments, i.e., an increase in basal and ligand-induced AR activity and a decrease in fold induction by ligands (Fig. 2C). At a distinct threshold level of transfected AR (in this case, 5 ng), there was a marked (6-fold) increase in basal receptor activity. With increasing AR protein levels, there was a 10-fold decrease in the concentration of DHT required to give 50% maximal activity (EC50; Fig. 2C; Supplementary Data 2B and C).


Figure 2
View larger version (47K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. {alpha}SGT affects AR basal activity and sensitivity to ligand-dependent activation distinct from classic coregulators. A, effect of {alpha}SGT overexpression on AR activity. PC-3 cells were transfected with AR (2.5 ng), {alpha}SGT expression or empty vector control, and ARR3-tk-Luc (100 ng) and treated with vehicle control (ethanol) or DHT. Points, mean activity from six to eight independently transfected wells presented as relative light units (RLU); bars, SE. Numbers to the right show mean fold activation (activity divided by basal activity; ±SE) for the highest concentration of ligand; inset, basal activity in the presence of vehicle control (i.e., in the absence of exogenous ligand). Columns, basal activity presented as RLU; bars, SE. Immunoblots show {alpha}SGT antisera resolving native and transfected HA-tagged {alpha}SGT proteins in a parallel experiment. B, AR functional assays in C4-2B cells transfected with a specific {alpha}SGT siRNA or a nonspecific (N.S.) negative control. Points, mean expression of the androgen-responsive PSA gene as a ratio of glyceraldehyde-3-phosphate dehydrogenase determined by triplicate quantitative real-time PCR analysis 1 d after transfection; bars, SE. Immunoblot analysis of parallel samples shows knockdown of {alpha}SGT, but not of AR or actin, by the specific siRNA compared with nonspecific control. C, effect of increasing amounts of transfected AR on transcriptional activity in PC-3 cells done as in A. Immunoblot was done with AR antisera. The EC50 (ligand concentration required for 50% maximal activity) calculated from transactivation data for each amount of transfected AR (bottom). D, effect of {alpha}SGT overexpression on ER transcriptional activity in PC-3 cells, essentially as in A, transfected with an ER expression vector (1–5 ng), ERE-tk-luc (100 ng), and either {alpha}SGT expression or empty vector control, and treated with estradiol (E2).

 
The effects of {alpha}SGT on AR are distinct from that of classic receptor coregulators such as the p160 coactivator GRIP1, and transforming growth factor ß1–induced 1 (TGFB1I1/Hic5/ARA55), which increase basal and maximal induction but have a less dramatic effect on fold induction and do not alter the sensitivity to ligand (i.e., EC50; Supplementary Data 2D). In addition, {alpha}SGT overexpression did not affect basal ER{alpha} activity or the responsiveness of ER{alpha} to estradiol (Fig. 2D), suggesting that it may exhibit specificity for different steroid receptors as with the other TPR proteins, and arguing against a ubiquitous effect of {alpha}SGT on cellular signaling or the Hsp70/Hsp90 chaperone machinery.

{alpha}SGT acts in the cytoplasm to affect AR subcellular distribution. Recent evidence suggests that TPR chaperones such as FKBP52 may be involved in the shuttling of steroid receptors between the cytoplasm and nucleus (10, 21, 26). We therefore investigated the effects of {alpha}SGT overexpression on AR cellular localization and receptor redistribution by ligands (Fig. 3 ). In untreated cells, the AR was cytoplasmic with diffuse weak nuclear staining. However, coexpression of {alpha}SGT almost completely eliminated nuclear AR in the absence of ligands, and maintained a predominantly cytoplasmic distribution of the receptor even after treatment with 0.1 nmol/L of DHT. In contrast, treatment with a saturating concentration of DHT (1 nmol/L) resulted in nuclear localization of AR in either the absence or presence of exogenous {alpha}SGT. Transfected {alpha}SGT was exclusively cytoplasmic in all cases. Indeed, chromatin immunoprecipitation failed to detect {alpha}SGT at the endogenous AR-responsive PSA promoter in C4-2B cells (Supplementary Data 3), and had no effect on the activity of a constitutively nuclear and constitutively active AR variant truncated ligand-binding domain at residue 709 (data not shown). These results are consistent with {alpha}SGT affecting AR function in the cytoplasm rather than altering the capacity of the receptor per se to generate a competent transcriptional complex.


