Androgen receptor mRNA was translated in vitro, and androgen- and antiandrogen-bound receptor complexes were studied using limited proteolytic digestion by trypsin. Partial proteolysis of androgen-bound receptor protein resulted in a 29-kDa proteolysis-resisting fragment, whereas antiandrogen binding stabilised a 35-kDa fragment. Both fragments contain the entire ligand binding domain, and the 35-kDa fragment extended into the hinge region of the receptor. Several antiandrogens show agonistic properties for a mutated androgen receptor (LNCaP cell variant); trypsin digestion of antiandrogen-bound mutated receptor also resulted in a 29-kDa fragment. Our results point to an important difference between antiandrogens and antagonists of other steroid hormone receptors. Antiandrogens result in protection of both the hinge region and C-terminus of the androgen receptor against proteolytic attack, whereas other studies showed that antiestrogens and antiprogestagens expose the C-terminal end of the ligand binding domain of their respective receptors to protease. Differences in conformation of the hinge region distinguish androgen-bound from antiandrogen-bound receptor complexes, which represents an important feature of antiandrogen action

Kuil, C.W. (Cor)

Mulder, E. (Eppo)

Erasmus University Digital Repository

EISEVIER Molecular and Cellular Endocrinology 102 (1994) Rl-R5 n C E ibcular and + . -If Rapid Paper Mechanism of antiandrog~n action: conformational changes of the receptor C.W. Kuil *, E. Mulder Department of Endocrinology & Reproduct&, Erasmus Universiir Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, Netherlands (Received 7 January 1994, accepted 4 February 1994) Abstract Androgen receptor mRNA was translated in vitro, and androgen- and antiandrogen-bound receptor complexes were studied using limited proteotytic digestion by trypsin. Partial proteolysis of androgen-Lund receptor proteiu resulted in a 29-kDa proteolysis-resisting fragment, whereas antiandrogen binding stabilised a 3%kDa fragment. Both fragments contain the entire ligand binding domain, and the 35kDa fragment extended into the hinge region of the receptor. Several antiandrogens show agonistic properties for a mutated androgen receptor (LNCaP cell variant); trypsin digestion of antiandrogen-bound mutated receptor also resulted in a 29-kDa fragment. Our results point to an important difference between antiandrogens and antagonists of other steroid hormone receptors. Antiandrogens result in protection of both the hinge region and C-terminus of the androgen receptor against proteolytic attack, whereas other studies showed that ~tiestrogens and antiprogestagens expose the C-terminal end of the ligand binding domain of their respective receptors to protease. Differences in conformation of the hinge region distinguish androgen-bound from antiandrogen-bound receptor complexes, which represents au important feature of antiandro- gen action. lvey words: Androgen receptor; Antiandrogen; LNCaP cell; Prostate; Steroid hormone 1. Introduction The study of the mechanism of antiandrogen action is of great interest, not only because of the therapeutic potential of antiandrogens, but also because these compounds are important tools to elucidate the molec- ular mechanism of action of androgens. Androgens initiate effects in target cells through a ligand-activated transcription factor, the androgen receptor (AR). Like all members of the steroid hormone receptor family, the AR binds to its responsive element after a ligand- dependent activation process and interacts with the transcription complex to regulate gene transcription (Carson-Jurica et al., 1990; Smith and Toft, 1993). Antiandrogens compete with androgens for binding to the AR, but binding of antagonists does not result in full transfo~ation of the receptor to a transc~ption- ally active form. Several mechanisms for the resulting inhibitory effect have been postulated, and recently a * Corresponding author. Tel.: 3110~08733~ Fax: 3110-43~832. subdivision of antagonists in two distinct