Characterization of Transactivational Property and Coactivator Mediation of Rat Mineralocorticoid Receptor Activation Function-1 (AF-1) (original) (raw)

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1Pharmacological Research Department Teikoku Hormone Manufacturing Company, Ltd. (H.F.) Tokyo, Japan 107-8522

2Institute of Molecular and Cellular Biosciences The University of Tokyo (H.F., H.K., S.K.) Tokyo, Japan 113-0032

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2Institute of Molecular and Cellular Biosciences The University of Tokyo (H.F., H.K., S.K.) Tokyo, Japan 113-0032

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2Institute of Molecular and Cellular Biosciences The University of Tokyo (H.F., H.K., S.K.) Tokyo, Japan 113-0032

*Address requests for reprints to: Dr. Shigeaki Kato, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1–1-1, Bunkyo-ku, Tokyo 113-0032, Japan.

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Revision received:

03 February 2000

Accepted:

23 February 2000

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Hiroaki Fuse, Hirochika Kitagawa, Shigeaki Kato, Characterization of Transactivational Property and Coactivator Mediation of Rat Mineralocorticoid Receptor Activation Function-1 (AF-1), Molecular Endocrinology, Volume 14, Issue 6, 1 June 2000, Pages 889–899, https://doi.org/10.1210/mend.14.6.0467
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Abstract

The autonomous activation function-2 (AF-2) in the mineralocorticoid receptor (MR) E/F domain is known to play a major role in the ligand-induced transactivation function of MR; however, it remained unclear about the transactivation function of its A/B domain. We therefore tried to characterize the MR A/B domain as the AF-1 and further studied the actions of known coactivators for AF-2 in the E/F ligand-binding domain in the function of the MR A/B domain. Deletion analyses of rat and human MRs revealed that the A/B domains harbor a transactivation function acting as AF-1. The MR mutant (E959Q) with a point mutation in helix 12, which causes a complete loss of MR AF-2 activity, still retained ligand-induced transactivation function, indicating a significant role for AF-1 in the full activity of the ligand-induced MR function. Among the coactivators tested to potentiate the MR AF-2, TIF2 and p300 potentiated the MR AF-1 through two different core regions [amino acids (a.a.) 1–169, a.a. 451–603] and exhibited functional interactions with the MR A/B domain in the cultured cells. However, such interactions were undetectable in a yeast and in an in vitro glutathione-_S_-transferase pull-down assay, indicating that the functional interaction of TIF2 and p300 with the MR A/B domain to support the MR AF-1 activity require some unknown nuclear factor(s) or a proper modification of the A/B domain in the cells.

INTRODUCTION

The actions of mineralocorticoid in the homeostasis of ion balance in target tissues are believed to be exerted through the transcriptional control of target genes by its nuclear receptor (1, 2). Mineralocorticoid receptor (MR) is a member of the superfamily of steroid/thyroid hormone nuclear receptors, which act as a ligand-inducible transcription factor (3, 4). Based on similarities in structure and function with the detail analysis of ERα functional domains, the nuclear receptor proteins including MR are considered to be divided into functional domains designated domains A through F (E). The DNA-binding domain is mapped to the well conserved middle region (C domain) of the receptor. The less conserved C-terminal E/F domain is responsible for the ligand binding. For the ligand-induced transactivation, the N-terminal A/B domain and the C-terminal E/F domain are required, and their properties in transactivation are distinct and cell-type specific (5). Although the autonomous activation function-1 (AF-1) in the A/B domain itself is constitutively active, it is considered to be suppressed by ligand-unbound E/F domain. Ligand binding evokes the AF-2 function and simultaneously releases this suppression to restore the AF-1 function.

Previous observations that the AF-1 and AF-2 activities of steroid hormone receptors are transcriptionally squelched/interfered with by each other suggested the presence of common coactivators mediating the AF-1 and -2 activities for the basic transcription machinery (6). Recently, putative coactivators interacting with and activating the AF-2 activities of many nuclear receptors in a ligand-dependent fashion have been identified, and they include the TIF2/SRC-1 family proteins (7, 8), TIF1 (9), ARA70 (10), RIP140 (11), PGC-1 (12), Smad 3 (13), SRA (14), and many others (reviewed in Ref. 4). CBP/p300 proteins, a general transcriptional integrator, are also shown to enhance the AF-2 activities (15). More recently, another class of coactivator complex, DRIP/TRAP, has been reported to potentiate the ligand-induced function of the AF-2 of VDR and TR (4, 16, 17). In addition, components of the TFIID complex, such as TAFs and TFIIB, have been demonstrated to act as nuclear receptor coactivators (18). However, it remains unclear whether the reported coactivators potentiate the AF-1 activities of nuclear receptors. Moreover, as there is no highly conserved region in the A/B domain encompassing the AF-1 among the nuclear receptors, unlike the well conserved region (helix 12) in the E/F domain serving as an interface for the reported coactivator interactions, it is possible that there are AF-1 coactivators specific for each of the nuclear receptors.

In this respect, MR is of interest to study the AF-1 function, since among nuclear receptors their A/B domains are poorly conserved and hence are supposed to recruit a particular set of coactivators. Although previous studies suggested a possible role for the A/B domain in ligand selectivity of the MR, the transactivation function of the A/B domain itself was not studied in detail (19). In contrast, more recently, a contribution of the A/B domain to the ligand-induced transactivation of MR was suggested with the MR deletion mutants (20). The present study was hence undertaken to assess the transactivation function of the MR A/B domain in regard to the actions of known AF-2 coactivators on the MR A/B domain. Consequently, we found a significant AF-1 activity in the A/B domains of human and rat MRs. Although the activities of the AF-1, as well as the AF-2 of MR, were potentiated in the presence of either p300 or TIF2, no direct interaction of their coactivators with the MR A/B domains was detected, suggesting the presence of an unknown coactivator directly associating with the MR A/B domain to fully support the MR AF-1 activity.

