FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2000741688> ?p ?o ?g. }

• W2000741688 endingPage "8068" @default.
• W2000741688 startingPage "8060" @default.
• W2000741688 abstract "Androgens drive sex differentiation, bone and muscle development, and promote growth of hormone-dependent cancers by binding the nuclear androgen receptor (AR), which recruits coactivators to responsive genes. Most nuclear receptors recruit steroid receptor coactivators (SRCs) to their ligand binding domain (LBD) using a leucine-rich motif (LXXLL). AR is believed to recruit unique coactivators to its LBD using an aromatic-rich motif (FXXLF) while recruiting SRCs to its N-terminal domain (NTD) through an alternate mechanism. Here, we report that the AR-LBD interacts with both FXXLF motifs and a subset of LXXLL motifs and that contacts with these LXXLL motifs are both necessary and sufficient for SRC-mediated AR regulation of transcription. Crystal structures of the activated AR in complex with both recruitment motifs reveal that side chains unique to the AR-LBD rearrange to bind either the bulky FXXLF motifs or the more compact LXXLL motifs and that AR utilizes subsidiary contacts with LXXLL flanking sequences to discriminate between LXXLL motifs. Androgens drive sex differentiation, bone and muscle development, and promote growth of hormone-dependent cancers by binding the nuclear androgen receptor (AR), which recruits coactivators to responsive genes. Most nuclear receptors recruit steroid receptor coactivators (SRCs) to their ligand binding domain (LBD) using a leucine-rich motif (LXXLL). AR is believed to recruit unique coactivators to its LBD using an aromatic-rich motif (FXXLF) while recruiting SRCs to its N-terminal domain (NTD) through an alternate mechanism. Here, we report that the AR-LBD interacts with both FXXLF motifs and a subset of LXXLL motifs and that contacts with these LXXLL motifs are both necessary and sufficient for SRC-mediated AR regulation of transcription. Crystal structures of the activated AR in complex with both recruitment motifs reveal that side chains unique to the AR-LBD rearrange to bind either the bulky FXXLF motifs or the more compact LXXLL motifs and that AR utilizes subsidiary contacts with LXXLL flanking sequences to discriminate between LXXLL motifs. The cellular effects of the hormone 5-α-dihydrotestosterone (DHT) 1The abbreviations used are: DHT, 5-α-dihydrotestosterone; AR, androgen receptor; NR, nuclear receptor; Fmoc, N-(9-fluorenyl)methoxycarbonyl; SRC, steroid receptor coactivator; LBD, ligand binding domain; NTD, N-terminal domain; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; GST, glutathione S-transferase; DBD, DNA binding domain; CMV, cytomegalovirus; TR, thyroid receptor; AF, activation function.1The abbreviations used are: DHT, 5-α-dihydrotestosterone; AR, androgen receptor; NR, nuclear receptor; Fmoc, N-(9-fluorenyl)methoxycarbonyl; SRC, steroid receptor coactivator; LBD, ligand binding domain; NTD, N-terminal domain; MMTV, mouse mammary tumor virus; LTR, long terminal repeat; GST, glutathione S-transferase; DBD, DNA binding domain; CMV, cytomegalovirus; TR, thyroid receptor; AF, activation function. are mediated by the androgen receptor (AR), a member of the nuclear hormone receptor superfamily (1Lee H.J. Chang C. Cell Mol. Life Sci. 2003; 60: 1613-1622Crossref PubMed Scopus (139) Google Scholar). AR is absolutely required for normal male development, plays a variety of important roles in metabolism and homeostasis in adult men and women (2Liu P.Y. Death A.K. Handelsman D.J. Endocr. Rev. 2003; 24: 313-340Crossref PubMed Scopus (613) Google Scholar, 3Legros J.J. Charlier C. Bouillon G. Plomteux G. Ann. Endocrinol. (Paris). 2003; 64: 136PubMed Google Scholar), and is required for prostate cancer growth. Consequently, AR is a major target for pharmaceutical development and the recognized target for existing prostate cancer therapies, including androgen withdrawal and antiandrogens (1Lee H.J. Chang C. Cell Mol. Life Sci. 2003; 60: 1613-1622Crossref PubMed Scopus (139) Google Scholar, 4Gregory C.W. He B. Johnson R.T. Ford O.H. Mohler J.L. French F.S. Wilson E.M. Cancer Res. 2001; 61: 4315-4319PubMed Google Scholar, 5Culig Z. Klocker H. Bartsch G. Hobisch A. Endocr. Relat. Cancer. 