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FXXLF and WXXLF Sequences Mediate the NH2-terminal Interaction with the Ligand Binding Domain of the Androgen Receptor

2000; Elsevier BV; Volume: 275; Issue: 30 Linguagem: Inglês

10.1074/jbc.m002807200

ISSN

1083-351X

Autores

Bin He, Jon Kemppainen, Elizabeth M. Wilson,

Tópico(s)

Prostate Cancer Treatment and Research

Resumo

The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH2-terminal region which is not conserved. Recent biochemical and crystallographic studies revealed the molecular basis of AF2 is hormone-dependent recruitment of LXXLL motif-containing coactivators, including the p160 family, to a hydrophobic cleft in the ligand binding domain. Our previous studies demonstrated that AF2 in the androgen receptor (AR) binds only weakly to LXXLL motif-containing coactivators and instead mediates an androgen-dependent interaction with the AR NH2-terminal domain required for its physiological function. Here we demonstrate in a mammalian two-hybrid assay, glutathione S-transferase fusion protein binding studies, and functional assays that two predicted α-helical regions that are similar, but functionally distinct from the p160 coactivator interaction sequence, mediate the androgen-dependent, NH2- and carboxyl-terminal interaction. FXXLF in the AR NH2-terminal domain with the sequence23FQNLF27 mediates interaction with AF2 and is the predominant androgen-dependent interaction site. This FXXLF sequence and a second NH2-terminal WXXLF sequence 433WHTLF437 interact with different regions of the ligand binding domain to stabilize the hormone-receptor complex and may compete with AF2 recruitment of LXXLL motif-containing coactivators. The results suggest a unique mechanism for AR-mediated transcriptional activation. The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH2-terminal region which is not conserved. Recent biochemical and crystallographic studies revealed the molecular basis of AF2 is hormone-dependent recruitment of LXXLL motif-containing coactivators, including the p160 family, to a hydrophobic cleft in the ligand binding domain. Our previous studies demonstrated that AF2 in the androgen receptor (AR) binds only weakly to LXXLL motif-containing coactivators and instead mediates an androgen-dependent interaction with the AR NH2-terminal domain required for its physiological function. Here we demonstrate in a mammalian two-hybrid assay, glutathione S-transferase fusion protein binding studies, and functional assays that two predicted α-helical regions that are similar, but functionally distinct from the p160 coactivator interaction sequence, mediate the androgen-dependent, NH2- and carboxyl-terminal interaction. FXXLF in the AR NH2-terminal domain with the sequence23FQNLF27 mediates interaction with AF2 and is the predominant androgen-dependent interaction site. This FXXLF sequence and a second NH2-terminal WXXLF sequence 433WHTLF437 interact with different regions of the ligand binding domain to stabilize the hormone-receptor complex and may compete with AF2 recruitment of LXXLL motif-containing coactivators. The results suggest a unique mechanism for AR-mediated transcriptional activation. activation function 2 androgen receptor ligand binding domain NH2-terminal and carboxyl-terminal Chinese hamster ovary mouse mammary tumor virus polymerase chain reaction dihydrotestosterone glutathione S-transferase transcriptional mediator/intermediary factor 2 Nuclear receptors facilitate ligand-dependent increases of gene transcription by direct interactions with nuclear coactivators. p160 coactivators have histone acetyltransferase activity (1Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1059) Google Scholar) and interact with nuclear receptors through their ligand binding and NH2-terminal regions (2Glass C.K. Rose D.W. Rosenfeld M.G. Cur. Opin. Cell Biol. 1997; 9: 222-232Crossref PubMed Scopus (600) Google Scholar, 3McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1650) Google Scholar, 4Webb P. Nguyen P. Shinsako J. Anderson C. Feng W. Nguyen M.P. Chen D. Huang S.M. Subramanian S. McKinerney E. Katzenellenbogen B.S. Stallcup M.R. Kushner P.J. Mol. Endocrinol. 