Figure 3
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. {alpha}SGT affects AR subcellular distribution. A, confocal imaging microscopy of AR (green) and {alpha}SGT (red) in transfected cells treated with the indicated concentration of DHT or with vehicle control for 24 h. Nuclei stained with Hoescht dye (blue). B, transfected cells from three independent experiments (as in A) were scored (blinded to category) as exhibiting predominantly nuclear (N > C) or cytoplasmic (C ≥ N) AR localization by manual counting. Columns, mean percentage of transfected cells in each category from three independent experiments; bars, SE.

 
Changes in {alpha}SGT and AR levels during the progression of human PCa. Immunohistochemistry showed almost exclusive cytoplasmic staining in a range of normal mouse tissues (Supplementary Data 4). In the prostate, {alpha}SGT was confined to the cytoplasm of luminal epithelial cells with no evidence of expression in stromal cells (Fig. 4A ). In contrast, AR is predominantly nuclear in mouse and human prostatic epithelial cells, and is also present in smooth muscle and fibroblast stromal cells (27). To investigate the role of {alpha}SGT in AR function in vivo, we undertook quantitative immunohistochemistry of these two proteins in a cohort of human prostate epithelium (Fig. 4A) using previously described methods (14). A correlation between AR and {alpha}SGT immunoreactivity was observed in primary tumors (R = 0.469, P = 0.009; Fig. 4B) but not in metastatic disease (R = 0.081, P = 0.562; Fig. 4C), reflecting changes in the expression of one or both proteins with disease progression. Compared with nonmalignant prostate samples and primary tumors, the mean and median levels of {alpha}SGT immunoreactivity were significantly lower in metastatic disease. In contrast, nuclear AR immunostaining was significantly higher (Fig. 4B–D). When immunostaining was considered pairwise, there was a significant increase in the ratio of AR/{alpha}SGT immunoreactivity in metastatic (2.1 ± 0.4) compared with primary tumor samples (0.79 ± 0.09; P = 0.003) and nonmalignant prostate cells (0.99 ± 0.12; P = 0.040; Fig. 4D). The AR/{alpha}SGT ratio was significantly greater (P = 0.023) in metastases from patients who received hormone ablation or hormone ablation plus chemotherapy (3.62 ± 0.78) than in metastases from untreated patients (1.68 ± 0.18), supporting the notion of further adaptation in a low hormone environment.


Figure 4
View larger version (50K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Differential expression of AR and {alpha}SGT with prostate cancer progression. A, immunohistochemistry with {alpha}SGT and AR U407 antisera. Left, an example of {alpha}SGT immunostaining in mouse prostate and competition by a 5x molar excess of specific blocking peptide. Right, two examples of immunohistochemistry with {alpha}SGT and AR U407 antisera in nonmalignant prostate and in primary and metastatic prostate cancers. B, comparison of AR and {alpha}SGT immunohistochemistry in 30 primary human prostate tumors. Immunoreactivity of each antisera in epithelial cells (stroma was excluded) was measured by quantitative video image analysis on 20 contiguous fields for each sample. Video image measurements of integrated optical density (IOD) and the total area (TA) analyzed were used to derive the mean integrated optical density (MIOD = IOD / TA), which equates to the mean immunoreactivity per unit area (MIOD) in each sample. Dashed red lines, the mean MIOD (values in brackets) determined for each antisera. The coefficient (R) and probability (P) of correlation between AR and {alpha}SGT immunoreactivity (Spearman's rho test) are shown. C, comparison of AR and {alpha}SGT levels in 54 metastatic human prostate tumors presented as in B. Solid black lines, mean values in metastatic samples. Dashed red lines are from B and are shown for comparison. D, quantification of {alpha}SGT and AR immunoreactivity in 32 primary prostate cancers, 64 metastatic lesions, and 30 nonmalignant prostate controls. The ratio of AR/{alpha}SGT was calculated for those samples in which sufficient informative immunoreactive area was available for both antisera, and represents the data in B and C.