2002; 9: 155-170Crossref PubMed Scopus (195) Google Scholar, 6Santos A.F. Huang H. Tindall D.J. Steroids. 2004; 69: 79-85Crossref PubMed Scopus (62) Google Scholar). It is nonetheless desirable to obtain new antiandrogens that spare patients from harmful side-effects and inhibit AR action in secondary hormone-resistant prostate cancer, where AR action becomes sensitized to low levels of androgens or existing antiandrogens (6Santos A.F. Huang H. Tindall D.J. Steroids. 2004; 69: 79-85Crossref PubMed Scopus (62) Google Scholar, 7Balk S.P. Urology. 2002; 60 (discussion 138-139): 132-138Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Improved understanding of AR signaling pathways will facilitate development of these compounds.Like most nuclear receptors (NRs), AR activity depends on interactions with members of the steroid receptor coactivator (SRC) family (1Lee H.J. Chang C. Cell Mol. Life Sci. 2003; 60: 1613-1622Crossref PubMed Scopus (139) Google Scholar, 8Ding X.F. Anderson C.M. Ma H. Hong H. Uht R.M. Kushner P.J. Stallcup M.R. Mol. Endocrinol. 1998; 12: 302-313Crossref PubMed Google Scholar, 9Berrevoets C.A. Doesburg P. Steketee K. Trapman J. Brinkmann A.O. Mol. Endocrinol. 1998; 12: 1172-1183Crossref PubMed Google Scholar). Several lines of evidence indicate that AR contacts with SRCs are important in prostate cancer. First, androgens promote SRC recruitment to the androgen-regulated prostate-specific antigen promoter, and this event is inhibited by the antiandrogen flutamide (10Shang Y. Myers M. Brown M. Mol. Cell. 2002; 9: 601-610Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar). Second, exogenous SRC2 (GRIP1/TIF2) promotes the androgen-dependent progression from the G1 to S phase in LNCaP prostate tumor cells, in a manner that requires specific AR contact (10Shang Y. Myers M. Brown M. Mol. Cell. 2002; 9: 601-610Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar). Third, SRCs often become expressed at high levels in prostate cancers (5Culig Z. Klocker H. Bartsch G. Hobisch A. Endocr. Relat. Cancer. 2002; 9: 155-170Crossref PubMed Scopus (195) Google Scholar). Finally, AR contacts with SRCs mediate hormone-independent AR signaling in conditions that resemble secondary prostate cancer (11Gregory C.W. Fei X. Ponguta L.A. He B. Bill H.M. French F.S. Wilson E.M. J. Biol. Chem. 2004; 279: 7119-7130Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 12Blaszczyk N. Masri B.A. Mawji N.R. Ueda T. McAlinden G. Duncan C.P. Bruchovsky N. Schweikert H.U. Schnabel D. Jones E.C. Sadar M.D. Clin. Cancer Res. 2004; 10: 1860-1869Crossref PubMed Scopus (52) Google Scholar). Thus, strategies to inhibit AR contacts with SRCs could be useful in blocking prostate cancer cell growth.For many NRs, overall transcriptional activity stems mostly from the hormone-dependent activation function (AF-2) within the NRs ligand binding domain (LBD), and involves interaction between a conserved hydrophobic cleft on the surface of the LBD and short leucine-rich hydrophobic motifs (NR boxes, consensus LXXLL motif) reiterated within each SRC (13Needham M. Raines S. McPheat J. Stacey C. Ellston J. Hoare S. Parker M. J. Steroid Biochem. Mol. Biol. 2000; 72: 35-46Crossref PubMed Scopus (72) Google Scholar, 14Leo C. Chen J.D. Gene (Amst.). 2000; 245: 1-11Crossref PubMed Scopus (436) Google Scholar). In contrast, current models of AR action suggest that AR activity stems from a potent hormone-independent activation function, AF-1, within the N-terminal domain (NTD) of the AR and emphasize the role of contacts between NTD and glutamine-rich sequences within the SRC C terminus in SRC recruitment (15Alen P. Claessens F. Schoenmakers E. Swinnen J.V. Verhoeven G. Rombauts W. Peeters B. Mol. Endocrinol. 1999; 13: 117-128Crossref PubMed Scopus (115) Google Scholar, 16He B. Lee L.W. Minges J.T. Wilson E.M. J. Biol. Chem. 2002; 277: 25631-25639Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Ma H. Hong H. Huang S.M. Irvine R.A. Webb P. Kushner P.J. Coetzee G.A. Stallcup M.R. Mol. Cell. Biol. 1999; 19: 6164-6173Crossref PubMed Scopus (214) Google Scholar, 18Christiaens V. Bevan C.L. Callewaert L. Haelens A. Verrijdt G. Rombauts W. Claessens F. J. Biol. Chem. 2002; 277: 49230-49237Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 19Powell S.M. Christiaens V. Voulgaraki D. Waxman J. Claessens F. Bevan C.L. Endocr. Relat. Cancer. 