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• W2077854579 abstract "The basis for specificity of gene regulation by steroid hormone receptors remains an important problem in the study of steroid hormone action. One possible mechanism for steroid specificity is the difference in DNA binding characteristics of the receptors, although they share a high homology in their DNA-binding domains. Indeed, the androgen-specific expression of, for example, the probasin (PB) gene can be explained by the presence of an androgen response element (ARE) in its promoter (PB-ARE-2), specifically recognized by the androgen and not by the glucocorticoid receptor. Three residues in the DNA-binding domain of the AR were identified as main determinants for its high affinity for the PB-ARE-2. In addition, the direct repeat nature of this ARE seems to prohibit high affinity binding by the glucocorticoid receptor. This is confirmed by the fact that several imperfect direct repeats of the 5′-TGTTCT-3′ core recognition sequence are recognized by the androgen receptor and not by the glucocorticoid receptor. Up to now, only differences between the androgen and glucocorticoid receptor in the transcription activation functions were invoked to explain the specificity of their genomic actions. In the present study, we describe the influence of the DNA-binding domain on the specificity of androgen action. The novelty of our working hypothesis resides in the demonstration of the capacity of the AR-DNA-binding domain to recognize elements with a direct repeat structure. The basis for specificity of gene regulation by steroid hormone receptors remains an important problem in the study of steroid hormone action. One possible mechanism for steroid specificity is the difference in DNA binding characteristics of the receptors, although they share a high homology in their DNA-binding domains. Indeed, the androgen-specific expression of, for example, the probasin (PB) gene can be explained by the presence of an androgen response element (ARE) in its promoter (PB-ARE-2), specifically recognized by the androgen and not by the glucocorticoid receptor. Three residues in the DNA-binding domain of the AR were identified as main determinants for its high affinity for the PB-ARE-2. In addition, the direct repeat nature of this ARE seems to prohibit high affinity binding by the glucocorticoid receptor. This is confirmed by the fact that several imperfect direct repeats of the 5′-TGTTCT-3′ core recognition sequence are recognized by the androgen receptor and not by the glucocorticoid receptor. Up to now, only differences between the androgen and glucocorticoid receptor in the transcription activation functions were invoked to explain the specificity of their genomic actions. In the present study, we describe the influence of the DNA-binding domain on the specificity of androgen action. The novelty of our working hypothesis resides in the demonstration of the capacity of the AR-DNA-binding domain to recognize elements with a direct repeat structure. androgen receptor glucocorticoid receptor androgen response element C-terminal extension probasin DNA-binding domain nuclear localization signal direct repeat cytomegalovirus Dulbecco's modified Eagle's medium 9-cis retinoic acid receptor thyroid receptor Steroid hormones are important endocrine messengers that activate their receptors, which translocate to the cell nucleus and regulate gene expression mainly after interaction with DNA sequences, called response elements (1.Evans R. Science. 1988; 240: 889-895Crossref PubMed Scopus (6276) Google Scholar, 2.Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2838) Google Scholar). The steroid receptors are a subfamily of the nuclear receptor superfamily, a large group of structurally homologous transcription factors. A problem with the explanation of the specificity of these hormone responses arose when several studies pointed out that the class I receptors (androgen receptor (AR),1 glucocorticoid receptor (GR), progesterone receptor, and mineralocorticoid receptor) have identical consensus response elements (3.Nordeen S.K. Suh B.J. Kuhnel B. Hutchison C. Mol. Endocrinol. 1990; 4: 1866-1873Crossref PubMed Scopus (169) Google Scholar, 4.Roche P. Hoare S. Parker M. Mol. Endocrinol. 