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Reconstitution of the N-terminal Transcription Activation Function of Human Mineralocorticoid Receptor in a Defective Human Glucocorticoid Receptor*

  • Manjapra V. Govindan
    Correspondence
    To whom correspondence should be addressed. Tel.: 418-691-5532; Fax: 418-691-5439;
    Affiliations
    Centre Recherche Hôtel-Dieu Québec and Laval University, Côte du Palais, Québec G1R 2J6, Canada
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  • Nalini Warriar
    Footnotes
    Affiliations
    Centre Recherche Hôtel-Dieu Québec and Laval University, Côte du Palais, Québec G1R 2J6, Canada
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  • Author Footnotes
    * This work was supported in part by Medical Research Council Grant MT-14098 (to M.V.G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.This paper is dedicated to Dr. Paul Loew-Beer of Loba Chimie, Vienna, Austria, for his undaunted support and generosity.
    § Recipient of a studentship from the Foundation of Laval University.
Open AccessPublished:September 18, 1998DOI:https://doi.org/10.1074/jbc.273.38.24439
      N-terminal sequences involved in transcription activation by the human mineralocorticoid receptor (hMR) have yet to be defined. We have addressed this issue and generated overlapping internal deletion mutants hMRΔ59–162, hMRΔ59–247, hMRΔ59–328, hMRΔ162–247, hMRΔ247–328,hMRΔ247–382, and hMRΔ328–382 with intact DNA-binding and hormone-binding domains. A second set of mutant receptors with unique BglII sites was generated to facilitate the isolations of fragments. Immunodetection with anti-hMR peptide antibodies and hormone-binding assays showed that the mutations did not affect the expression of the receptors or ability to bind aldosterone. Distribution of aldosterone binding activity of wild type and deletion mutants expressed in HeLa cells was predominantly nuclear. Furthermore, deletion of sequences between 59 and 390 did not affect DNA binding activity. Transfection studies with HeLa cells revealed a region around residue 247 that was crucial for normal receptor function. Deletion of amino acids 59–162 did not affect the transcriptional activity of the hMR. However, deletion of sequences 247–382 and 328–382 markedly decreased the transcription activation function. The induction of the reporter gene by the chimera hGRΔ71–262/hMR328–382 was 2-fold higher than with the wild type hGR, but 200-fold when compared with hGRΔ71–262, indicating that the AF-1 domain is located between positions 328 and 382 in the hMR.
      hMR
      human mineralocorticoid receptor
      hGR
      human glucocorticoid receptor
      hER
      human estradiol receptor
      HBD
      hormone-binding domain
      DBD
      DNA-binding domain
      MMTV
      mouse mammary tumor virus
      CAT
      chloramphenicol acetyltransferase
      PBS
      phosphate-buffered saline
      DCC-FBS
      fetal bovine serum stripped with dextran-coated charcoal
      MRE
      mineralocorticoid response element.
      In humans, the biologically active mineralocorticoid aldosterone maintains the homeostasis of ion balance principally in the kidney, but also in the gut, salivary, and sweat glands. The evolution of the modular structure of nuclear receptors into distinct domains is largely the consequence of complex requirements that arose during cellular growth and development. The structures of the human mineralocorticoid receptor (hMR)1 and the human glucocorticoid receptor (hGR) are very similar. The N-terminal region in this family of nuclear receptors is of variable length and contains a transactivation function, the AF-1 (
      • Arriza J.L.
      • Weinberger C.
      • Cerelli G.
      • Glaser T.M.
      • Handelin B.L.
      • Hausman D.E.
      • Evans R.M.
      ). In the absence of a hormone-binding domain (HBD), this region is constitutively active. The N-terminal regions of hGR and hMR have only 15% homology, and it is this domain that is responsible for differences in target gene specificity.
      In the hGR, the N-terminal activation domain is 185 amino acids long and a 58-amino acid peptide that is almost as active as the intact region has been identified (
      • Dahlman-Wright K.
      • Almlof T.
      • McEwan I.J.
      • Gustafsson J.-A.
      • Wright A.P.H.
      ). The experiments with hGR were, however, performed with constructs containing segments of the hGR expressed in yeast and not in mammalian cells, and the cooperative function of the hGR HBD was not taken into consideration in these analyses. Such regions in the hMR remain to be defined. Our experiments were performed in animal cells in culture, and the intact DNA-binding domain (DBD) and HBD of the hMR or hGR were used in transfection studies.