1998; 12: 1605-1618Crossref PubMed Scopus (0) Google Scholar). Additional acetyltransferases p300/cAMP response element binding protein and p300/cAMP response element binding protein associated factor interact with the p160 coactivators (5Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1262) Google Scholar, 6Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1922) Google Scholar, 7Torchia J. Glass C. Rosenfeld M.G. Curr. Opin. Cell Biol. 1998; 10: 373-383Crossref PubMed Scopus (511) Google Scholar). Nuclear receptors also interact with multiprotein complexes referred to as thyroid hormone receptor-associated proteins (8Fondell J.D. Brunel F. Hisatake K. Roeder R.G. Mol. Cell. Biol. 1996; 16: 281-287Crossref PubMed Google Scholar), activator-recruited cofactor (9Naar A.M. Beaurang P.A. Zhou S. Abraham S. Solomon W. Tjian R. Nature. 1999; 398: 828-832Crossref PubMed Scopus (371) Google Scholar), or vitamin D receptor-interacting protein complex (10Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Naar A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Crossref PubMed Scopus (631) Google Scholar). The p160 coactivators and at least one of the thyroid hormone receptor-associated proteins/activator-recruited cofactor/vitamin D receptor-interacting protein subunit 205 interact in a ligand-dependent manner with activation function 2 (AF2)1 in the ligand binding domain (LBD) of nuclear receptors through the consensus sequence LXXLL, where L is leucine and X is any amino acid (11Darimont 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 (828) Google Scholar, 12Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1681) Google Scholar, 13Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1769) Google Scholar, 14Torchia J. Rose D.W. Inostroza J. Kmei Y. Westin S. Glass C. Rosenfeld M. Nature. 1997; 382: 677-684Crossref Scopus (1105) Google Scholar, 15McInerney 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 (526) Google Scholar, 16Voegel J.J. Heine M.J.S. Tini M. Vivat V. Chambon P. Gronemeyer H. EMBO J. 1998; 17: 507-519Crossref PubMed Scopus (429) Google Scholar). Crystal structures of nuclear receptor LBDs have shown that a hydrophobic cleft within a multilayered α-helical structure serves as the LXXLL coactivator binding surface AF2 (17Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1060) Google Scholar, 18Feng W. Ribeiro R.C.J. Wagner R.L. Nguyen H. Apriletti J.W. Fletterick R.J. Baxter J.D. Kushner P.J. West B.L. Science. 1998; 280: 1747-1749Crossref PubMed Scopus (514) Google Scholar). In the estrogen receptor, agonist binding positions helix 12 over the binding cavity to complete the AF2 surface (19Freedman L.P. Cell. 1999; 97: 5-8Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 20Wurtz J.M. Bourguet W. Renaud J.P. Vivat V. Chambon P. Moras D. Gronemeyer H. Nat. Struct. Biol. 1996; 3: 87-94Crossref PubMed Scopus (679) Google Scholar), whereas binding of an antagonist such as 4-hydroxytamoxifen displaces helix 12 (21Brzozowski A.M. Pike A.C.W. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.A. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2932) Google Scholar) causing an LXXLL-like sequence in helix 12 to mimic and thereby block coactivator binding (22Shiau 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 (2241) Google Scholar). It has become apparent that the AF2 region overlaps with regions that serve as the binding site for a variety of LXXLL-related sequences as recently shown for corepressor binding (23Nagy L. Kao H.Y. Love J.D. Li C. Banayo E. Gooch J.T. Krishna V. Chatterjee K. Evans R.M. Schwabe J.W.R. Genes Dev. 1999; 13: 3209-3216Crossref PubMed Scopus (345) Google Scholar, 24Perissi V. Staszewski L.M. McInerney E.M. Kurokawa R. Krones A. Rose D.W. Lambert M.H. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1999; 13: 3198-3208Crossref PubMed Scopus (423) Google Scholar, 25Hu X. Lazar M.A. Nature. 1999; 402: 93-96Crossref PubMed Scopus (524) Google Scholar). Furthermore, we demonstrated that the AR AF2 region mediates an androgen-dependent NH2-terminal/carboxyl-terminal (N/C) interaction (26He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Therefore we investigated the possibility that AF2 in the AR LBD interacts with an LXXLL-related sequence in the NH2-terminal domain. In this report, evidence is presented that sequences similar to but distinct from the LXXLL core sequence mediate a direct interaction between the NH2-terminal and carboxyl-terminal regions of AR. The FXXLF core sequence 23FQNLF27 in the AR NH2-terminal domain binds AF2 in the carboxyl-terminal region in an androgen-dependent manner. In addition, a second motif in the NH2-terminal region WXXLF with the sequence 433WHTLF437 binds to a region of the LBD outside of AF2. Interaction of these NH2-terminal sequences with the LBD slows the dissociation rate of bound androgen. Sequence specificity was indicated since FXXLF could not be functionally replaced by an LXXLL core sequence. Mammalian two-hybrid N/C interaction assays were performed in Chinese hamster ovary (CHO) cells as described previously (27Langley E. Zhou Z. Wilson E.M. J. Biol. Chem. 1995; 270: 29983-29990Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 28Langley E. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 1998; 273: 92-101Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). VPAR-(1–660) codes for the herpes simplex virus VP16 protein transactivation domain residues 411–456 expressed as a fusion protein with AR NH2-terminal and DNA binding domain residues 1–660. GALAR-(624–919) codes for a fusion protein of the Saccharomyces cerevisiae GAL4 DNA binding domain residues 1–147 and AR LBD residues 624–919. Deletions within VPAR-(1–660) (Δ179–199, Δ394–405, and Δ429–439) were created by two polymerase chain reactions (PCR) using oligonucleotide primers with appropriate deletions. The reporter vector G5E1bLuc contained five GAL4 DNA-binding sites and the luciferase reporter coding region (29Lillie J.W. Green M.R. Nature. 1989; 338: 39-44Crossref PubMed Scopus (471) Google Scholar). CHO cells were plated at 0.425 × 106/6-cm dish and transfected using DEAE-dextran (27Langley E. Zhou Z. Wilson E.M. J. Biol. Chem. 1995; 270: 29983-29990Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 28Langley E. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 1998; 273: 92-101Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Cells were incubated for 24 h in the absence and presence of increasing concentrations of dihydrotestosterone (DHT) as indicated and harvested in lysis buffer (Ligand Pharmaceuticals). Luciferase activity was determined using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego). For the assessment of AR transcriptional activity, monkey kidney CV1 cells maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), 20 mm Hepes, pH 7.2 (DMEM-H), and antibiotics were transiently transfected by plating 0.425 × 106 cells/6-cm dish. Wild-type and mutant pCMVhAR DNA (10–50 ng/dish) were precipitated with 5 μg of mouse mammary tumor virus promoter region (MMTV)-luciferase reporter vector (5 μg) using calcium phosphate (30Kemppainen J.A. Langley E. Wong C.I. Bobseine K. Kelce W.R. Wilson E.M. Mol. Endocrinol. 1999; 13: 440-445Crossref PubMed Scopus (0) Google Scholar). Cells were incubated for 24 or 48 h with the indicated concentrations of hormones and harvested and assayed in lysis buffer as described above. VPAR-(1–660) containing Δ329–381, Δ382–429, Δ430–499, or Δ429–439 was digested with AflII and KpnI, and the resulting inserts were ligated into pCMVhARL26A/F27A digested with the same enzymes. PCMVhARΔ339–499, Δ9–28 (27Langley E. Zhou Z. Wilson E.M. J. Biol. Chem. 1995; 270: 29983-29990Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 31Zhou Z.X. Lane M.V. Kemppainen J.A. French F.S. Wilson E.M. Mol. Endocrinol. 1995; 9: 208-218Crossref PubMed Google Scholar), and AR-(507–919) (32Simental J.A. Sar M. Lane M.V. French F.S. Wilson E.M. J. Biol. Chem. 1991; 266: 510-518Abstract Full Text PDF PubMed Google Scholar) were previously described. PCR mutagenesis was used to create single and double amino acid mutations in the23FXXLF27 and433WXXLF437 domains. L26A/F27AΔ339–499 was prepared by ligating pCMVhARΔ339–499 digested with BglII/AflII with the insert from pCMVhARL26A/F27A using the same enzymes. Apparent equilibrium binding affinity was determined in whole cell binding assays at 37 °C by plating monkey kidney COS1 cells (0.2 × 106/well of 12-well tissue culture plates) and transfecting 0.1 μg of pCMVhAR wild-type and mutant DNA using DEAE-dextran (26He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Twenty four h after transfection, cells were incubated with increasing concentrations of [3H]R1881 from 0.1 to 5 nmin the presence and absence of a 100-fold excess unlabeled R1881. Cells were incubated at 37 °C for 2 h, washed, and harvested in 0.5 ml of 2% SDS, 10% glycerol, and 10 mm Tris, pH 6.8. Radioactivity was determined by scintillation counting. For determination of dissociation rates of [3H]R1881, COS cells were plated at 0.4 × 106 cells/well in 6-well plates and transfected with 2 μg of pCMVhAR DNA/well using DEAE-dextran. Cells were incubated for 2 h at 37 °C with 5 nm [3H]R1881 in the presence and absence of a 100-fold excess unlabeled R1881. Dissociation was started by the addition of 50 μm unlabeled R1881, and the cells were incubated for