1992; 6: 2229-2235Crossref PubMed Scopus (184) Google Scholar) and that their DNA-binding domains were highly conserved (5.Umesono K. Evans R. Cell. 1989; 57: 1139-1146Abstract Full Text PDF PubMed Scopus (715) Google Scholar). This contrasts with the fact that the in vivo expression of several genes is specifically controlled by only one steroid hormone (6.Truss M. Beato M. Endocrine Rev. 1993; 14: 459-479Crossref PubMed Scopus (0) Google Scholar). Several possible mechanisms have been described to explain the steroid specificity of transcriptional control, e.g. steroid metabolism, tissue-specific receptor presence (7.Funder J.W. Science. 1993; 259: 1132-1133Crossref PubMed Scopus (156) Google Scholar), influence of coactivator complexes (8.Xu L. Glass C.K. Rosenfeld M.G. Curr. Opin. Genet. Dev. 1999; 9: 140-147Crossref PubMed Scopus (806) Google Scholar), and chromatin structure (9.Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 10.Wolffe A. Hayes J.J. Nucleic Acids Res. 1999; 27: 711-720Crossref PubMed Scopus (431) Google Scholar). In addition, more recent reports indicate that the AR on the one hand and the GR, progesterone receptor, and mineralocorticoid receptor on the other exhibit different DNA binding characteristics (11.Rundlett S.E. Miesfeld R.L. Mol. Cell. Endocrinol. 1995; 109: 1-10Crossref PubMed Scopus (51) Google Scholar, 12.Claessens F. Alen P. Devos A. Peeters B. Verhoeven G. Rombauts W. J. Biol. Chem. 1996; 271: 19013-19016Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13.Zhou Z. Cordons J. Brown T. J. Biol. Chem. 1997; 272: 8227-8235Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 14.Verrijdt G. Schoenmakers E. Alen P. Haelens A. Peeters B. Rombauts W. Claessens F. Mol. Endocrinol. 1999; 13: 1558-1570Crossref PubMed Google Scholar, 15.Haelens A. Verrijdt G. Schoenmakers E. Alen P. Peeters B. Rombauts B. Claessens F. Mol. Cell. Endocrinol. 1999; 153: 91-102Crossref PubMed Scopus (39) Google Scholar). One AR-specific response element was found in the promoter of the rat probasin gene (PB-ARE-2) (12.Claessens F. Alen P. Devos A. Peeters B. Verhoeven G. Rombauts W. J. Biol. Chem. 1996; 271: 19013-19016Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 16.Rennie P. Bruchovsky N. Leco K. Sheppard P. McQueen S. Cheng H. Snoek R. Hamel H. Bock M. Chang C. Liao S. Cattini P.A. Matusik R. Mol. Endocrinol. 1993; 7: 23-36Crossref PubMed Scopus (215) Google Scholar, 17.Kasper S. Rennie P.S. Bruchovsky N. Sheppard P.C. Cheng H. Lin L. Shiu R.P.C. Matusik R.J. J. Biol. Chem. 1994; 269: 31763-31769Abstract Full Text PDF PubMed Google Scholar). Probasin is an androgen-regulated protein exclusively expressed in the dorsolateral epithelium of the prostate (18.Spence A.M. Sheppard P.C. Davie J.R. Matuo Y. Nishi N. Mckeehan W.L. Dodd J.G. Matusik R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7843-7847Crossref PubMed Scopus (79) Google Scholar). Two cis-acting androgen response elements (PB-ARE-1 and PB-ARE-2) were identified in the promoter and were shown to be necessary for its androgen regulation. The core DNA-binding domain (DBD) of the nuclear receptors is composed of two zinc finger modules (19.Freedman L.P. Endocr. Rev. 1992; 13: 129-145Crossref PubMed Scopus (241) Google Scholar). The N-terminal zinc finger is involved in specific DNA interaction, whereas the C-terminal zinc finger mainly provides DNA-dependent dimerizations (20.Luisi B. Xu W. Otwinski Z. Freedman L. Yamamoto K. Sigler P. Nature. 1991; 352: 497-505Crossref PubMed Scopus (1210) Google Scholar). Our earlier results indicated that residues in the second zinc finger and a C-terminal extension (CTE) of 12 amino acids determine the difference in the PB-ARE-2 binding between the AR and the GR (21.Schoenmakers E. Alen P. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. Biochem. J. 1999; 314: 515-521Crossref Google Scholar). This was the first clear indication of a direct involvement of the CTE residues in the specificity of DNA binding for steroid receptors. The CTE described here overlaps with the T-box described for other members of the nuclear receptor family (e.g. 9-cis retinoic acid receptor (RXR), thyroid receptor (TR), and nerve growth factor-inducible protein B) (22.Rastinejad F. Perlmann T. Evans R. Sigler P. Nature. 