      Although 94% of the 68 amino acids in the centrally located DBD of hMR and hGR are identical, the HBD has only 57% homology. The hMR contains an additional 24 amino acids including a sequence of 4 glutamines and 8 prolines encoded by repetitive nucleotide elements (
      • Arriza J.L.
      • Weinberger C.
      • Cerelli G.
      • Glaser T.M.
      • Handelin B.L.
      • Hausman D.E.
      • Evans R.M.
      ). The C terminus of the HBD also contains a hormone-dependent transcription activation function (AF-2).
      In this paper, we have identified hMR sequences crucial for the transactivation function by first generating receptor mutants with unique BglII sites. In order to identify the hMR sequences capable of conferring activity upon a hGR that lacks transcriptional activity, we have constructed a series of hGR·hMR chimeras. To map the exact domain responsible for transactivation function of hMR, the chimeras were introduced into receptor-deficient CV-1 cells and sequences in the hMR (amino acids 328–382) were identified.

      DISCUSSION

      This paper describes the functional mapping of the N-terminal AF-1 domain in the hMR. Using deletion studies, we have identified a 54-amino acid sequence in the hMR that is essential for enhancing transactivation. Activation domains with variable chemical and structural properties influence the capacity of transcription factors to mediate cell specific responses from promoters of various responsive genes (
      • Pearce D.
      • Yamamoto K.R.
      ,
      • Arriza J.L.
      • Simerly R.B.
      • Swanson L.W.
      • Evans R.M.
      ). The AF-1 region in the hER (
      • Tora L.
      • White J.
      • Brou C.
      • Tasset D.
      • Webster N.
      • Scheer E.
      • Chambon P.
      ) is distinct from the AF-2 situated in the HBD. In the hPR, the 91-amino acid N-terminal transactivation domain is rich in proline residues (
      • Meyer M.-E.
      • Quirin-Stricker C.
      • Lerouge T.
      • Bocquel M.-T.
      • Gronemeyer H.
      ,
      • Mitchell P.J.
      • Tjian R.
      ); in the hGR, the 41 amino acid region contains clusters of acidic residues (
      • Dahlman-Wright K.
      • Almlof T.
      • McEwan I.J.
      • Gustafsson J.-A.
      • Wright A.P.H.
      ). The AF-1 domain in the hPR also interacts with an intermediary transcription factor (
      • Shemshedini L.
      • Ji J.W.
      • Brou C.
      • Chambon P.
      • Gronemeyer H.
      ), while the hGR AF-1 interacts directly with a component of the basal transcription machinery (
      • McEwan I.J.
      • Wright A.P.H.
      • Dahlman-Wright K.
      • Gustafsson J.-A.
      ,
      • Wright A.P.H.
      • McEwan I.J.
      • Dahlman-Wright K.
      • Gustafsson J.-A.
      ). This reflects a further functional distinction between the various domains within the same family of nuclear receptors.
      The structurally similar hGR and hMR both complex with heat shock protein (hsp) 90 (
      • Hollenberg S.M.
      • Weinberger C.
      • Ong E.S.
      • Cerelli G.
      • Ore A.
      • Lebo R.
      • Thompson E.B.
      • Rosenfeld M.G.
      • Evans R.M.
      ,
      • Hollenberg S.M.
      • Giguere V.
      • Segui P.
      • Evans R.M.
      ,
      • Fang Y.
      • Fliss A.E.
      • Robins D.M.
      • Caplan A.J.
      ,
      • Bohen S.
      • Yamamoto K.R.
      ,
      • Nemoto T.
      • Ohara-Nemoto Y.
      • Denis M.
      • Gustafsson J-A.
      ). Thus, despite the 94% similarity between the DBD of the hGR and hMR and the 57% homology of HBD, the N-terminal accounts for specific cellular response to glucocorticoids and mineralocorticoids. When amino acids between 76 and 261 of hGR are deleted, there is a dramatic decline in AF-1 function and a chimera containing repeats of this domain (hGR76–261) activates transcription 3–4-fold (
      • Gronemeyer H.
      • Turcotte B.
      • Stricker C.
      • Bocquel M.
      • Meyer M.-E.
      • Krozowski Z.
      • Jeltsch J.M.
      • Lerouge T.
      • Garnier J.M.
      • Chambon P.
      ). The chicken progesterone receptor PRa (78 kDa) induces the progesterone responsive ovalbumin-CAT chimeric gene, while PRb (108 kDa) failed to induce the model gene in transient transfection assays (
      • Hollenberg S.M
      • Evans R.M.