increasing times at 37 °C up to 3 h, washed once, and harvested in SDS buffer as described above, with radioactivity determined by scintillation counting. GlutathioneS-transferase (GST) fusion vectors GSTAR-(1–660) and GSTAR-(1–565) were prepared as described previously (26He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). pGEX5X-1AR-(1–660) was digested with XhoI (blunt) and SmaI and religated to make GSTAR-(1–36). pGEX5X-1AR-(1–660) was digested with AflII/XhoI, blunt-ended, and religated to make GSTAR-(1–173). pGEX5X-1AR-(1–660) was digested with SacI/XhoI, blunt-ended, and religated to make GSTAR-(1–333). pGEX-3XAR-(1–566) was digested with BamHI/AflII, blunt-ended, and religated to make GSTAR-(174–566). GALAR-(1–660)-L26A/F27A was digested with BamHI/AflII, and the insert was ligated into pGEX-5X-1AR-(1–660) digested with BamHI/AflII to make GSTAR-(1–660)-L26A/F27A. GSTAR-(1–660)-L26A/F27A was digested with KpnI/AflII and ligated with the insert from VPAR-(1–660)-Δ429–439 digested with KpnI/AflII to make GSTAR-(1–660)-L26A/F27A-(Δ429–439). AR NH2-terminal residues 344–566 were PCR-amplified from VPAR-(1–660) with the appropriate deletions, digested at BamHI/XhoI primer sites, and ligated into pGEX-4T-1 to make GSTAR-(344–566), GSTAR-(344–566)-Δ339–381, GSTAR-(344–566)-Δ382–429, GSTAR-(344–566)-Δ430–499, and GSTAR-(344–566)-Δ429–439. GSTTIF2 expressing the central TIF2 amino acid residues 624–1141 containing three LXXLL motifs was described previously (26He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). pcDNA3-AR-(624–919)-E897K, V716R, K720A, and V889M were prepared by digesting GALD-H containing the appropriate mutation with BamHI/XbaI and ligating the fragment into pcDNA3HA digested with BamHI/XbaI. GST fusion protein binding studies were performed essentially as described previously (26He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). GST fusion proteins with different regions of the human AR NH2-terminal region were expressed from XL1-Blue Escherichia coli cells treated with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h during log phase growth. Glutathione-agarose beads (Amersham Pharmacia Biotech) were incubated for 1 h at 4 °C with sonicated bacterial supernatants containing the GST-AR fusion proteins. Beads were washed with 0.5% Nonidet P-40, 1 mm EDTA, 0.1m NaCl, 0.02 m Tris, pH 8.0, and combined with 25 μCi of [35S]methionine (NEN Life Science Products)-labeled human AR LBD (AR amino acid residues 624–919) using TNT T7 Quick-coupled Transcription/Translation System (Promega) and incubated for 2 h at 4 °C in the absence or presence of 0.2 μm DHT. Beads were washed, eluted with SDS, and analyzed on 12% acrylamide gels containing SDS. The androgen-dependent interaction between the AR NH2-terminal and carboxyl-terminal (N/C) domains occurs in the regions of predicted α-helices 3, 4, and 12 that comprise AF2 of the LBD, overlapping with the binding site for p160 coactivators (26He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Because LXXLL motifs mediate the interaction of p160 coactivators with AF2 of nuclear receptors (11Darimont 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 (828) Google Scholar, 12Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1681) Google Scholar, 13Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1769) Google Scholar, 14Torchia J. Rose D.W. Inostroza J. Kmei Y. Westin S. Glass C. Rosenfeld M. Nature. 1997; 382: 677-684Crossref Scopus (1105) Google Scholar, 15McInerney 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 (526) Google Scholar, 16Voegel J.J. Heine M.J.S. Tini M. Vivat V. Chambon P. Gronemeyer H. EMBO J. 1998; 17: 507-519Crossref PubMed Scopus (429) Google Scholar), it raised the possibility that an LXXLL-like motif (13Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1769) Google Scholar, 14Torchia J. Rose D.W. Inostroza J. Kmei Y. Westin S. Glass C. Rosenfeld M. Nature. 