 
{alpha}SGT level affects AR sensitivity to nonclassic ligands. The weak-agonist activities of nonclassic steroids such as progesterone and estradiol, and of receptor antagonists such as hydroxyflutamide, have been implicated in the maintenance of AR function following androgen ablation (2). Overexpression of {alpha}SGT resulted in a substantial decrease in the capacity of the adrenal androgen androstenedione and the synthetic progestin medroxyprogesterone acetate to activate the receptor compared with DHT (Fig. 5A and B ; compare with Fig. 2A), and eliminated the capacity of progesterone and hydroxyflutamide to induce weak AR activity at comparable concentrations of ligand (Fig. 5C and D). These results imply that {alpha}SGT acts to maintain fidelity/specificity of AR activation by classic agonist ligands. A mutation in the ligand-binding domain of the AR in LNCaP and C4-2B cells allows estradiol, progesterone, medroxyprogesterone acetate, and hydroxyflutamide to exhibit full agonist activity on the receptor comparable to DHT.10 As expected, reduction in {alpha}SGT levels by RNA interference in C4-2B and LNCaP cells resulted in an increase in PSA gene expression with these ligands comparable to that observed with DHT (Supplementary Data 2E).


Figure 5
View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. {alpha}SGT expression affects AR sensitivity to nonclassic ligands. Effect of {alpha}SGT overexpression on AR transcriptional activity by nonclassic ligands. PC-3 cells were transfected with AR (2.5 ng), {alpha}SGT expression or empty vector control, and ARR3-tk-Luc (100 ng) and treated with vehicle control (ethanol) or ligands: A, androstenedione (ASD); B, medroxyprogesterone acetate (MPA); C, hydroxyflutamide (OHF); D, progesterone (PROG). Points, mean activity determined from six to eight independently transfected wells presented as relative light units (RLU); bars, SE. Insets, basal activity (i.e., in the presence of vehicle control only).

 
The {alpha}SGT TPR interacts with the AR hinge whereas the COOH-terminal region of the chaperone mediates dimerization. The TPR domains of steroid receptor–associated cochaperones form a conserved amphipathic channel that mediates binding to the COOH-terminal MEEVD peptide of Hsp90 and/or IEEVD of Hsp70 (11, 22). Using solid phase partial proteolysis and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprint analysis, the AR-{alpha}SGT interaction was mapped to the amino terminal 141 amino acids of {alpha}SGT and residues in the AR hinge encompassing amino acids 630 to 645 (data not shown). In silico docking analysis predicted that binding was mediated primarily through the interaction of {alpha}SGT residues 105 to 127, which are contained within the first two TPRs and AR hinge amino acids 638KLQEEGEA645 (Fig. 6A ). Significantly, this AR peptide sequence strongly resembles the EEVD-containing peptides of Hsp90 and Hsp70 (11, 22). Deletion of the 638KLQEEGEA645 peptide from the full-length AR (i.e., AR{Delta}638–646) resulted in increased basal activity and reduced fold activation by DHT compared with wild-type AR (Supplementary Data 2F), but did not alter overall transcriptional activity.


Figure 6
View larger version (49K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. {alpha}SGT interactions. A, {alpha}SGT is predicted to interact with a short peptide sequence in the AR hinge that structurally resembles the COOH-terminal EEVD peptides from Hsp70 and Hsp90. The AR hinge sequence is shown delineating sites of phosphorylation (P) and acetylation (Ac), and the AR peptide (green box) predicted to mediate interaction with {alpha}SGT. The molecular surface model is of the {alpha}SGT TPR domain colored according to electrostatic potential: red, negative; blue, positive. Geometric centers (green spheres) of in silico docking solutions of the AR hinge cluster with amino acids 105 to 127 of {alpha}SGT, which encompasses the first of the three TPR repeats. The top 50 docking solutions were clustered to produce a theoretical docked single AR peptide, depicted in ribbon format (inset), showing the predominant role of 638KLQEEGEA645 residues (green). B, {alpha}SGT dimerization. Top, mammalian two-hybrid assay mapping the {alpha}SGT dimerization to the first 80 amino acids in transfected COS-1 cells. Columns, mean activity from eight independently transfected wells; bars, SE. Bottom, immunoblot analysis done with {alpha}SGT antisera on increasing amounts of COS-1 cell lysates resolved by PAGE under nonreducing (native) conditions showing the existence of {alpha}SGT dimers. C, schematic of {alpha}SGT delineating the interaction site (if known) of its client proteins, with a brief description of known effects derived from {alpha}SGT interaction. D, model detailing the proposed effects of {alpha}SGT on AR maturation and transport to the nucleus. a, the {alpha}SGT dimer will facilitate recognition and efficient folding of the nascent AR by Hsp70 by providing both a platform for trimeric interaction between the three proteins and by enhancing the ATPase activity of Hsp70 and client recognition. b, {alpha}SGT interaction could allow efficient exchange of Hsp70 for Hsp90 during AR maturation and act to stabilize the apo-receptor/Hsp90 heterocomplex through trimeric interactions, thereby ensuring greater conformational integrity of apo-receptor pool and limiting passive nuclear transport or inappropriate ligand-mediated signaling. c, by facilitating weak association with microtubules, {alpha}SGT may further act to limit passive nuclear transport of the apo-receptor and to facilitate efficient tethering of the AR to microtubules following ligand-induced exchange of {alpha}SGT for FKBP52.