classes has been made (Reese and Katzenellenbogen, 1991; Klein- Hitpass et al., 1991; Gronemeyer et al., 1992). One class of antagonists does not, or does so with decreased efficiency, promote DNA binding of the receptor, whereas the other class of antagonists promotes DNA binding but induces an abnormal conformation of the ligand-binding domain. The latter class of antagonists may give rise to a partial agonistic effect, through a transcription activation function in the N-terminal do- main of the receptor. For the progesterone receptor it was recently shown that binding of progestagens and antiprogestagens re- sults in different susceptibility of the receptor to prote- olytic digestion. Antagonists induced protection of a smaher progesterone receptor fragment than did ago- nists (Allan et al., 1992a,b), and studies with antibodies indicated that a short region at the C-terminal end of the progesterone receptor is involved in this difference (Allan et al., 1992a; Weigel et al., 19921, The presented data concern differences in suscepti- bility of androgen- and antiandrogen-fund receptor complexes to proteolytic digestion. 0303-7207/94/$07.00  1994 Elsevier Science Ireland Ltd. All rights resewed SSDI 0303-7207(94)00035-8 R2 C. II! Kuil, E. Mulder / Molecular and Cellular Endocrinology 102 (1994) RI -R5 2. Materials and methods 2.3. In vitro transcription and translation 2.1. Materials RNA transcription kit and pBluescript II KS- were obtained from Stratagene (La Jolla, USA). Nuclease- treated rabbit reticulocyte lysate was purchased from Promega (Madison, USA). Lj3’S]methionine @+.a. > 1000 mCi/mM) was obtained from Amersham (Buckinghamshire, UK). Trypsin (type III), soybean trypsin inhibitor (type I-S>, goat-anti-mouse agarose and goat-anti-rabbit agarose were obtained from Sigma (St. Louis, USA). R1881 (methyltrienolone) was pur- chased from NEN (Boston, USA). Cyproterone acetate was a gift from Schering AG (Berlin, Germany), hy- droxyflutamide from Schering USA (Bloomfield, USA) and ICI 176.334 (“Casodex”) from ICI Pharmaceuti- cals (Macclesfield, UK). Dihydrotestosterone and testosterone were obtained from Steraloids (Wilton, USA). 2.2. Plasmid construction The coding sequence for the wild-type human AR (ARO) was excised from the expression vector pSVAR0 (Brinkmann et al., 1989) and subcloned in the Sal1 site of pBluescript to obtain pBSAR0. The recombinant pBSAR0 615-910 was obtained from pBSAR0 after digestion with Sac1 and religation. The recombinants were linearized with XhoI for transcription. The cod- ing sequence for the mutant LNCaP AR CARL) was subcloned between the Sal1 and BamHI sites of pBluescript (pBSARL). For linearization, the recombi- nant was digested with XbaI. A Both in vitro transcription and in vitro translation in the presence of r_-[3sS]methionine were performed ac- cording to manufacturer’s instruction. For in vitro tran- scription of pBSAR0 and pBSAR0 615-910, T, RNA polymerase was used to produce sense mRNA, whereas pBSARL was transcribed with T, RNA polymerase. 2.4. Limited proteolytic digestion of in vitro-produced receptors Two ~1 of labeled translation mix was pre-incubated for 1 h at room temperature with 3 ~1 of ligand solution diluted in water. For limited proteolytic diges- tion, 5 11 trypsin (40 pg/ml, dissolved in water) was added to the pre-incubation mix, followed by an incu- bation for 15 min at room temperature. After incuba- tion, 20 ~1 SDS sample buffer was added. Samples were boiled for 3 min, and 15 ~1 was loaded onto 0.1% (w/v) sodium dodecyl sulfate-12.5% (w/v> polyacryl- amide gels. After electrophoresis (Laemmli, 1970), the gels were vacuum-dried at 80°C for 45 min and autora- diography was performed overnight. 