1995; 375: 203-211Crossref PubMed Scopus (458) Google Scholar, 23.Lee M. Kliewer S. Provencal J. Wright P. Evans R. Science. 1993; 260: 1117-1120Crossref PubMed Scopus (247) Google Scholar, 24.Wilson T. Paulsen R. Padgett K. Milbrandt J. Science. 1992; 256: 107-110Crossref PubMed Scopus (276) Google Scholar). Structural studies showed that in these receptors the T-box residues form an α-helix, which interacts with the DNA phosphate backbone and which is also involved in the DNA-dependent heterodimerization. Structural studies of the estrogen receptor DBD and GR-DBD, however, did not clarify the involvement of this region in DNA binding (20.Luisi B. Xu W. Otwinski Z. Freedman L. Yamamoto K. Sigler P. Nature. 1991; 352: 497-505Crossref PubMed Scopus (1210) Google Scholar, 25.Schwabe J.W.R. Neuhaus D. Rhodes D. Nature. 1990; 348: 458-461Crossref PubMed Scopus (345) Google Scholar, 26.Härd T. Kellenbach E. Boelens R. Maler B. Dahlman K. Freedman L.P. Carlstedt-Duke J. Yamamoto K.R. Gustafsson J.Å. Kaptein R. Science. 1990; 249: 157-160Crossref PubMed Scopus (449) Google Scholar, 27.Remerowski M. Kellenbach E. Boelens R. van der Marel G. VanBoom J. Maler B. Yamamoto K. Kaptein R. Biochem. 1991; 30: 11620-11624Crossref PubMed Scopus (25) Google Scholar, 28.Schwabe J.W.R. Chapman L. Finch J.T. Rhodes D. Cell. 1993; 75: 567-578Abstract Full Text PDF PubMed Scopus (586) Google Scholar). The RXR and the TR bind in a “head-to-tail” orientation on a response element with a direct repeat structure (half-site, 5′-AGGTCA-3′), whereas the steroid receptors bind in a “head-to-head” orientation on a response element organized as an inverted repeat (half-site estrogen receptor, 5′-AGGTCA-3′; class I receptors, 5′-TGTTCT-3′). In the present study, the difference in DNA binding between the AR and the GR is analyzed in detail by determining the identity of the amino acids involved and the characteristics of the response element responsible for the exclusion of the GR from binding. All restriction and modifying enzymes were obtained from either Life Technologies, Inc., Amersham Pharmacia Biotech, Promega (Madison, WI), Takara Shuzo Co. Ltd. (Shiga, Japan), Eurogentec (Seraing, Belgium), or Roche Molecular Biochemicals. Oligonucleotides were synthesized on a Biosearch Cyclone DNA synthesizer (Milligen Corp., Bedford, MA) or purchased from Eurogentec. R1881 (methyltrienolone), dexamethasone, aldosterone, and progesterone were purchased from Sigma, [α-32P]dATP from Amersham Pharmacia Biotech, and X-Omat S x-ray films from Eastman Kodak Co. The cDNA encoding the rat AR was described by Chang et al. (29.Chang C. Kokontis J. Liao S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7211-7215Crossref PubMed Scopus (459) Google Scholar), and that for the rat GR was described by Hollenberg et al. (30.Hollenberg S. Giguère V. Segui P. Evans R. Cell. 1987; 49: 39-46Abstract Full Text PDF PubMed Scopus (331) Google Scholar). fAGA is derived from the full-size rat AR by swapping the AR-DBD for that of the rat GR (AR amino acids 537–619) (21.Schoenmakers E. Alen P. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. Biochem. J. 1999; 314: 515-521Crossref Google Scholar). The receptor constructs pCMV5-mAR, pCMV5-rat GR, pCMV5-AGA, and pCMV5-GAG were a kind gift from Dr. D. Robbins (University of Michigan Medical School). In the latter constructs (AGA and GAG), the DBD is exchanged between the AR and the GR (31.Scheller A. Hughes E. Golden K.L. Robins D.M. J. Biol. Chem. 1998; 273: 24216-24222Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The reporter constructs pPBARE2luc and pC3AREluc, described in Schoenmakers et al. (21.Schoenmakers E. Alen P. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. Biochem. J. 1999; 314: 515-521Crossref Google Scholar), each contain two copies of the corresponding ARE: PB-ARE-2, 5′-AGCTTAATAGGTTCTTGGAGTACTTTACGTCGA-3′; C3(1) ARE, 5′-AGCTTACATAGTACGTGATGTTCTCAAGGTCGA-3′ (the ARE hexamers are underlined). The pES vector was generated by the cloning of the SalI(blunt)/BglII tk-promoter fragment of pPBARE2luc into the SmaI/BglII site of pGL3-Basic (Promega). The PB-ARE-1 (5′-AGCTTTATGATAGCATCTTGTTCTTAGTGAGCT-3′; the ARE hexamers are underlined) is cloned in the SacI site of pES, generating pPBARE1luc. pPB2-IR1luc, pDRIluc, and pIDRluc are reporter constructs containing response elements derived from PB-ARE-2 or the perfect direct repeat of 5′-TGTTCT-3 (Table II). TheHindIII(blunt)/BamHI PB promoter fragment (–426 to + 28), a kind gift of Dr. R. Matusik (Vanderbilt Medical Center, Canada), was cloned into the SmaI/BglII site of the pGL3-Basic vector and is referred to as pPBluc.Table IIComparison of the KS values of AR1 and GR1 for mutated PB-ARE-2 sequences and direct repeatsNameDNA sequenceK SAR1GR1nm−7 0 −7PB-ARE-1ATAGCATCTTGTTCT45 (±6)81 (±9)PB-ARE-2GGTTCTTGGAGTACT23 (±5)165 (±10)C3(1)AREAGTACGTGATGTTCT5 (±1)21 (±3)PSA ARE-1AGCACTTGCTGTTCT26 (±4)27 (±3)PB2-IR1AGTACTTGAAGTACT10 (±3)30 (±10)PB2-IR2GGTTCTTGAAGAACC90 (±10)>1000PB2-DR2GGTTCTTGACCTTCT32 (±8)600 (±100)IDRTGTTCTTGAAGAACC>1000>1000DRTGTTCTTGATGTTCT70 (±20)>1000DRIGGTTCTTGATGTTCT33 (±2)330 (±90)DRI4TGTACTTGATGTTCT13 (±2)200 (±20)DRI2TGTTCTTGAGGTTCT>1000>1000DRI3TGTTCTTGAAGTTCT>1000>1000DRI5TGTTCTTGATGTACT110 (±20)>1000Sequences of the oligonucleotides mentioned in the text. TheK S of the dimeric binding complex of the DBD constructs was determined as described under “Experimental Procedures.” The hexamers are underlined. Open table in a new tab Sequences of the oligonucleotides mentioned in the text. TheK S of the dimeric binding complex of the DBD constructs was determined as described under “Experimental Procedures.” The hexamers are underlined. For the prokaryotic expression and purification of the steroid receptor DBDs, the corresponding receptor cDNA-fragments were amplified by polymerase chain reaction and subsequently cloned in the pGEX-2TK expression vector (Amersham Pharmacia Biotech). The fragments were expressed as glutathioneS-transferase fusion proteins in the E. coli BL21 strain. After thrombin cleavage to remove the glutathioneS-transferase fusion partner, the DBDs still contain a short foreign amino acid stretch at the N-terminal end (GSRRASV) as well as at the C-terminal end (IHRD). The AR1 construct consists of rat AR-DBD amino acids 533–637; the GR1 construct comprises the corresponding rat GR-DBD amino acids 432–533. The different mutated GR1 and AR1 constructs generated are described in Fig. 2. The different receptor fragments were purified as described by Schoenmakers et al. (21.Schoenmakers E. Alen P. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. Biochem. J. 1999; 314: 515-521Crossref Google Scholar). The protocol for the preparation of nuclear extracts has been described by Andrews and Faller (32.Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2207) Google Scholar) and was used with some modifications. Briefly, 106 COS-7 cells were plated in 10 cm Petri dishes and transfected with 2.5 μg of expression plasmid. The cells were incubated (1 h) before harvesting with 10−5m hormone, after which the medium was removed, and the cells were washed twice with 3 ml of ice-cold phosphate-buffered saline. The cells were collected in 1.5 ml of ice-cold phosphate-buffered saline per dish, transferred to an Eppendorf tube, and pelleted by centrifugation (10 s). The phosphate-buffered saline was removed, and the cells were resuspended in 400 μl of ice-cold buffer containing 10 mmHepes·KOH, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, and 0.2 mm phenylmethylsulfonyl fluoride. After 10 min of incubation on ice, the mixture was vortexed (30 s), and the nuclei were collected by a short spin in a microcentrifuge. The supernatant was removed, and the nuclei were resuspended in 100 μl of ice-cold high salt buffer: 20 mm Hepes·KOH, pH 7.9, 1.5 mmMgCl2, 420 mm KCl, 25% glycerol, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride. After 20 min of incubation on ice and a short vortexing (10 s), the extracts were cleared by centrifugation (12,000 ×g) at 4 °C for 2 min. The supernatant was then frozen in liquid nitrogen and stored at −80 °C. Western blotting was performed on these extracts as described (33.Alen 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). The double-stranded oligonucleotides containing the ARE sequences (described in Table II) were labeled with [α-32P]dATP by a fill-in reaction by the Klenow fragment of DNA-polymerase to a specific activity of 5000 cpm/fmol. The response elements with a direct repeat sequence organization are derived from the DR element 5′-AGCTTTCATTGTTCTTGATGTTCTGAATGAGCT-3′ (TableII). The dissociation constants were determined by means of gel shift assays as described by Schoenmakers et al. (21.Schoenmakers E. Alen P. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. Biochem. J. 1999; 314: 515-521Crossref Google Scholar). In short, a constant amount of labeled oligonucleotide was incubated with increasing amounts of purified receptor fragment until complete retardation of the probe was obtained. The K S value of each construct was calculated from the percentage of retarded probe with the formula of Hill kinetics based on at least three independent assays. For the gel shift assays with the cell extracts containing the full-size receptor, constant amounts (20,000 cpm) of labeled double-stranded oligonucleotides were incubated (20 min on ice) with equal amounts of nuclear extracts in 20 μl of binding buffer (10 mm Hepes·KOH, pH 7.9, 2.5 mmMgCl2, 0.05 mm EDTA, 10% glycerol, 1 μg of poly(dI-dC), 0.05% Triton X-100, l mm dithiothreitol). Subsequently, free and bound probe were separated by electrophoresis for 120 min at 4 V/cm in a 4% nondenaturing polyacrylamide gel. In competition gel shift assays, 300-fold excess of cold C3(1) ARE was incubated on ice with the receptor mixture for 10 min prior to the addition of the labeled oligonucleotide. Specific antibodies against the N-terminal part of the AR (34.Marivoet S. Hertogen M. Verhoeven G. Heyns W. J. Steroid Biochem. Mol. Biol. 1990; 37: 39-45Crossref PubMed Scopus (12) Google Scholar) and the GR (kindly provided by Prof. Gustafsson, Karolinska Institute, Stockholm, Sweden) were used as described (35.Gustafsson J.Å. Wikstrom A.C. Denis M. J. Steroid. Biochem. 1989; 34: 1-6Crossref Scopus (10) Google Scholar) to obtain a supershift. COS-7 cells were obtained from the American Type Culture Collection and were routinely maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) containing 1 g/liter glucose, supplemented with antibiotics (penicillin, 100 IU/ml; streptomycin, 100 μG/ml; Life Technologies) and 10% heat-inactivated fetal bovine serum. For the transient transfection assays, the cells were cultured in DMEM containing 5% dextran-coated, charcoal-stripped fetal bovine serum (DCC). Reporter plasmids and the expression vectors for the receptor constructs were co-transfected by means of the FuGENE 6 transfection reagent (Roche Molecular Biochemicals). A β-galactosidase expression plasmid (CMV-β-galactosidase, Stratagene) was used as internal control of the transfection efficiency. For the transfection assay 15 ng of reporter plasmid, together with 5 ng of receptor expression vector, 20 ng of CMV-β-galactosidase, and 0.1 μg of carrier DNA (pGEM-7), were mixed with 20 μl of DMEM containing 0.2 μl of FuGENE transfection reagent. The mixture was incubated for 30 min at room temperature and then added dropwise onto the cells (96-well culture dish containing 5 × 103 cells in 200 μl of DMEM). After overnight incubation at 37 °C, the medium was replaced by DMEM containing 5% DCC, and the cells were grown in the absence or presence of 1 nm R1881, dexamethasone, aldosterone, or progesterone for 48 h. Luciferase activity was measured with the luciferase assay system from Promega, and β-galactosidase activity was measured with the detection system from CLONTECH. The difference in PB-ARE-2 recognition between the AR and GR was first observed using only their DBDs (named AR1 and GR1) (12.Claessens F. Alen P. Devos A. Peeters B. Verhoeven G. Rombauts W. J. Biol. Chem. 1996; 271: 19013-19016Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 21.Schoenmakers E. Alen P. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. Biochem. J. 1999; 314: 515-521Crossref Google Scholar). To analyze the influence of the other receptor domains on the difference in DNA binding, chimerical full-size AR and GR constructs with swapped DBDs (AGA, GR-DBD residues 449–533; GAG, AR-DBD residues 551–637) (31.Scheller A. Hughes E. Golden K.L. Robins D.M. J. Biol. Chem. 1998; 273: 24216-24222Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), were used. The DNA binding capacities of these chimerical receptors were compared with these of the AR and GR in gel shift assays with nuclear extracts of transfected COS-7 cells. The C3(1) ARE was recognized by all receptor constructs with similar affinity (Fig. 1 A). The specificity of DNA binding was verified by competition assays using cold C3(1) ARE and by the appearance of a supershifted complex in the presence of a specific anti-AR or anti-GR antibody. The PB-ARE-2 was bound with high affinity by both AR-DBD-containing proteins AR and GAG. In contrast, neither the GR nor the AGA bound this ARE with high affinity, albeit that the antibodies induce a small amount of supershifted binding complexes. In transient cotransfection assays, the pC3AREluc reporter construct is induced 4–5-fold by all the chimerical receptors, indicating the functionality of the receptor proteins (Fig. 1 B). Most remarkable is the transactivation of the PB-ARE-2-containing reporter construct by GAG to a comparable level as the wild type AR, whereas the wild type GR does not enhance the transcription. The AGA chimera is still able to transactivate pPBARE2luc in response to androgens, although the induction factor (2.5) is very low. Western blot analysis of the chimeras (Fig. 1 C) shows a slightly higher level of expression of AGA and GAG compared with respectively AR and GR. In our earlier study, we demonstrated that the first zinc finger of the AR is not involved in the specific recognition of the PB-ARE-2 (21.Schoenmakers E. Alen P. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. Biochem. J. 1999; 314: 515-521Crossref Google Scholar). However, experiments with the chimerical DBD constructs G/A and A/G (Fig.2 and Ref. 21.Schoenmakers E. Alen P. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. Biochem. J. 1999; 314: 515-521Crossref Google Scholar) demonstrate that for the AR, residues of the hinge region as well as of the N-terminal part of the second zinc finger are involved. We therefore analyzed the importance of all residues that differ between the AR-DBD and the GR-DBD in these two regions. We have already described the necessity of a C-terminal extension of 12 amino acids for specific and high affinity binding of the AR to the PB-ARE-2 (21.Schoenmakers E. Alen P. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. Biochem. J. 1999; 314: 515-521Crossref Google Scholar). With the constructs AR28.1 and 28.2 (TableI), we could confirm the involvement of the 12 CTE residues in high affinity DNA binding. These constructs are the result of a deletion of residues forming the nuclear localization signal, which partially overlaps with the CTE (36.Jenster G. Trapman J. Brinkman A. Biochem. J. 1993; 293: 761-768Crossref PubMed Scopus (211) Google Scholar). Note that AR28.1 and 28.2, kindly provided by Dr. A. O. Brinkmann, are derived from the human AR, which differs from the rat AR at three residues in the extreme C terminus of the receptor fragment. Deletion constructs showed, however, that deletion of this part of the protein had no influence on the DNA binding by the rat AR-DBD (21.Schoenmakers E. Alen P. Verrijdt G. Peeters B. Verhoeven G. Rombauts W. Claessens F. Biochem. J. 1999; 314: 515-521Crossref Google Scholar). Six of the 12 CTE amino acids differ between the AR and the GR. Systematic mutations of the AR-specific residues to the GR homologues resulted in a number of AR1 mutants, schematically illustrated in Table I. The apparent dissociation constants of these constructs for the PB-ARE-2 and C3(1) ARE indicate that mainly the mutation of Gly-610 (and to a lower extent, mutation of Leu-617) abrogates the PB-ARE-2 binding.Table IMutations in the CTE and surrounding residues of the AR hinge region fragmentNameAmino acid sequenceK Snm599 CTE 637PB-ARE-2C3(1) AREAR1LRCYEAGMTLGARKLKKLGNLKLQEEGBNSSAGSPTED23 (±5)5 (±1)AR(28.1)*************——*************TT****E>1000120 (±5)AR(28.2)************——–M*********TT****E>1000>1000AR608N*********N*****************************14 (±3)5 (±1)AR610E***********E***************************340 (±60)4 (±1)AR614T***************T***********************16 (±1)4 (±1)AR617K/622Q******************K****Q***************60 (±10)4 (±1)AR618I/622Q*******************I***Q***************19 (±5)5 (±1)AR619K/622Q********************K**Q***************20 (±1)7 (±1)Schematic representation of the AR1 derived constructs with deletions or mutations in the CTE or the NLS with their K S for the PB-ARE-2 and the C3(1) ARE. Asterisks indicate that no residues have changed compared to AR1, dashes give the deleted residues, and mutated amino acids are named. The amino acids composing the NLS are indicated in italics. AR(28.1) and AR(28.2) are derived from the human AR, which has three different residues in the C-terminal part of the hinge region fragment, compared to the rat AR. Open table in a new tab Schematic representation of the AR1 derived constructs with deletions or mutations in the CTE or the NLS with their K S for the PB-ARE-2 and the C3(1) ARE. Asterisks indicate that no residues have changed compared to AR1, dashes give the deleted residues, and mutated amino acids are named. The amino acids composing the NLS are indicated in italics. AR(28.1) and AR(28.2) are derived from the human AR, which has three different residues in the C-terminal part of the hinge region fragment, compared to the rat AR. In the C-terminal part of the second zinc finger of the GR-DBD, an α-helix (amino acids 492–503) was described (20.Luisi B. Xu W. Otwinski Z. Freedman L. Yamamoto K. Sigler P. Nature. 1991; 352: 497-505Crossref PubMed Scopus (1210) Google Scholar, 26.Härd T. Kellenbach E. Boelens R. Maler B. Dahlman K. Freedman L.P. Carlstedt-Duke J. Yamamoto K.R. Gustafsson J.Å. Kaptein R. Science. 1990; 249: 157-160Crossref PubMed Scopus (449) Google Scholar). In the ARHm construct, all the nonhomologous residues of the α-helical structure in the C-terminal side in the AR-DBD (amino acids 594–605) were swapped for those of the GR (Fig. 2). This construct had DNA binding capacities similar to those of the wild type AR1, indicating that the putative helix in the C-terminal region of the second zinc finger had no influence on the difference in DNA sequence recognition between the AR and the GR. However, for high affinity DNA binding to PB-ARE-2, a correct conformation of the helix is necessary. This is illustrated by the mutation of Leu-599 of the AR to the GR homologue Tyr-497 (AR599Y) (Fig. 2). In the x-ray structure of the GR-DBD, Tyr-497 and Leu-501 are oriented toward each other. When a tyrosine is present at both places, as in AR599Y, steric hindrance might prevent the correct folding of the helix, influencing the overall DBD-conformation. We tested this hypothesis by combining the Leu-599 to Tyr with the Tyr-603 to Leu mutation in AR1. As expected, the AR599Y/603L regained its affinity for the PB-ARE-2 leading to the conclusion that Tyr-497 and Leu-501 in the GR are two complementary residues. This is in contrast with the mutated AR construct, AR599Y/610E, which has lost its affinity for PB-ARE-2. The exchange of another nonconserved residue in the putative α-helix of AR1, namely Glu-604, for that of the GR homologue glutamine had no effect on the DNA specificity. To identify the other residues in the second zinc finger necessary for the AR-specific PB-ARE-2 binding, we introduced the GR homologues in the AR1. However, none of the mutated AR1 constructs displayed a dramatic decrease in affinity for PB-ARE-2 or C3(1) ARE (data not shown). Therefore, we took an alternative approach based on the observation that the G/A chimera, containing only part of the second zinc finger and the hinge region of the AR, has a low affinity for the PB-ARE-2. In an attempt to restore the binding to the PB-ARE-2, mutations were introduced in G/A (Fig. 2). The apparent dissociation constants of these constructs indicate that Thr-585 (position 483 in GR1) might be involved in high affinity binding of the AR to PB-ARE-2. In TableII, synthetic oligonucleotides containing either inverted or direct repeats of the left or right half-site of the PB-ARE-2 are listed (PB2-IR1, PB2-IR2, and PB2-DR2). The apparent" @default.
• W2077854579 created "2016-06-24" @default.
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• W2077854579 title "Differences in DNA Binding Characteristics of the Androgen and Glucocorticoid Receptors Can Determine Hormone-specific Responses" @default.
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