2004; 11: 117-130Crossref PubMed Scopus (61) Google Scholar). The AR-LBD is proposed to bind LXXLL motifs weakly and, instead, bind preferentially to aromatic-rich motifs that are found within the AR NTD (FQNLF and WHTLF) and AR-specific coactivators such as ARA70 (16He B. Lee L.W. Minges J.T. Wilson E.M. J. Biol. Chem. 2002; 277: 25631-25639Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 20He B. Minges J.T. Lee L.W. Wilson E.M. J. Biol. Chem. 2002; 277: 10226-10235Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 21He B. Wilson E.M. Mol. Genet. Metab. 2002; 75: 293-298Crossref PubMed Scopus (78) Google Scholar, 22Zhou Z.X. He B. Hall S.H. Wilson E.M. French F.S. Mol. Endocrinol. 2002; 16: 287-300Crossref PubMed Scopus (43) Google Scholar, 23He B. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 2000; 275: 22986-22994Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). The intramolecular interactions between the LBD and the NTD FQNLF motif promote formation of head to tail dimers (N-C interaction), which render the AF-2 surface unavailable for direct cofactor contacts (21He B. Wilson E.M. Mol. Genet. Metab. 2002; 75: 293-298Crossref PubMed Scopus (78) Google Scholar). Together, the notion that AR AF-2 binds coactivators weakly, and the fact that it will be occluded by the N-C interaction, has led to the suggestion that AR AF-2 does not play an active role in SRC recruitment.Nonetheless, several lines of evidence suggest that AR AF-2 can contribute directly to coactivator recruitment in some contexts. First, the N-C interaction is required for optimal AR activity at some promoters, including those of probasin, prostate-specific antigen, and C3, but not at others, including those of the sex-limiting protein and the mouse mammary tumor virus-long terminal repeats (MMTV-LTRs) (16He B. Lee L.W. Minges J.T. Wilson E.M. J. Biol. Chem. 2002; 277: 25631-25639Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Thus, AF-2 may be available for coactivator contacts in some circumstances. Second, mutation of AR AF-2 recognition sequences within target coactivators inhibits AR coactivation (16He B. Lee L.W. Minges J.T. Wilson E.M. J. Biol. Chem. 2002; 277: 25631-25639Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 19Powell S.M. Christiaens V. Voulgaraki D. Waxman J. Claessens F. Bevan C.L. Endocr. Relat. Cancer. 2004; 11: 117-130Crossref PubMed Scopus (61) Google Scholar, 20He B. Minges J.T. Lee L.W. Wilson E.M. J. Biol. Chem. 2002; 277: 10226-10235Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Thus, mutation of FXXLF motifs within AR-specific coactivators such as ARA70 blocks their ability to interact with AR and potentiate AF-2 activity. More surprisingly, given the prevailing notion that AR AF-2 contacts with LXXLL motifs are weak, mutation of all three SRC LXXLL motifs inhibits AR coactivation when SRCs are overexpressed, when AR NTD FQNLF and WHTLF motifs are mutated, or when AR acts at promoters such as the MMTV-LTR.It is important to understand the overall significance of particular AR to coregulator contacts, and the mechanism of these interactions, to develop strategies to inhibit AR activity in prostate cancer. In this study, we examine AR AF-2 interactions with target coactivators. Our studies confirm that AR AF-2 binds FXXLF motifs, but also show that AR AF-2 binds a subset of SRC LXXLL motifs with higher affinity and, further, that the same LXXLL motifs are required to mediate AR AF-2 activity. Crystal structures of AR-LBD in complex with native FXXLF and LXXLL peptides reveal the structural basis for these unusual coactivator binding preferences and may suggest new approaches to drug design.EXPERIMENTAL PROCEDURESProtein Expression and Purification—AR-LBD (residues 663–919) was expressed in Escherichia coli and purified to homogeneity using a modified version of previously published protocols (24Matias P.M. Donner P. Coelho R. Thomaz M. Peixoto C. Macedo S. Otto N. Joschko S. Scholz P. Wegg A. Basler S. Schafer M. Egner U. Carrondo M.A. J. Biol. Chem. 2000; 275: 26164-26171Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). Bacterial cell preparations were grown at ambient or lower temperatures to high optical density at 600 nm (>1.00) in 2× LB supplemented with DHT. AR-LBD protein was expressed by induction