 
The interaction of {alpha}SGT with its client proteins seems to be mediated by overlapping binding sites centered on the TPR repeat (see below). The clear exception is the first 80 amino acids, which according to native page and mammalian two-hybrid analyses, form an {alpha}SGT dimerization interface (Fig. 6B), as has been previously suggested (25). In this manner, a dimer of {alpha}SGT could simultaneously interact with two identical or nonidentical client proteins via its TPR domains.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Little is known about the cellular role and function of {alpha}SGT. This is surprising given that {alpha}SGT has been highly conserved in evolution from yeast to humans, has been implicated in cell division and apoptosis (28, 29), and has the potential to affect the movement, localization, and functional response of a broad range of important signaling molecules of mammalian and viral origin (refs. 5, 25, 30–36; Fig. 6C).

Although the four known steroid receptor–associated TPR proteins, FKBP51, FKBP52, PP5, and Cyp40 exhibit a similar affinity for Hsp90 in vitro (11), their presence and function in a mature Hsp90/steroid receptor heterocomplex varies according to their relative abundance, the particular receptor, cellular location, and whether the receptor resides in a ligand-bound state. For example, the relative cellular levels of FKBP51 and FKBP52 affect the affinity of glucocorticoid receptors for ligands both in vitro and in vivo (37), yet FKBP52 overexpression does not appreciably affect the binding affinity of the AR, instead causing a left-shift in the dose-response curve and an increase in receptor transactivation activity (23, 38). Conversely, we observed that overexpression of {alpha}SGT results in a right-shift in the dose-response and a decrease in receptor transactivation activity. Whereas the AR/FBKP52 interaction is enhanced by the addition of ligands (23), we have shown that DHT decreases the interaction of AR with {alpha}SGT, suggesting that these two cochaperones may act at different points in AR signaling. Importantly, {alpha}SGT did not affect the ligand responsiveness of ER{alpha} arguing against the ubiquitous effect of {alpha}SGT on the Hsp70/Hsp90 chaperone system.

Whereas {alpha}SGT is unique among the related TPR proteins in that it lacks a peptidyl-prolyl isomerase (PPIase) domain implicated in protein folding, its cochaperone functions could nonetheless affect the capacity of the AR to bind and respond to ligand. The refolding capacity and weak ATPase activity of Hsp70, which provides the free energy for chaperone function and the cyclic association and dissociation of client proteins, are enhanced by interaction with {alpha}SGT leading to a higher affinity of Hsp70 for substrate proteins and more efficient protein folding (39). Consequently, yeast that lack {alpha}SGT exhibit a 50-fold reduction in recovery from heat shock compared with the native yeast strain (35). By enhancing the ATPase activity of Hsp70, and/or Hsp90 (22), which favors their ADP-dependent association with client molecules, {alpha}SGT could affect the ligand-binding capacity of the AR via the maturation pathway, stabilize the receptors interaction with Hsp90, or limit the pool of misfolded receptors with inappropriate activities. By ensuring the folding quality and control of cytoplasmic AR, this may explain how {alpha}SGT overexpression limits receptor activity and nuclear transport in the absence of ligand, and prevents inappropriate responses to weak androgens and nonclassic ligands. Conversely, our data suggests that when {alpha}SGT is limiting, as is the case when AR levels are increased, misfolded receptors can exhibit aberrant responses. It has previously been shown that AR overexpression allows nonsteroidal AR antagonists such as bicalutamide to induce significant AR agonist activity (40).