2.5. Immunoprecipitation Labeled translation mix (20 ~1) was hormone-treated and digested with trypsin as indicated above. After digestion, soybean trypsin inhibitor was supplemented to a final concentration of 200 pg/ml. Goat-anti-rabbit or goat-anti-mouse agarose (100 ~1, diluted 1:4 in phosphate-buffered saline (PBS)) was incubated for 2 h at 4°C with 1 ~1 of the indicated polyclonal rabbit or B _ - 61 .,I:.. : ;:. * ! :d, : Fig. 1. Limited proteolytic digestion of in vitro-produced AR (A) and AR0 615-910 (B) bound with different androgens and antiandrogens. Lane 1: no trypsin added. Lane 2: control digestion without steroid (-1. Lanes 3-7: 10 nM R1881, 1 PM cyproterone acetate (CA), 10 PM hydroxyfiutamide (OH-F), 10 WM ICI 176.334 (ICI 3341, and 10 nM dihydrotestosterone (DHT), respectively. Molecular mass markers are indicated at the right. * indicates a non-specific band. C. W. Kuil, E. Mulder / Molecular and Cellular Endocridogy 102 (1994) RI 4.5 R3 monoclonal mouse antiserum. The polyclonal anti- serum SP066 (epitope amino acids 892-910) and mon- oclonal antiserum F52 (epitope amino acids 593-612) were described previously (Zegers et al., 1991; Veld- scholte et al., 1992). Following this incubation, the resin was washed three times with 1 ml PBS and added to the limited proteolytic digest of the receptor. After incubation for 2 h at 4 “C, the resin was washed three times with 1 ml PBS, and 25 ~1 of sample buffer was added. Electrophoresis was performed as described above. 3. Results and discussion Limited proteolytic digestion of [ 35S]methionine la- beled, in vitro-produced AR was used to detect confar- mational changes upon androgen or antiandrogen binding to the receptor. The in vitro-produced AR showed steroid binding properties similar to those ob- served for AR isolated from mammalian cells (Kuiper et al., 1993). After incubation of the AR with androgen or antiandrogen, a limited amount of trypsin was added and the digestion products were analyzed by denatur- ing gel electrophoresis. Either in the absence of ligand (Fig. 1A) or in the presence of a steroid with no affinity for the AR (dexamethasone; result not shown), the AR was completely digested to fragments that were unde- tectable with electrophoresis. Proteolytic digestion of AR incubated either with the synthetic androgen R1881 or the natural ligands dihydrotestosterone and testos- terone, resulted in a 29-kDa proteolysis-resisting frag- ment (Fig. lA, result for testosterone not shown). Incubation of AR with the antiandrogens cyproterone acetate, hydroxyflutamide, or ICI 176.334 before tryp- tic digestion resulted in stabilisation of a 35kDa frag- ment (Fig. 1A). The concentrations of the different ligands used varied according to their differences in relative binding affinities of the ligands for the AR (Veldscholte et al., 1992). Formation of a 29-kDa fragment was the result of binding of an agonist, whereas stabilisation of a 35-kDa fragment indicated binding of an antagonist. When increasing concentrations of the antiandrogen ICI 176.334 were added together with a constant level of R1881, digestion with trypsin resulted in less an amount of the 29-kDa fragment and an increased amount of the 35-kDa fragment (Fig. 2). As predicted from the relative binding affinities of the ligands, 50% binding inhibition of 1 nM R1881 occurred at 1 PM ICI 176.334. When the concentration of R1881 was in- creased, the 29-kDa fragment reappeared (Fig. 2). Several in vitro-produced AR fragments, including a 35-kDa fragment, were already present before the start of the proteolytic digestion, probably due to alternative translation initiation. Therefore, the effect of trypsin R1881 (nM) - - 1 1 1 1 1 - 100 ICI 334(uM) - - - 0.01 0.1 1 10 10 10 kDa Fig. 2. Competition of 1 nM R1881 with increasing levels of ICI 176.334 (ICI 334). Ligands were bound to the in vitro-produced AR before digestion with trypsin. Lane 1: no trypsin added. Lane 2: control digestion without steroid. Lanes 3-9: indicated levels of R1881 and/or ICI 176.334. Molecular mass markers are indicated at the right. * indicates a non-specific band. on the 35-kDa fragment was studied in detail in sepa- rate experiments. A mutant AR cDNA, deleted of the N-terminal and DNA-binding domains (AR0 615-9101, was translated in vitro into a predominant 35-kDa protein (Fig. 1B). Also for this truncated receptor protein, binding