with isopropyl 1-thio-β-d-galactopyranoside for 14–16 h at 15 °C before harvest and cell lysis by freeze-thawing and mild sonication. Purification involved an initial affinity chromatography step using a glutathione-Sepharose column, followed by thrombin cleavage of the GST affinity tag. Finally cation exchange chromatography with Sepharose SP afforded the purified protein. Our procedures differ from published work in that we use Sepharose SP for the second purification step instead of Fractogel SO3, which does not retain AR in our experiments.Peptide Library Synthesis—Coregulator peptides consisting of 20 amino acids with the general motif of C XXXXXXXL XX(L/A)(L/A)XXXXXXX were constructed, where C is cysteine, L is leucine, A is alanine, and X is any amino acid. The sequences of all the coregulator peptides were obtained from human isoform candidate genes (SRC1/AAC50305, SRC2/Q15596, SRC3/Q9Y6Q9, and ARA70/Q13772). The peptides were synthesized in parallel using standard Fmoc chemistry in 48-well synthesis blocks (FlexChem System, Robbins). Preloaded Wang (Novagen) resin was deprotected with 20% piperidine in dimethylformamide. The next amino acid was then coupled using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (2.38 eq. wt.), Fmoc-protected amino acid (2.5 eq. wt.), and diisopropylethylamine (5 eq. wt.) in anhydrous dimethylformamide. Coupling efficiency was monitored by the Kaiser test. Synthesis then proceeded through a cycle of deprotection and coupling steps until the peptides were completely synthesized. The completed peptides were cleaved from the resin with concomitant side-chain deprotection (81% trifluoroacetic acid, 5% phenol, 5% thioanisole, 2.5% ethanedithiol, 3% water, 2% dimethylsulfide, 1.5% ammonium iodide), and crude product was dried down using a SpeedVac (GeneVac). Reversed-phase chromatography followed by mass spectrometry (matrix-assisted laser desorption ionization time-of-flight/electrospray ionization) was used to purify the peptides. The purified peptides were then lyophilized. A thiol-reactive fluorophore, 5-iodoacetamidofluorescein (Molecular Probes), was then coupled to the N-terminal cysteine following the manufacturer's protocol. Labeled peptide was isolated using reversed-phase chromatography and mass spectrometry. Peptides were quantified using UV spectroscopy. Purity was assessed using liquid chromatography/mass spectrometry.Peptide Binding Assay—Using a BiomekFX in the Center for Advanced Technology, AR-LBD was serially diluted from 100 μm to 0.002 μm in binding buffer (50 mm sodium phosphate, 150 mm NaCl, pH 7.2, 1 mm dithiothreitol, 1 mm EDTA, 0.01% Nonidet P-40, 10% glycerol) containing 150 μm ligand (dihydroxytestosterone) in 96-well plates. Then 10 μl of diluted protein was added to 10 μl of fluorescent coregulator peptide (20 nm) in 384-well plates yielding final protein concentrations of 50–0.001 μm and 10 nm fluorescent peptide concentration. The samples were allowed to equilibrate for 30 min. Binding was then measured using fluorescence polarization (excitation λ, 485 nm; emission λ, 530 nm) on an Analyst AD plate reader (Molecular Devices). Two independent experiments were assayed for each state in quadruplicate. Data were analyzed using SigmaPlot 8.0 (SPSS, Chicago, IL), and the Kd values were obtained by fitting data to the equation, y = min + (max - min)/1 + (x/Kd)⁁Hillslope).GST Pull-down Assays—Full-length SRC-2 (amino acids 1–1462) and AR NTD-DBD (amino acids 1–660) was expressed in a coupled transcription/translation system (TnT, Promega). AR-LBD (amino acids 646–919), or AR-LBD mutants, were expressed in E. coli strain BL21 as a GST fusion protein and attached to glutathione beads according to the manufacturer's protocol (Amersham Biosciences). Binding assays were performed by mixing glutathione-linked Sepharose beads containing 4 μg of GST fusion protein (estimated by Coomassie Plus protein assay reagent, Pierce) with 2 μl of 35S-labeled SRC-2 or AR NTD-DBD in 20 mm HEPES, 150 mm KCl, 25 mm MgCl2, 10% glycerol, 1 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, 20 μg/ml bovine serum albumin, and protease inhibitors containing to a final volume of 150 μl. The bead mix was shaken at 4 °C for 1.5 h, washed three times in 200 μl of binding buffer. The bound proteins were resuspended in