There is also accumulating evidence that {alpha}SGT mediates correct trafficking and/or prevents inappropriate movement and activity of several important signaling molecules. Interaction of {alpha}SGT with the HIV-1 encoded Vpu protein facilitates redistribution of HIV Gag to points of accumulation at the plasma membrane, resulting in more efficient budding of viral particles from infected cells (31). {alpha}SGT may also aid in the transport of the growth hormone receptor from the endoplasmic reticulum to the plasma membrane (34), and the secretion and activity of myostatin (32). Preferential interaction of the glucocorticoid receptor/Hsp90 heterocomplex with FKBP52 in the cytoplasm following dexamethasone binding is one of the earliest events in receptor activation (12). By stabilizing the glucocorticoid receptor/Hsp90 interaction and tethering the receptor via its PPIase domain to the cytoplasmic dynein, the motor protein that mediates retrograde movement of proteins along microtubules, FKBP52 promotes nuclear transport and enhances receptor transactivation (10, 21, 26). Considering the androgen-insensitive phenotype of FKBP52 knockout mice, a similar mechanism likely exists for the AR, and importantly, the AR nuclear targeting sequence lies directly adjacent to or overlaps the {alpha}SGT binding site identified in the current study. It is possible that by acting to retain apo-AR in a "persistent" complex with Hsp90, the PPIase-less {alpha}SGT will uncouple the receptor from the dynein transport machinery mediated by the related TPR proteins, thereby preventing inappropriate cytoplasmic aggregation and movement of the receptor to the nucleus in the absence of a specific hormonal signal. That deletion of the {alpha}SGT binding site recapitulates only one effect of {alpha}SGT knockdown (i.e., increased basal but not an overall increase in receptor function), supports the notion of common or overlapping binding sites in the AR hinge for different TPR proteins, each contributing to distinct aspects of receptor signaling. We have shown that DHT causes the dissociation of {alpha}SGT from the AR, and conversely, that {alpha}SGT overexpression decreases the capacity of DHT to mediate receptor transport to the nucleus. Mutations in the AR nuclear targeting sequence have been shown to result in delayed ligand-dependent nuclear transport, cytoplasmic aggregation of the receptor in a complex with Hsp70 and other chaperones, and nuclear-clearing of the apo-AR similar to that seen in the current study with overexpression of {alpha}SGT (18).

Cytoplasmic {alpha}SGT is associated with microtubules and actin filaments, but does not form an integral part of the cytoskeleton (31). Microtubule-targeting by {alpha}SGT could contribute to cytoplasmic retention of apo-AR, and may facilitate efficient exchange of {alpha}SGT for dynein-linked TPR proteins following ligand binding and the subsequent receptor redistribution to the nucleus. This hypothesis is consistent with the emerging dependence of steroid receptors on an intact cytoskeleton for nuclear translocation (10, 12). Importantly, the redistribution of {alpha}SGT observed with the collapse of the microtubule network during cell division and by chemical means (31), suggests that microtubule-targeting agents will disrupt {alpha}SGT regulation of its clients. This may explain the efficacy of the chemotherapeutic agent, docetaxel, in patients with PCa following the failure of conventional androgen ablation (41).

Collectively, {alpha}SGT may act to ensure the quality of Hsp70/Hsp90-dependent receptor maturation, prevent inappropriate cytoplasmic aggregation, stabilize the apo-AR in a persistent chaperone heterocomplex in the cytoplasm in order to prevent inappropriate movement of the receptor to the nucleus in the absence of a specific hormonal signal, and/or mediate the efficient exchange of TPR proteins following agonist binding (Fig. 6D). Evolutionarily, the maintenance of a cytoplasmic AR might stem from the receptor's capacity to weakly activate androgen-regulated genes in the absence of ligand, or in response to extraneous steroids or low concentrations of cognate ligands, which could be undesirable depending on the cellular and/or developmental context.