      ). This indicates the presence of a tissue-specific signal located in the N terminus. This domain of PR can be replaced with the AF-1 domain of hGR (
      • Govindan M.V.
      ). These experiments show that the AF-1 modular function can be translocated to a defective receptor. Furthermore, the modular function of sequences between residues 1 and 131 of hGR, is similar to the function of mutant hGRΔ2 (Δ115–130) (
      • Dahlman-Wright K.
      • Almlof T.
      • McEwan I.J.
      • Gustafsson J.-A.
      • Wright A.P.H.
      ). Deletion of the acidic region in the hGR referred to as transcription activation unit tau-1, reduces the transcriptional activation capacity (
      • Hollenberg S.M.
      • Giguere V.
      • Segui P.
      • Evans R.M.
      ,
      • Hollenberg S.M
      • Evans R.M.
      ).
      We have generated chimeras in order to examine the function of sequences involved in transcriptional activity. When fused to deletion mutant hGRΔ71–262 with intact hormone and DNA-binding capabilities, hMR328–382 and hMR247–382confer transactivation capacity upon hGRΔ71–262 in the presence of dexamethasone. Sequences located N-terminal to residue 247 in the hMR are not particularly efficient in conferring activity, while C-terminal sequences confer activity upon the defective hGRΔ76–261. Our previous analyses have shown that the hMR sequences between amino acids 148 and 390 were involved in transcription regulation.2 When the BglIIsite at position 383 in the hMR, which replaces an alanine with a serine, is introduced, transcriptional activity increases. It appears as though the Ala-383 is required for normal transcriptional activity by the hMR. One reason could be the interaction with other cellular factors that act as coactivators/corepressors. Three important aspects are evident from these studies. (i) when compared with hGR wild type, sequences situated N-terminal to 247 in the hMR are inhibitory, but show no remarkable function when compared with defective hGRΔ76–261; (ii) the activity of sequences located C-terminal to 247 is similar to the wild type, or 20-fold higher when compared with hGRΔ76–261; and (iii) hMR residues between 328 and 382 are much more efficient in inducing CAT activity than hGR sequences between residues 76 and 261.
      The hMRΔ59–247 increases the transcription of MMTV-CAT 2.6-fold (Fig. 2, lane 6). In fact, this mutant is more active than wild type hMR, and the constitutive level is 50% that of wild type hMR with aldosterone. We propose that this activity is modulated by negative response elements or by factors that inhibit transcription activation and name it the hMR repressive function (hMR RF-1). Elimination of polyglutamine stretches between amino acids 168 and 221 located upstream of the activation domain (
      • Jenster G.
      • Van der Korput H.A.G.M.
      • Van Vroonhoven C.
      • Van der Kwast T.H.
      • Trapman J.
      • Brinkmann A.O.
      ,
      • Simental J.A.
      • Sar M.
      • Lane M.V.
      • French F.S.
      • Wilson E.M.
      ) in the rat AR (
      • Chamberlain N.
      • Driver E.D.
      • Miesfeld R.L.
      ) results in a receptor with increased activity. Super-receptor activity is also observed with the hGR HBD substitution mutant C638S (
      • Yu C.
      • Warriar N.
      • Govindan M.V.
      ).
      Further deletions in the hMR show a region around amino acid 247 that is pivotal to transcriptional activity and also delineates two regions: a stimulatory function between residues 247 and 382 and an inhibitory function between residues 59 and 247. A negative regulator of the hER, when mutated, increases transcriptional activity (
      • McDonnell D.P.
      • Vegeto E.
      • O'Malley B.
      ). Chimera analysis shows that the region between amino acids 247 and 382 in the hMR itself consists of more than one distinct domain. Sequences between 247 and 382 are distinct from 328–382 since hGRΔ71–262/hMR247–382 is less efficient than wild type hGR in transcription activation, while the chimeric receptor hGRΔ71–262/hMR328–382 induces MMTV-CAT 2-fold. The AF-1 domain in the hGR is phosphorylated at four major sites: Thr-171, Ser-224, Ser-232, and Ser-246. Ser-246 is phosphorylated by a c-Jun N-terminal kinase, and this phosphorylation inhibits hGR transcriptional activation (
      • Rogatsky I.
      • Logan S.K.
      • Garabedian M.J.
      ). Although the hMR is also a phosphoprotein (
      • Alnemri E.S.
      • Maksymowych A.B.
      • Robertson N.M
      • Litwack G.