SDS-PAGE loading buffer, separated by using 10% SDS-polyacrylamide gel electrophoresis, and visualized by autoradiography.Cell Culture and Transfection Assays—HeLa, DU145, and CV-1 cells were maintained in Dulbecco's modified Eagle's medium H-21 4.5 g/liter glucose, containing 10% steroid depleted fetal bovine serum (Invitrogen), 2 mm glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin. For transfection, cells were collected and resuspended in Dulbecco's phosphate-buffered saline (0.5 ml/4.5 × 107 cells) containing 0.1% dextrose, and typically 4 μg of luciferase reporter plasmid, 1 μg of AR expression vector or empty vector control, and 2 μg of pCMV-β-galactosidase. Cells were electroporated at 240 V and 960 microfarads, transferred to fresh media, and plated into 12-well plates. After incubation for 24 h at 37 °C with androgen or vehicle, cells were collected, and pellets were lysed by addition of 150 μl of 100 mm Tris-HCl, pH 7.8, containing 0.1% Triton X-100.For transfections with full-length AR, the reporter gene utilized the Mouse Mammary Tumor Virus promoter fused to luciferase. For transfections with GAL-AR-LBD, GAL-TR LBD, and GAL-CBP fusions, the reporter contained five GAL4 response elements upstream of a minimal promoter. LUC and β-galactosidase activities were measured using the Luciferase Assay System (Promega) and Galacto-Light Plus β-galactosidase reporter gene assay system (Applied Biosystems), according to the manufacturer's instructions.Crystallization, Structure Determination, and Refinement—The complexes of SRC2–2, SRC2–3, SRC3–2, and ARA70 peptides and AR-LBD were prepared by mixing at 0 °C for 2 h, with variable ratios of peptide (3–10 mm) and protein (at about 4.5 mg/ml). Crystals were obtained by vapor diffusion methods (sitting-drop technique) using crystal screens from Hampton. The protein-peptide complex solution was mixed with the reservoir solution (0.8 m sodium citrate, 0.1 m Tris, pH 7.5 or pH 8.0), and concentrated against 300 μl of the reservoir. Crystals appeared after 1 day and grew to maximal dimensions after 4 days. After 4 days these crystals started to crack, so new crystallization trials were necessary to find additives that would stabilize the crystals. 0.3 μl of either 2.0 m NaCl, 1.0 m LiCl2, or 0.1 m EDTA were added to a 1-μl protein plus a -1-μl reservoir drop to stabilize AR-LBD crystals at room temperature.Crystals for either AR-DHT or AR-DHT-peptide were transferred to a new drop containing 10% (v/v) of glycerol for cryoprotection. The crystals were then flash-cooled using liquid nitrogen and measured using the synchrotron radiation at the 8.3.1 beam line at the Advanced Light Source (Berkeley). Crystals containing SRC2-3, SRC2-2, and SRC3-2 diffracted to 2.07, 1.66, and 2.7 Å, respectively. Cocrystals of ARA70 peptide with AR-LBD were also grown, and a complete data set was obtained at 2.3 Å resolution. All the crystals belong to space group P212121 (orthorhombic) and contain one molecule per asymmetric unit.The diffraction data were integrated and scaled using the computer program ELVES (ucxray.berkeley.edu/~jamesh/elves/) (25Holton J. Alber T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1537-1542Crossref PubMed Scopus (199) Google Scholar). Molecular replacement solutions for all AR-LBD peptide structures were obtained using rotation and translation functions from Crystallography & NMR Systems (CNS, cns.csb.yale.edu/v1.1/) (26Brunger A.T. Adams P.D. Rice L.M. Curr. Opin. Struct. Biol. 1998; 8: 606-611Crossref PubMed Scopus (81) Google Scholar).The first electron maps calculated after the rigid body refinement that followed the molecular replacement displayed clear electron density for the peptides. During the improvement of the protein model, the Fourier maps revealed better electron density for more flanking residues of the peptides. The electron density for the peptide was always modeled as a short α-helix. However, refinement of the SRC2-2 peptide as an α-helix was unsuccessful as such peptide does not adopt such helical conformation on the AR-LBD AF2 surface. Further SRC2-2 model building and refinement were not pursued as an α-helix. A composite omit map not including the peptides was calculated in the last steps of refinement for overcoming phase bias for each one of the complexes. This