1997; 382: 677-684Crossref Scopus (1105) Google Scholar, 15McInerney 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 (526) Google Scholar) in the AR NH2-terminal region has a similar function to mediate the N/C interaction. Sequence analysis of the AR NH2-terminal regions previously implicated in the N/C interaction (27Langley E. Zhou Z. Wilson E.M. J. Biol. Chem. 1995; 270: 29983-29990Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) revealed four predicted amphipathic α-helices that resemble LXXLL core sequences at residues 21–34, 351–359, 395–405, and 432–434 with another predicted outside these regions (33Alen P. Claessens F. Verhoeven G. Rombauts W. Peeters B. Mol. Cell. Biol. 1999; 19: 6085-6097Crossref PubMed Scopus (217) Google Scholar) at residues 177–201. We tested wild-type, deletion, and single amino acid mutations of these α-helical regions in a variety of assays. These included the mammalian two-hybrid N/C interaction assay performed in CHO cells and functional assays that included the effects of the mutations on [3H]androgen dissociation rate and transcriptional activation. In vitro domain interactions were also directly tested using E. coli-expressed GST fusion proteins. For the mammalian two-hybrid assay, wild-type and mutant VPAR-(1–660) coding for the AR NH2-terminal and DNA binding domains were cotransfected with GALAR-(624–919) expressing the LBD (Fig.1). Similar expression levels of wild-type and mutant VPAR-(1–660) vectors were verified by immunoblot analysis using AR52 antibody (data not shown). Also all of the VPAR-(1–660) constructs, when cotransfected with GALAR-(1–503) coding for just the NH2-terminal domain, resulted in 2.2–3.2-fold induction of luciferase activity indicative of the NH2-terminal/NH2-terminal AR interaction previously reported (27Langley E. Zhou Z. Wilson E.M. J. Biol. Chem. 1995; 270: 29983-29990Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar), thus confirming similar expression levels of the VPAR-(1–660) vectors. Wild-type and mutant VPAR-(1–660) coding for the AR NH2-terminal and DNA binding regions were coexpressed in the mammalian two-hybrid assay with GALAR-(624–919) coding for the AR LBD. The VPAR-(1–660) fragment with wild-type AR sequence induced 38 ± 19-fold luciferase activity relative to the no hormone control (Fig. 1). Deletion of NH2-terminal residues 179–199, 394–405, or 429–439 had no significant effect on the interaction (Fig. 1). In contrast, Δ9–28 reduced the interaction to only a 1.5-fold increase over the no hormone control and Δ339–499 to 10-fold relative to the control (Fig. 1) (27Langley E. Zhou Z. Wilson E.M. J. Biol. Chem. 1995; 270: 29983-29990Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). These results suggested interactions between the LBD and residues 9–28 and 339–499. Because23FQNLF27 lies within the 9–28 region and resembles the LXXLL core sequence, it was investigated further by mutagenesis. Changing phenylalanine 23 to alanine (F23A) or leucine 26 and phenylalanine 27 to alanine (L26A/F27A) reduced the N/C interaction to 1.6 ± 0.2-fold over background levels, whereas a flanking mutation of glutamine 28 to alanine (Q28A) resulted in an interaction similar to that of wild-type AR (Fig. 1). Changing both phenylalanine residues in 23FQNLF27 to leucine (F23L/F27L) to recreate a consensus LXXLL sequence resulted in only a 2-fold induction of luciferase activity (Fig. 1). These results indicated that an FXXLF motif was required in the N/C interaction. Specificity of the23FXXLF27 interaction with the AR LBD was indicated by the greatly reduced interaction when FXXLF was substituted by LXXLL. The requirement for the FXXLF motif in the N/C interaction was also investigated using AR NH2- and carboxyl-terminal fragments in transient transfection assays using the MMTV-luciferase reporter. As previously reported, the AR DNA binding and ligand binding domain fragment AR-(507–919) had negligible transcriptional activity in the presence of androgen (Fig. 2) indicative of the lack of AF2 transcriptional activity (32Simental J.A. Sar M. Lane M.V. French F.S. Wilson E.M. J. Biol. Chem. 1991; 266: 510-518Abstract Full Text PDF PubMed Google Scholar) and weak recruitment of p160 coactivators by the AR AF2 region (26He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). In cotransfection studies, wild-type NH2-terminal domain residues AR-(1–503) interacted with AR-(507–919) to stimulate an 18–28-fold increase in luciferase activity (Fig. 2). In contrast, NH2-terminal fragment AR-(1–503) with residues 14–150 deleted (Δ14–150) or with the mutated sequence22FQNAA27 (L26A/F27A) failed to interact with AR-(507–919), supporting an important role for23FQNLF27 in the N/C interaction. Androgen-induced luciferase activity was 6–12-fold with deletion of NH2-terminal residues 142–337 comprising the AR transactivation domain (Δ142–337) which could have resulted from reduced transactivation by AR rather than a decrease in the N/C interaction as previously suggested (27Langley E. Zhou Z. Wilson E.M. J. Biol. Chem. 1995; 270: 29983-29990Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Δ339–499 