      ), it is not known which residues are implicated. The predominant accumulation of the specific aldosterone binding activity of wild type and deletion mutants of hMR in the nucleosol showed that nuclear localization and specific interaction with aldosterone were not affected by these deletions. DNA binding experiments demonstrated that the abilities to bind DNA were also not affected by the deletion of sequences between 59 and 390.
      The hER has two independent nonacidic transcriptional activation functions whose activities are cell type-dependent (
      • Shemshedini L.
      • Ji J.W.
      • Brou C.
      • Chambon P.
      • Gronemeyer H.
      ). It remains to be seen whether a similar cell-typical function can be attributed to the hMR sequences 59–247, 247–382, and 328–382. A third autonomous activation domain within the hER has been identified between amino acids 282 and 351, which are active in both yeast and mammalian cells (
      • Norris J.D.
      • Fan D.
      • Kerner S.A.
      • McDonnell D.P.
      ). In the hPR, the third AF-3, which is ligand-independent, is located in the DBD (
      • Schwerk B.
      • Klotzbucher M.
      • Sachs M.
      • Ulber V.
      • Klein-Hitpass L.
      ). The steroidogenic receptor cofactor-1 (SRC-1) enhances hormone-stimulated estradiol receptor transcriptional activity (
      • Smith C.L.
      • Nawaz Z
      • O'Malley B.
      ,
      • Le Douarin B.
      • Zechel C.
      • Garnier J.-M.
      • Lutz Y.
      • Tora L.
      • Pierrat B.
      • Heery D.
      • Gronemeyer H.
      • Chambon P.
      • Losson R.
      ,
      • vom Baur E.
      • Zechel C.
      • Heery D.
      • Heine M.J.S.
      • Garnier J.-M.
      • Chambon P.
      • Losson R.
      ). The mutation of a lysine residue in the HBD of the hER reduces the ability of the receptor to bind SRC-1 but has no affect on RIP140 protein (
      • Henttu P.M.A.
      • Kalkhoven E.
      • Parker M.G.
      ). The interaction of cloned factors such as SRC-1 and RIP140 with the hMR remains to be established. Our preliminary results indicate that interactions of hormone and hormone receptor complexes with DNA, and the AF-2 domain with coactivators such as SRC-1 and GRIP, are not sufficient to induce transcription by AF-1-defective mutants.
      M. V. Govindan and N. Warriar, manuscript in preparation.
      In conclusion, we have identified a 54-amino acid region in the hMR that has higher activity than the intact receptor and is able to confer activity upon a hGR deletion mutant that has no activity. Studies are under way in our laboratory to determine associated factors using the yeast two-hybrid system.

      ACKNOWLEDGEMENTS

      We thank Dr. Carl Séguin and Dr. John Grose for critically reading the manuscript and helpful suggestions.

      REFERENCES

        • Arriza J.L.
        • Weinberger C.
        • Cerelli G.
        • Glaser T.M.
        • Handelin B.L.
        • Hausman D.E.
        • Evans R.M.
        Science. 1987; 237: 268-275
        • Dahlman-Wright K.
        • Almlof T.
        • McEwan I.J.
        • Gustafsson J.-A.
        • Wright A.P.H.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1619-1623
        • Maniatis T.
        • Fritsch E.
        • Sambrook J.
        Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989
        • Govindan M.V.
        Mol. Endocrinol. 1990; 4: 417-427
        • Govindan M.V.
        • Leclerc S.
        • Roy R.
        • Rathanaswamy P.
        • Xie B.
        J. Steroid Biochem. 1991; 39: 91-103
        • Warriar N.W., Yu, C.
        • Govindan M.V.
        J. Biol. Chem. 1994; 269: 29010-29015
        • Yu C.
        • Warriar N.
        • Govindan M.V.
        Biochemistry. 1995; 34: 14163-14173
        • Giguere V.
        • Hollenberg S.M.
        • Rosenfeld M.G.
        • Evans R.M.
        Cell. 1986; 46: 645-652
        • Warriar N., Yu, C.
        • Pagé N.
        • Govindan M.V.
        J. Biol. Chem. 1994; 269: 29016-29023
        • Warriar N.
        • Pagé N.
        • Govindan M.V.
        Biochemistry. 1994; 33: 12837-12843
        • Pagé N.
        • Warriar N.
        • Govindan M.V.
        Am. J. Physiol. 1994; 267: L464-L474
        • Cidlowski J.A.
        • Cidlowski N.B.
        Endocrinology. 1981; 109: 1975-1982
        • Pearce D.
        • Yamamoto K.R.
        Science. 1993; 259: 1161-1165
        • Arriza J.L.
        • Simerly R.B.
        • Swanson L.W.