In summary, the current study indicates that the equilibrium between cytoplasmic and nuclear localization of the AR in PCa cells, and the sensitivity of the receptor to activation by ligand, will depend in part on the relative cellular levels of both AR and {alpha}SGT. {alpha}SGT can therefore be considered a molecular rheostat of androgen signaling in prostate epithelial cells, and thus, a contributor to the homeostatic control of the action of androgen. The increased AR level often observed in metastatic PCa, and recently shown to be predictive of progression in localized disease (2, 27, 40), might overwhelm the capacity of limiting cellular {alpha}SGT to (a) ensure AR conformational quality and appropriate cellular localization, (b) buffer basal and ligand-independent receptor transactivation, and/or (c) limit AR's response to androgenic and nonsteroidogenic ligands. Equally, decreased levels of {alpha}SGT could mimic these effects without any identifiable change in AR expression in an individual tumor. In either case, our findings identify a novel mechanism to explain how the AR can continue to signal in PCa cells in a low hormonal environment.


   Acknowledgments
 
Grant support: National Health and Medical Research Council of Australia (ID no. 299048, W.D. Tilley), The Prostate Cancer Foundation (G.A. Coetzee), The U.S. Department of Defense (W81XWH-04-1-0017, G. Buchanan; W81XWH-04-1-0049 and W81XWH-04-1-0823, G.A. Coetzee), and the NIH/National Cancer Institute (R01CA84890 and R01CA109147, G.A. Coetzee). G. Buchanan is a recipient of a National Health and Medical Research Council C.J. Martin Biomedical Fellowship.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Paul F. Lambert, D. Marrocco, M. Lee, E.F. Need, A. Ochnik, M.A. Pickering, M. Yang, and K. Murti provided technical assistance. The authors thank Professor John Funder for his critique, Dr. Mike Stallcup for GRIP1 and Hic5 vectors, and Dr. Howard Shen for valuable discussions and pCMV:AR{Delta}638-646.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