map was calculated omitting 5% of the total model allowing a better tracing of the peptide and permitted to visualize more residues that were not visible in the 2Fo - Fc map. Model building was done using the program QUANTA (Accelrys Software, www.accelrys.com/quanta/) monitored using the free R factor. Calculation of the electron density maps and crystallographic refinement was performed with CNS using the target parameters of Engh and Huber (27Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2537) Google Scholar). Several cycles of model building, conjugate gradient minimization, and simulated annealing using CNS resulted in structures with good stereochemistry. A Ramachandran plot shows that most of the residues fall into the most favored or additionally favored regions. The statistics for data collection and refinement of each one of the data sets can be found in Table I.Table IStatistics for data collection and refinementAR-SRC2-3AR-SRC2-2 (non-helical)AR-SRC3 (RAC3)AR-ARA70Molecules/asymmetric unit1111Space groupP212121P212121P212121P212121Cell constants a/b/c (Å)54.49/67.37/70.5255.60/67.58/69.3253.06/66.83/71.0755.68/66.42/68.25Resolution (Å)2.071.662.72.3Reflections measured393,765511,617375,686458,173Unique reflections16,41635,22117,75313,713Overall completeness (%)97.291.79092.8Outermost shell completeness (%)94.388.083.885.2R merge (%)aR merge (%) = Σhkl|〈I〉 - I|/Σhkl|I|4.465.55Reflections used refinement15,91532,2606,15110,881Resolution range (Å)24-2.0725-1.6625-2.724-2.3R factor (%)bR factor (%) = Σhkl‖Fo| - |Fc‖/Σhkl|Fo|19.821.125.322.8R free (%)cThe R free set contained 5% of total data23.224.831.525.8Number of water molecules160361100106Matthews coefficient2.1572.1162.1002.104Solvent content (%)434241.540Ramachandran plot most favored (%)93928292Ramachandran plot allowed (%)77178a R merge (%) = Σhkl|〈I〉 - I|/Σhkl|I|b R factor (%) = Σhkl‖Fo| - |Fc‖/Σhkl|Fo|c The R free set contained 5% of total data Open table in a new tab The structures have been deposited with the Protein Data Bank (PDB) and assigned the following ID numbers: AR·DHT·SRC2-3, PDB 1T63, RCSB RCSB022358; AR·DHT·ARA70, PDB 1T5Z, RCSB RCSB022354; AR·DHT·SRC2-2, PDB 1T65, RCSB RCSB022360; and AR·DHT·SRC3-2, PDB 1XJ7, RCSB RCSB030414.RESULTSAR AF-2 Binds SRC-2 NR Boxes 1 and 3 with High Affinity—To understand the unusual spectrum of AR AF-2 coactivator interactions, we measured binding of the AR-LBD to a library composed of NR boxes from known coactivating proteins, including both SRCs and AR specific coactivators (Fig. 1A). Such peptides are known to bind to other NRs with equal affinity to the full-length coactivator (28Darimont B.D. Wagner R.L. Apriletti J.W. Stallcup M.R. Kushner P.J. Baxter J.D. Fletterick R.J. Yamamoto K.R. Genes Dev. 1998; 12: 3343-3356Crossref PubMed Scopus (824) Google Scholar). AR-LBD interacted to varying degrees with all of the peptides containing an LXXLL motif tested except the first NR box of ARA70. As expected, AR-LBD interacted with FXXLF sequences present in ARA70 and the AR NTD (21He B. Wilson E.M. Mol. Genet. Metab. 2002; 75: 293-298Crossref PubMed Scopus (78) Google Scholar, 29Bourguet W. Andry V. Iltis C. Klaholz B. Potier N. Van Dorsselaer A. Chambon P. Gronemeyer H. Moras D. Protein Expression Purif. 2000; 19: 284-288Crossref PubMed Scopus (37) Google Scholar) fairly strongly with measurable dissociation constants of 33 ± 3.3 and 38 ± 3.8 μm, respectively. Surprisingly, AR also recognized a subset of NR boxes from the SRC family (30He B. Wilson E.M. Mol. Cell. Biol. 2003; 23: 2135-2150Crossref PubMed Scopus (86) Google Scholar). Specifically, peptides of the first (SRC2-1, Kd = 13 ± 2.1 μm) and third (SRC2-3, Kd = 15 ± 1.2 μm) NR boxes of SRC-2 (GRIP1/TiF-2/N-CoA-2) bound strongly to AR, followed in affinity by FXXLF motifs. The second NR box of SRC3 (RAC3/p/CIP/p300/CBP-interacting protein) was also recruited to AR (Kd = 39 ± 5 μm). The remaining NR boxes from SRC-1, SRC-2, and NTD weakly interacted with AR either nonspecifically or with binding affinities above the assay range (>40 μm). Control experiments with the same sequences in which LXXLL or FXXLF had been converted to LXXAA or FXXAA revealed the binding was dependent upon the intact triad of hydrophobic amino acids (not shown). This substitution has been shown previously to abolish interactions with NR (31McInerney E.M. Rose D.W. Flynn S.E. Westin S. Mullen T.M. Krones A. Inostroza J. Torchia J. Nolte R.T. Assa-Munt N. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1998; 12: 3357-3368Crossref PubMed Scopus (521) Google Scholar).Pull-down experiments confirmed that the AR-LBD bound SRC2 strongly, as opposed to the AR NTD or NTD-DBD (Fig. 1B). Furthermore, AR-LBD interactions with SRC2 were inhibited by mutation of SRC2 boxes 2 and 3 (Fig. 1C), or by increasing concentrations of SRC2-3 peptide (Fig. 1D). Thus, AR-LBD binds FXXLF motifs but also binds a subset of classic NR box peptides with comparable or higher affinities. Moreover, the preference of AR for individual LXXLL motifs is different from that observed with other NRs, such as the estrogen receptor and thyroid receptors (TRs), which bind box 2 in each of the three SRCs with high affinity (28Darimont B.D. Wagner R.L. Apriletti J.W. Stallcup M.R. Kushner P.J. Baxter J.D. Fletterick R.J. Yamamoto K.R. Genes Dev. 1998; 12: 3343-3356Crossref PubMed Scopus (824) Google Scholar, 32Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2224) Google Scholar, 33Ribeiro R.C. Apriletti J.W. West B.L. Wagner R.L. Fletterick R.J. Schaufele F. Baxter J.D. Ann. N. Y. Acad. Sci. 1995; 758: 366-389Crossref PubMed Scopus (64) Google Scholar, 34Moore J.M. Galicia S.J. McReynolds A.C. Nguyen N.H. Scanlan T.S. Guy R.K. J. Biol. Chem. 2004; 279: 27584-27590Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar).AR-dependent Transactivation Requires SRC2 Boxes 1 and 3—Next, we examined the ability of SRC2 to coactivate isolated AR AF-2 and requirements for individual LXXLL motifs in this effect. As expected, a fusion protein containing the AR-LBD (amino acids 646–919) linked to the yeast GAL4 DNA binding function conferred androgen-dependent transcriptional activity on a GAL4-responsive reporter in several cell types, and simultaneous expression of SRC2 strongly enhanced AR AF-2 activity (Fig. 2A). Overall, AR AF-2 activity was more potent than that of AR AF-1 in HeLa and DU145, particularly in the presence of SRC2, and about 20–30% as potent as that induced by TR and estrogen receptorα LBDs, which bind a wider range of SRCs (see supplemental material). As expected from prior results, AF-1 dominates signaling in CV-1 cells, the effects of AF-1 and AF-2 are balanced in DU145 cells, and AF-2 dominates in HeLa cells (35Slagsvold T. Kraus I. Bentzen T. Palvimo J. Saatcioglu F. Mol. Endocrinol. 2000; 14: 1603-1617Crossref PubMed Scopus (38) Google Scholar, 36Wang Q. Lu J. Yong E.L. J. Biol. Chem. 2001; 276: 7493-7499Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Thus, our results are consistent with the notion that AR AF-2 is potent (35Slagsvold T. Kraus I. Bentzen T. Palvimo J. Saatcioglu F. Mol. Endocrinol. 2000; 14: 1603-1617Crossref PubMed Scopus (38) Google Scholar, 36Wang Q. Lu J. Yong E.L. J. Biol. Chem. 2001; 276: 7493-7499Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and contradict the notion that AR AF-2 has little or no intrinsic activity.Fig. 2Transcriptional activation by AR, AR-NTD, and AR-LBD constructs and the enhancement of activation by SRC constructs. A, transcriptional activation of a GAL4-luciferase reporter construct by fusions of GAL4 DNA binding domain with AR-NTD AF-1 or LBD AF-2 domains in three cell lines. In all cell lines, AR-LBD induces signaling in response to DHT, and this effect is enhanced by expression of SRC2. The level of AR-NTD-driven expression varies from cell line to cell line but remains constant in the presence or absence of both DHT and SRC2. B, the effects of mutation of SRC2 NR boxes 1 through 3 upon signaling by GAL4-AR-LBD constructs from a GAL-driven lucifera" @default.
• W2000741688 created "2016-06-24" @default.
• W2000741688 creator A5008278245 @default.
• W2000741688 creator A5019228061 @default.
• W2000741688 creator A5031784533 @default.
• W2000741688 creator A5047363333 @default.
• W2000741688 creator A5053305571 @default.
• W2000741688 creator A5053307838 @default.
• W2000741688 creator A5076185585 @default.
• W2000741688 creator A5076885571 @default.
• W2000741688 creator A5083296600 @default.
• W2000741688 creator A5085871491 @default.
• W2000741688 date "2005-03-01" @default.
• W2000741688 modified "2023-09-29" @default.