of either R1881 or dihydrotestos- terone resulted in formation of a 29-kDa fragment upon proteolytic digestion, whereas the antiandrogens predominantly stabilised the 35-kDa fragment. These results show that formation of the 29-kDa fragment is not dependent on interaction of the ligand binding domain with the N-terminal and DNA-binding do- mains. From these experiments it can be concluded that trypsin treatment of androgen- or antiandrogen-bound AR results in different proteolysis-resisting fragments which suggest a different structural conformation. Comparable, but not similar, observations have been made for the progesterone and estrogen receptors (Vegeto et al., 1992; Allan et al., 1992a; Beekman et al., 1993). In contrast with the results found for the AR, antagonist binding to these receptors resulted in smaller proteolysis-resisting fragments than obtained after agonist binding. Immunoprecipitation was performed to determine which part of the AR was removed upon conversion of the 35-kDa fragment into the 29-kDa fragment. After incubation of the full-length AR with ligand, followed by limited proteolytic digestion with trypsin, the frag- ments resisting proteolysis were immunoprecipitated with different antisera (Fig. 3). Neither the 35-kDa fragment nor the 29-kDa fragment could be immuno- precipitated with the antiserum F52 which recognizes an epitope in the DNA-binding domain (Zegers et al., 1991). Therefore, this epitope appears not to be pre- sent in either fragment. The antiserum SP066, raised against a peptide at the C-terminus of the AR, recog- nizes both fragments, which indicates that the differ- ence in size of the 29-kDa and 35-kDa fragments is not located at the C-terminus of the AR. R4 C. W; Kuil, E. Mulder /Molecular and Cellular Endocrinology 102 (1994) RI -R5 These results indicate that, in the AR, a part of the hinge region is protected against degradation by trypsin in the presence of antiandrogens. This is in contrast with the results obtained for the progesterone recep- tor, where the C-terminus was only retained during proteolytic digestion in the presence of an agonist (AIlan et al., 1992a; Weigel et al., 1992). Another study (Vegeto et al., 1992) showed that a truncated proges- terone receptor, with a C-terminal deletion of 42 amino acid residues, could not bind a progestagen but still bound the antiprogestagen RU486. In addition, RU486 was able to act as an agonist in a transcription activa- tion assay. LNCaP prostate tumor cells contain an AR (ARL) with a single amino acid change in the steroid binding domain (codon 868; Thr-Ala (Veldscholte et al., 1990)). Both cyproterone acetate and hydroxyflutamide act as agonists for the ARL but ICI 176.334 still behaves as an anti~drogen with the ARL (Velds~holte et al., 19921. ARL is therefore a useful tool to study the mechanism of action of antagonists. The in vitro-pro- duced ARL was treated with the same androgens and antiandrogens as described above for the wild-type AR (Fig. 4). For R1881, dihydrotestosterone and ICI 176.334, the results for ARL were comparable with those obtained with the wild-type AR. However, the antiandrogens cyproterone acetate and hydroxyflu- tamide, which gave rise to a 35kDa fragment with the wild-type AR, both induced formation of a 29-kDa fragment with the ARL. As these antiandrogens act as agonists for the ARL, it can be assumed that the single amino acid change in the ARL made it possible for R1881 OH-F nnnn kDa - 110 - 61 - 35 -. _ -- L” Fig. 3. Immunoprecipitation of AR fragments with different antisera. After treatment with vehicle (-; lanes 3-41, 100 nM R1881 (lanes 5-6) or 100 nM hydroxyflutamide (OH-F, lanes 7 and 81, the AR was digested with trypsin. Lanes 1 and 2 were controls without trypsin. After digestion, immunopre~ipitation was performed with the mono- cional antiserum F52 (lanes 1,3,5 and 7) or the polyclonal antiserum SP066 (lanes 2, 4, 6 and 8). Molecular mass markers are indicated at the right. kDa - 110 - 61 * - 35 * 20 Fig. 4. Limited proteolytic digestion of in