5 http://swissmodel.expasy.org//SWISS-MODEL.html Back

6 http://www.cmbi.kun.nl/whatif/ Back

7 http://amber.scripps.edu/ Back

8 http://www.cqfb.fct.unl.pt/bioin/chemera/Chemera/Bgg_Algorithm.html Back

9 http://www.povray.org/ Back

10 G. Buchanan and W.D. Tilley, unpublished observations. Back

Received 5/ 4/07. Revised 7/ 3/07. Accepted 8/ 6/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Feldman BJ, Feldman D. The development of androgen-independent prostate cancer. Nat Rev 2001;1:34–45.
  2. Scher HI, Buchanan G, Gerald W, Butler LM, Tilley WD. Targeting the androgen receptor: improving outcomes for castration-resistant prostate cancer. Endocr Relat Cancer 2004;11:459–76.[Abstract/Free Full Text]
  3. Sun K, Montana V, Chellappa K, et al. Phosphorylation of a conserved serine in the deoxyribonucleic acid binding domain of nuclear receptors alters intracellular localization. Mol Endocrinol 2007;21:1297–311.[Abstract/Free Full Text]
  4. Cheung J, Smith DF. Molecular chaperone interactions with steroid receptors: an update. Mol Endocrinol 2000;14:939–46.[Free Full Text]
  5. Fonte V, Kapulkin V, Taft A, Fluet A, Friedman D, Link CD. Interaction of intracellular ß amyloid peptide with chaperone proteins. Proc Natl Acad Sci U S A 2002;99:9439–44.[Abstract/Free Full Text]
  6. Whitesell L, Lindquist SL. HSP90 and the chaperoning of cancer. Nat Rev Cancer 2005;5:761–72.[CrossRef][Medline]
  7. Auluck PK, Chan HY, Trojanowski JQ, Lee VM, Bonini NM. Chaperone suppression of {alpha}-synuclein toxicity in a Drosophila model for Parkinson's disease. Science 2002;295:865–8.[Abstract/Free Full Text]
  8. Waza M, Adachi H, Katsuno M, et al. 17-AAG, an Hsp90 inhibitor, ameliorates polyglutamine-mediated motor neuron degeneration. Nat Med 2005;11:1088–95.[CrossRef][Medline]
  9. Kamal A, Thao L, Sensintaffar J, et al. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature 2003;425:407–10.[CrossRef][Medline]
  10. Pratt WB, Galigniana MD, Harrell JM, DeFranco DB. Role of hsp90 and the hsp90-binding immunophilins in signalling protein movement. Cell Signal 2004;16:857–72.[CrossRef][Medline]
  11. Pratt WB, Toft DO. Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr Rev 1997;18:306–60.[Abstract/Free Full Text]
  12. Davies TH, Ning YM, Sanchez ER. A new first step in activation of steroid receptors: hormone-induced switching of FKBP51 and FKBP52 immunophilins. J Biol Chem 2002;277:4597–600.[Abstract/Free Full Text]
  13. Holzbeierlein J, Lal P, LaTulippe E, et al. Gene expression analysis of human prostate carcinoma during hormonal therapy identifies androgen-responsive genes and mechanisms of therapy resistance. Am J Pathol 2004;164:217–27.[Abstract/Free Full Text]
  14. Buchanan G, Birrell SN, Peters AA, et al. Decreased androgen receptor levels and receptor function in breast cancer contribute to the failure of response to medroxyprogesterone acetate. Cancer Res 2005;65:8487–96.[Abstract/Free Full Text]
  15. Buchanan G, Yang M, Cheong A, et al. Structural and functional consequences of glutamine tract variation in the androgen receptor. Hum Mol Genet 2004;13:1677–92.[Abstract/Free Full Text]
  16. Jia L, Choong CS, Ricciardelli C, Kim J, Tilley WD, Coetzee GA. Androgen receptor signaling: mechanism of interleukin-6 inhibition. Cancer Res 2004;64:2619–26.[Abstract/Free Full Text]
  17. Gioeli D, Black BE, Gordon V, et al. Stress kinase signaling regulates androgen receptor phosphorylation, transcription, and localization. Mol Endocrinol 2006;20:503–15.[Abstract/Free Full Text]
  18. Thomas M, Dadgar N, Aphale A, et al. Androgen receptor acetylation site mutations cause trafficking defects, misfolding, and aggregation similar to expanded glutamine tracts. J Biol Chem 2004;279:8389–95.[Abstract/Free Full Text]
  19. Chen S, Xu Y, Yuan X, Bubley GJ, Balk SP. Androgen receptor phosphorylation and stabilization in prostate cancer by cyclin-dependent kinase 1. Proc Natl Acad Sci U S A 2006;103:15969–74.[Abstract/Free Full Text]
  20. Black BE, Paschal BM. Intranuclear organization and function of the androgen receptor. Trends Endocrinol Metab 2004;15:411–7.[CrossRef][Medline]
  21. Davies TH, Sanchez ER. FKBP52. Int J Biochem Cell Biol 2005;37:42–7.[CrossRef][Medline]
  22. Scheufler C, Brinker A, Bourenkov G, et al. Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70–90 multichaperone machine. Cell 2000;101:199–210.[CrossRef][Medline]
  23. Cheung-Flynn J, Prapapanich V, Cox MB, Riggs DL, Suarez-Quian C, Smith DF. Physiological role for the cochaperone FKBP52 in androgen receptor signaling. Mol Endocrinol 2005;19:1654–66.[Abstract/Free Full Text]