• W2000741688 title "The Molecular Mechanisms of Coactivator Utilization in Ligand-dependent Transactivation by the Androgen Receptor" @default.
• W2000741688 cites W1977159265 @default.
• W2000741688 cites W1988275180 @default.
• W2000741688 cites W1995115168 @default.
• W2000741688 cites W1996127793 @default.
• W2000741688 cites W2009616108 @default.
• W2000741688 cites W2011119018 @default.
• W2000741688 cites W2012460741 @default.
• W2000741688 cites W2013905016 @default.
• W2000741688 cites W2018368054 @default.
• W2000741688 cites W2033281378 @default.
• W2000741688 cites W2033837346 @default.
• W2000741688 cites W2036441940 @default.
• W2000741688 cites W2039585420 @default.
• W2000741688 cites W2042095971 @default.
• W2000741688 cites W2042741202 @default.
• W2000741688 cites W2043852388 @default.
• W2000741688 cites W2044481770 @default.
• W2000741688 cites W2054191752 @default.
• W2000741688 cites W2059344207 @default.
• W2000741688 cites W2059815463 @default.
• W2000741688 cites W2082610063 @default.
• W2000741688 cites W2102892939 @default.
• W2000741688 cites W2108296383 @default.
• W2000741688 cites W2111650618 @default.
• W2000741688 cites W2113606874 @default.
• W2000741688 cites W2116275717 @default.
• W2000741688 cites W2119963782 @default.
• W2000741688 cites W2124174146 @default.
• W2000741688 cites W2136294116 @default.
• W2000741688 cites W2138120234 @default.
• W2000741688 cites W2141686420 @default.
• W2000741688 cites W2150319350 @default.
• W2000741688 cites W2151286904 @default.
• W2000741688 cites W2151873398 @default.
• W2000741688 cites W2151922331 @default.
• W2000741688 cites W2164472870 @default.
• W2000741688 cites W2172024571 @default.
• W2000741688 doi "https://doi.org/10.1074/jbc.m407046200" @default.
• W2000741688 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15563469" @default.
• W2000741688 hasPublicationYear "2005" @default.
• W2000741688 type Work @default.
• W2000741688 sameAs 2000741688 @default.
• W2000741688 citedByCount "140" @default.
• W2000741688 countsByYear W20007416882012 @default.
• W2000741688 countsByYear W20007416882013 @default.
• W2000741688 countsByYear W20007416882014 @default.
• W2000741688 countsByYear W20007416882015 @default.
• W2000741688 countsByYear W20007416882016 @default.
• W2000741688 countsByYear W20007416882017 @default.
• W2000741688 countsByYear W20007416882018 @default.
• W2000741688 countsByYear W20007416882019 @default.
• W2000741688 countsByYear W20007416882020 @default.
• W2000741688 countsByYear W20007416882021 @default.
• W2000741688 countsByYear W20007416882022 @default.
• W2000741688 countsByYear W20007416882023 @default.
• W2000741688 crossrefType "journal-article" @default.
• W2000741688 hasAuthorship W2000741688A5008278245 @default.
• W2000741688 hasAuthorship W2000741688A5019228061 @default.
• W2000741688 hasAuthorship W2000741688A5031784533 @default.
• W2000741688 hasAuthorship W2000741688A5047363333 @default.
• W2000741688 hasAuthorship W2000741688A5053305571 @default.
• W2000741688 hasAuthorship W2000741688A5053307838 @default.
• W2000741688 hasAuthorship W2000741688A5076185585 @default.
• W2000741688 hasAuthorship W2000741688A5076885571 @default.
• W2000741688 hasAuthorship W2000741688A5083296600 @default.
• W2000741688 hasAuthorship W2000741688A5085871491 @default.
• W2000741688 hasBestOaLocation W20007416881 @default.
• W2000741688 hasConcept C104317684 @default.
• W2000741688 hasConcept C121608353 @default.
• W2000741688 hasConcept C12554922 @default.
• W2000741688 hasConcept C1292079 @default.
• W2000741688 hasConcept C185592680 @default.
• W2000741688 hasConcept C203966977 @default.
• W2000741688 hasConcept C2777911890 @default.
• W2000741688 hasConcept C2780192828 @default.
• W2000741688 hasConcept C54355233 @default.
• W2000741688 hasConcept C55493867 @default.
• W2000741688 hasConcept C61367390 @default.
• W2000741688 hasConcept C71315377 @default.
• W2000741688 hasConcept C77445288 @default.
• W2000741688 hasConcept C86339819 @default.
• W2000741688 hasConcept C86803240 @default.
• W2000741688 hasConcept C95444343 @default.

• next