vitro-produced mutant ARL bound with different androgens and antiandrogens. Lane I: no trypsin added. Lane 2: control digestion without steroid (-I. Lanes 3-7: 10 nM R1881,l &I cyproterone acetate (CA), 10 PM hydroxy- flutamide (OH-F), 10 PM ICI 176.334 (ICI 3341, and 10 nM dihy- drotestosterone (DHT), respectively. Molecular mass markers are indicated at the right. * indicates a non-specific hand. both antiandrogens to induce a comparable conforma- tion as formed after the interaction of an androgen with the wild-type AR. In conclusion, androgens and antiandrogens induce a different change in the conformation of the AR as detected by proteolytic digestion. This appears to in- volve the hinge region of the receptor, which is in contrast with studies on other steroid receptors. Allan et al. (1992a) proposed a general action of steroid receptor antagonists in preventing the formation of a transcriptionally competent conformation of the C- terminal end of the ligand binding domain. Conforma- tional changes in the C-terminal end of the AR were not detected by trypsin treatment although several consensus sites for trypsin digestion are present. The results therefore suggest a difference in mechanism of antiandrogen action compared to other steroid hor- mone receptor antagonists. Acknowledgements We thank Dr. J.A. Grootegoed and Dr. J. Veld- scholte for helpful discussions and critical reading of the manu~ript. References Allan, G.F., Leng, X., Tsai, S.Y., Weigel, N.L., Edwards, D.P., Tsai, M.J. and 0 Malley, B.W. (1992aI. J. Biol. Chem. 267, 19513- 19520. Allan, G.F., Tsai, S.Y., Tsai, M.-J. and 0’ Malley, B.W. (1992b). Proc. Nat. Acad. Sci. USA 89, 11750-11754. Baniahmad, A. and Tsai, M.-J. (1993). J. Ceil. Biochem. 51, 351-1.56. Smith, D.F. and Toft, D.O. (1993). Mol. Endocrinol. 7,4-H. Beckman, J.M., Aflan, G.F., Tsai, S.Y., Tsai, M.-J. and O’MalIey, Vegeto, E., Allan, G.F., Schrader, W.T., Tsai, M.-J., McDonnell, B.W. (1993). Mol. Endocrinol. 7, 1266-1274. D.P. and G’Maliey, B.W. (1992). Cell 69, 703-713. Brinkmann, A.O., Faber, P.W., van Rooij, H.C.J., &riper, G.G.J.M., Veldscholte, J., Ris-Stalpers, C., Kuiper, G.G.J.M., Jenster, G., Ris, C., Klaassen, P., van der Korput, J.A.G.M., Voorhorst, Berrevoets, C., Claassen, E., Van Rooij, H.C.J., Trapman, J., M.M., van Laar, J.H., Mulder, E. and Trapman, J. (1989). J. Brinkmann, A.O. and Mulder, E. (1990). Biochem. Biophys. Res. Steroid Biochem. 34, 307-310. Commun. 173,534~540. Carson-Jurica, M.A., Schr%ler, W.T. and O’Malley, B. (1990). En- doer. Rev. l&201-220. Gronemeyer, H., Benhamou, B., Berry, M., Bocquel, M.T., Gofflo, D., Garcia, T., Lerouge, T., Metzger, D., Meyer, M.E., Tora, L., Vergezac, A. and Chambon, P. (1992). J. Steroid B&hem. Mol. Biol. 41, 217-221. Veldschotte, J., Berrevoets, CA., Brinkmann, A.O., Grootegoed, J.A. and Mulder, E, (1992). Bi~hemist~ 31,2393-2399. Veldscholte, J., Berrevoets, C.A., Zegers, N.D., van der Kwast, Th.H., Grootegoed, J.A. and Mulder, E. (1992). Biochemistry 31, 7422-7430. Klein-Hitpass, L., Cato, A.C.B., Henderson, D. and Ryffel, G.U. (1991). Nucleic Acids Res. 19, 1227-1234. Kuiper, G.G.J.M., de Ruiter, P.E., Trapman, J., Jenster, G. and Brinkmann, A.0.(1993). Biochem. J. 296, 161-167. Laemmli, U.K. (1970). Nature 227, 680-685. Reese, J.C. and Katzenellenbogen, B. (1991). Nucleic Acids Res. 19, 6595-6602. Weigel, N.L., Beck, C.A., Estes, P.A., Prendergast, P., Altmann, M., Christensen, K. and Edwards, D.P. (1992). Mol. Endocrinol. 6, 1585-1597. Zegers, N.D., Ctaassen, E., Neelen, C., Mulder, E., van Laar, J. Voorhorst, M., Berrevoets, C.A., Brinkmann, A.O., van der Kwast, Th.H., Ruizeveld de Winter, J.A., Trapman, J. and Boersma, W.J.A. (1991). Biochim. Biophys. Acta 1073, 23-32. C. K Kuil, E. Mulder /Molecular and Cellular Endocrinology 102 (1994) RI-R5 R5 

Mechanism of antiandrogen action: Conformational changes of the receptor

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Mechanism of antiandrogen action: Conformational changes of the receptor

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