  24. Yong W, Yang Z, Periyasamy S, et al. Essential role for co-chaperone Fkbp52 but not Fkbp51 in androgen receptor-mediated signaling and physiology. J Biol Chem 2007;282:5026–36.[Abstract/Free Full Text]
  25. Liou ST, Wang C. Small glutamine-rich tetratricopeptide repeat-containing protein is composed of three structural units with distinct functions. Arch Biochem Biophys 2005;435:253–63.[CrossRef][Medline]
  26. Silverstein AM, Galigniana MD, Kanelakis KC, Radanyi C, Renoir JM, Pratt WB. Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor, hsp90, and cytoplasmic dynein. J Biol Chem 1999;274:36980–6.[Abstract/Free Full Text]
  27. Ricciardelli C, Choong CS, Buchanan G, et al. Androgen receptor levels in prostate cancer epithelial and peritumoral stromal cells identify non-organ confined disease. Prostate 2005;63:19–28.[CrossRef][Medline]
  28. Wang H, Shen H, Wang Y, et al. Overexpression of small glutamine-rich TPR-containing protein promotes apoptosis in 7721 cells. FEBS Lett 2005;579:1279–84.[CrossRef][Medline]
  29. Winnefeld M, Rommelaere J, Cziepluch C. The human small glutamine-rich TPR-containing protein is required for progress through cell division. Exp Cell Res 2004;293:43–57.[CrossRef][Medline]
  30. Cziepluch C, Kordes E, Poirey R, Grewenig A, Rommelaere J, Jauniaux JC. Identification of a novel cellular TPR-containing protein, SGT, that interacts with the nonstructural protein NS1 of parvovirus H-1. J Virol 1998;72:4149–56.[Abstract/Free Full Text]
  31. Handley MA, Paddock S, Dall A, Panganiban AT. Association of Vpu-binding protein with microtubules and Vpu-dependent redistribution of HIV-1 Gag protein. Virology 2001;291:198–207.[CrossRef][Medline]
  32. Wang H, Zhang Q, Zhu D. hSGT interacts with the N-terminal region of myostatin. Biochem Biophys Res Commun 2003;311:877–83.[CrossRef][Medline]
  33. Natochin M, Campbell TN, Barren B, et al. Characterization of the G alpha(s) regulator cysteine string protein. J Biol Chem 2005;280:30236–41.[Abstract/Free Full Text]
  34. Schantl JA, Roza M, De Jong AP, Strous GJ. Small glutamine-rich tetratricopeptide repeat-containing protein (SGT) interacts with the ubiquitin-dependent endocytosis (UbE) motif of the growth hormone receptor. Biochem J 2003;373:855–63.[CrossRef][Medline]
  35. Angeletti PC, Walker D, Panganiban AT. Small glutamine-rich protein/viral protein U-binding protein is a novel cochaperone that affects heat shock protein 70 activity. Cell Stress Chaperones 2002;7:258–68.[CrossRef][Medline]
  36. Fielding BC, Gunalan V, Tan TH, et al. Severe acute respiratory syndrome coronavirus protein 7a interacts with hSGT. Biochem Biophys Res Commun 2006;343:1201–8.[CrossRef][Medline]
  37. Denny WB, Valentine DL, Reynolds PD, Smith DF, Scammell JG. Squirrel monkey immunophilin FKBP51 is a potent inhibitor of glucocorticoid receptor binding. Endocrinology 2000;141:4107–13.[Abstract/Free Full Text]
  38. Febbo PG, Lowenberg M, Thorner AR, Brown M, Loda M, Golub TR. Androgen mediated regulation and functional implications of fkbp51 expression in prostate cancer. J Urol 2005;173:1772–7.[CrossRef][Medline]
  39. Tobaben S, Thakur P, Fernandez-Chacon R, Sudhof TC, Rettig J, Stahl B. A trimeric protein complex functions as a synaptic chaperone machine. Neuron 2001;31:987–99.[CrossRef][Medline]
  40. Chen CD, Welsbie DS, Tran C, et al. Molecular determinants of resistance to antiandrogen therapy. Nat Med 2004;10:33–9.[CrossRef][Medline]
  41. Pienta KJ, Smith DC. Advances in prostate cancer chemotherapy: a new era begins. CA Cancer J Clin 2005;55:300–18.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
E. F. Need, H. I. Scher, A. A. Peters, N. L. Moore, A. Cheong, C. J. Ryan, G. A. Wittert, V. R. Marshall, W. D. Tilley, and G. Buchanan
A Novel Androgen Receptor Amino Terminal Region Reveals Two Classes of Amino/Carboxyl Interaction-Deficient Variants with Divergent Capacity to Activate Responsive Sites in Chromatin
Endocrinology, June 1, 2009; 150(6): 2674 - 2682.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
M.O. Goodarzi, N. Xu, J. Cui, X. Guo, Y.I. Chen, and R. Azziz
Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), a candidate gene for polycystic ovary syndrome
Hum. Reprod., May 1, 2008; 23(5): 1214 - 1219.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Buchanan, G.
Right arrow Articles by Tilley, W. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Buchanan, G.
Right arrow Articles by Tilley, W. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online