        • Evans R.M.
        Neuron. 1988; 1: 887-900
        • Tora L.
        • White J.
        • Brou C.
        • Tasset D.
        • Webster N.
        • Scheer E.
        • Chambon P.
        Cell. 1989; 59: 477-487
        • Meyer M.-E.
        • Quirin-Stricker C.
        • Lerouge T.
        • Bocquel M.-T.
        • Gronemeyer H.
        J. Biol. Chem. 1992; 267: 10882-10887
        • Mitchell P.J.
        • Tjian R.
        Science. 1989; 245: 371-378
        • Shemshedini L.
        • Ji J.W.
        • Brou C.
        • Chambon P.
        • Gronemeyer H.
        J. Biol. Chem. 1992; 267: 1834-1839
        • McEwan I.J.
        • Wright A.P.H.
        • Dahlman-Wright K.
        • Gustafsson J.-A.
        Mol. Cell. Biol. 1993; 13: 399-407
        • Wright A.P.H.
        • McEwan I.J.
        • Dahlman-Wright K.
        • Gustafsson J.-A.
        Mol. Endocrinol. 1991; 5: 1366-1372
        • Hollenberg S.M.
        • Weinberger C.
        • Ong E.S.
        • Cerelli G.
        • Ore A.
        • Lebo R.
        • Thompson E.B.
        • Rosenfeld M.G.
        • Evans R.M.
        Nature. 1985; 318: 635-641
        • Hollenberg S.M.
        • Giguere V.
        • Segui P.
        • Evans R.M.
        Cell. 1987; 49: 39-46
        • Fang Y.
        • Fliss A.E.
        • Robins D.M.
        • Caplan A.J.
        J. Biol. Chem. 1996; 271: 28697-28702
        • Bohen S.
        • Yamamoto K.R.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11424-11428
        • Nemoto T.
        • Ohara-Nemoto Y.
        • Denis M.
        • Gustafsson J-A.
        Biochemistry. 1990; 29: 1880-1886
        • Gronemeyer H.
        • Turcotte B.
        • Stricker C.
        • Bocquel M.
        • Meyer M.-E.
        • Krozowski Z.
        • Jeltsch J.M.
        • Lerouge T.
        • Garnier J.M.
        • Chambon P.
        EMBO J. 1987; 6: 3985-3994
        • Hollenberg S.M
        • Evans R.M.
        Cell. 1988; 55: 899-906
        • Jenster G.
        • Van der Korput H.A.G.M.
        • Van Vroonhoven C.
        • Van der Kwast T.H.
        • Trapman J.
        • Brinkmann A.O.
        Mol. Endocrinol. 1991; 5: 1396-1404
        • Simental J.A.
        • Sar M.
        • Lane M.V.
        • French F.S.
        • Wilson E.M.
        J. Biol. Chem. 1991; 266: 510-518
        • Chamberlain N.
        • Driver E.D.
        • Miesfeld R.L.
        Nucleic Acids Res. 1994; 22: 3181-3186
        • McDonnell D.P.
        • Vegeto E.
        • O'Malley B.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10563-10567
        • Rogatsky I.
        • Logan S.K.
        • Garabedian M.J.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2050-2055
        • Alnemri E.S.
        • Maksymowych A.B.
        • Robertson N.M
        • Litwack G.
        J. Biol. Chem. 1991; 266: 18072-18081
        • Norris J.D.
        • Fan D.
        • Kerner S.A.
        • McDonnell D.P.
        Mol. Endocrinol. 1997; 11: 747-754
        • Schwerk B.
        • Klotzbucher M.
        • Sachs M.
        • Ulber V.
        • Klein-Hitpass L.
        J. Biol. Chem. 1995; 270: 21331-21338
        • Smith C.L.
        • Nawaz Z
        • O'Malley B.
        Mol. Endocrinol. 1997; 11: 657-666
        • Le Douarin B.
        • Zechel C.
        • Garnier J.-M.
        • Lutz Y.
        • Tora L.
        • Pierrat B.
        • Heery D.
        • Gronemeyer H.
        • Chambon P.
        • Losson R.
        EMBO J. 1995; 14: 2020-2033
        • vom Baur E.
        • Zechel C.
        • Heery D.
        • Heine M.J.S.
        • Garnier J.-M.
        • Chambon P.
        • Losson R.
        EMBO J. 1996; 15: 110-124
        • Henttu P.M.A.
        • Kalkhoven E.
        • Parker M.G.
        Mol. Cell. Biol. 1997; 17: 1832-1839