also reduced the interaction but less effectively than did the L26A/F27A mutation (Fig.2). The results support the requirement for23FQNLF27 in the N/C interaction and the presence of a second interaction site between residues 339 and 499. Similar results using fusion proteins of the AR NH2-terminal region linked to the VP16 transactivation domain supported the role of these two regions in the N/C interaction (Fig. 2). To establish a functional effect of the NH2-terminal mutations on AR activity and to obtain additional evidence for the putative second interacting site, we measured the androgen dissociation rate using the synthetic radiolabeled androgen [3H]R1881. These studies were based on previous studies that certain mutations in AF2 of the AR LBD cause androgen insensitivity by disrupting the N/C interaction. Although the equilibrium androgen binding affinity was unaffected by these mutations, the dissociation rate of bound androgen increased suggesting a corresponding increase in association rate (26He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 28Langley E. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 1998; 273: 92-101Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The results supported a role for the N/C interaction in slowing the androgen dissociation rate in wild-type AR (31Zhou Z.X. Lane M.V. Kemppainen J.A. French F.S. Wilson E.M. Mol. Endocrinol. 1995; 9: 208-218Crossref PubMed Google Scholar). Additional evidence that the N/C interaction influences AR ligand binding kinetics is that coexpression of the DNA binding domain and LBD fragment AR-(507–919) with NH2-terminal fragment AR-(1–660) slows the dissociation of [3H]R1881 by 2-fold (28Langley E. Kemppainen J.A. Wilson E.M. J. Biol. Chem. 1998; 273: 92-101Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). In contrast, no effect was seen by coexpression of the nuclear receptor coactivators transcriptional mediator/intermediary factor 2 (TIF2), vitamin D receptor-interacting protein 205, amplified in breast cancer-1 (AIB1) or protein inhibitor of activated signal transducer and activator of transcription-1 (PIAS1), each of which contains multiple consensus LXXLL motifs (10Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Naar A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Crossref PubMed Scopus (631) Google Scholar, 34Voegel J.J. Heine M.J.S. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (950) Google Scholar, 35Anzick S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.Y. Sauter G. Kallioniemi O.P. Trent J.M. Meltzer P.S. Science. 1997; 277: 965-968Crossref PubMed Scopus (1430) Google Scholar, 36Tan J.A. Hall S.A. Hamil K.G. Grossman G. Petrusz P. Liao J. Shuai K. French F.S. Mol. Endocrinol. 2000; 14: 14-26Crossref PubMed Scopus (86) Google Scholar). Dissociation of [3H]R1881 from the carboxyl-terminal AR-(507–919) fragment (half-time of dissociation t½ of 43 ± 3 min, see Fig. 4) was unchanged with t½ of 42 ± 4 min at 37 °C when coexpressed with each of these coactivators (data not shown). The results are consistent with a weak interaction of these coactivators with the AR AF2 region compared with the interaction with the AR NH2-terminal domain (26He B. Kemppainen J.A. Voegel J.J. Gronemeyer H. Wilson E.M. J. Biol. Chem. 1999; 274: 37219-37225Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) and suggest a correspondingly higher apparent binding activity of the23FXXLF27 core sequence for AF2 compared with the LXXLL motif. The functional significance of the NH2-terminal FXXLF core sequence was also evident by increased dissociation rates of bound androgen with the introduction of mutations in the FXXLF region in full-length AR. Δ9–28, F23A, or L26A/F27A increased the dissociation rate of [3H]R1881 at 37 °C to t½ of 72–74 min compared with t½ of 144 ± 24 min for wild-type AR (Fig.3). In contrast, mutation of the flanking carboxyl glutamine (Q28A) had no effect on dissociation rate (t½ of 162 ± 24 min) (Fig. 3) or N/C interaction (Fig. 1). Furthermore, changing23FQNLF27 to the consensus LXXLL sequence (F23L/F27L) increased the dissociation rate from t½ of 144 ± 24 min for wild-type AR to t½ of 84 ± 9 min (Fig. 3), which was similar to t½ 74 ± 5 min for F23A and L26A/F27A, supporting that an LXXLL motif is much less effective than FXXLF in mediating the N/C interaction. None of these mutations or those described below changed significantly the apparent equilibrium binding affinity of [3H]R1881 that ranged from 0

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