RESULTS

Identification of the Transactivation Function (AF-1) in the N-Terminal Domain of MR

MR has a relatively long A/B domain, but the importance of this domain in terms of transactivation remained unclear, since there is discrepancy in the function of the MR A/B domain among the previous reports (19–21). Studies of the MR deletion mutants supposed that the A/B domain of MR does not contribute to the ligand-induced transactivation function of MR (19, 21). However, more recently, the MR A/B domain was reported to harbor intrinsic transactivation activity (20). To test the latter possibility that the MR A/B domain exhibits a transactivation function (AF-1), we first prepared rat MR (rMR) deletion mutants, which retain only either the A/B or the E/F domain (Fig. 1A). The transactivation functions of the deletion mutants and the wild type of rMR were compared in COS-1 cells by a transient expression assay [chloramphenicol acetyltransferase (CAT) assay] using GRE2-tk-CAT as a reporter gene (Fig. 1B). Although the maximum activation was seen at 100 nm, a near maximum dose (10 nm) of aldosterone was added to the medium to induce the ligand-induced transactivation of the full-length MR and MR C-DE/F and confirmed the ligand-induced transactivation function of MR. In COS-1 cells, the MR A/B-C exhibited about half the transactivation activity of the full-length MR. The ligand-induced activity of AF-2 in the rMR E/F domain was as potent as that in the A/B domain. Likewise, intrinsic transactivation activity was detected also in the A/B domain of human MR (hMR) (data not shown). Thus, these results clearly indicate that the A/B domains of rMR and hMR serve as the AF-1, as in other nuclear receptors (5, 22–24). Note that the expression levels of the MR deletion mutants are the same when estimated by a Western blot analysis (Fig. 1C). These findings indicate that MR harbors the MR AF-1 activity as reported in many other nuclear receptors (5, 25, 26).

Fig. 1.

The A/B Domains of rMR Have a Transactivation Function (AF-1) A, Scheme of rMR A/B domain and E/F domain deletion mutants fused to FLAG in their N-terminal ends. MR A/B-C contains a.a.1–680 and lacks the E/F domain. MR C-DE/F contains a.a. 604–981 and lacks the A/B domain. Wild-type MR is shown at the bottom. B, Transcriptional activities of MR deletion mutants in COS-1 cells. Each of the receptor expression vectors (1 μg) was transfected together with 3 μg of GRE2-tk-CAT reporter plasmid, in the absence (−) and presence (+) of 10 nm of aldosterone. CAT assay was performed as described in Materials and Methods. Each value represents the mean ± se of three individual transfections and is shown as fold induction from the background activity of the reporter plasmid. C, Western blot analysis of the MR deletion mutants. A portion of the cell extracts used for the CAT assay was analyzed by a Western blotting with a specific antibody to FLAG as described in Materials and Methods.

Transactivation Function of AF-1 in Full-Length MR

To confirm the transactivation function (AF-1) in the MR A/B domain, we destroyed the AF-2 function by introducing a point mutation in helix 12 of the full-length rMR, since the point mutation in helix 12 of the full-length hERα completely impaired its AF-2 function without affecting the AF-1 (26). The helix 12 of many nuclear receptors contains two hydrophobic amino acid boxes and several negatively charged amino acids around these boxes, and these serve as a direct target for ligand-dependent coactivators such as SRC-1/TIF2 family proteins (27, 28). Because the MR helix 12 contains only one negatively charged amino acid (Glu959) in the conserved amino acid sequences, we displaced this Glu959 into electrically neutral Gln by site-directed mutagenesis (E959Q-mutant as depicted in Fig. 2A). MR C-DE/F exhibited ligand-induced transactivation in a dose-dependent manner (Fig. 2B; MR C-DE/F), whereas the point-mutation in helix 12 of the AF-2 (MRE959Q C-DE/F) caused a loss of transactivation by aldosterone even at 10 nm, suggesting that this mutation completely impairs the MR AF-2 function. However, despite such a mutation, the full-length MR still remained potent in ligand-induced transactivation but with about half the activity of the wild-type MR (compare MRE959Q with MR in Fig. 2B), clearly indicating a significant role for AF-1 in the ligand-induced transactivation of MR.

Fig. 2.

Ligand-Induced AF-1 Function in the Full-Length MR A, Scheme of rMR AF-2 AD core point-mutants. Glu959 of the rMR was replaced with Gln (E959Q point-mutation). This mutation was introduced to the MR C-DE/F deletion mutant (MRE959Q C-DE/F) and the full-length MR (MRE959Q). B, The ligand-induced transactivation function of MR AF-2 AD core point-mutants (left) was compared with that of wild-type (right). Each of the receptor expression vectors (1μ g) was transfected to COS-1 cells, together with 3 μg of GRE2-tk-CAT reporter plasmid in the presence of increasing concentrations (0–10 nm) of aldosterone. CAT assay was performed as described in Materials and Methods. Each value represents the mean ± se as in Fig. 1.

Core Regions Responsible for the MR AF-1 Activity

We further delineated the core regions responsible for the MR AF-1 activity. The transcriptional activities of a series of deletion mutants of the MR A/B-C (left panel in Fig. 3) were tested in COS-1 cells. The expression levels of the deletion mutants were confirmed by a Western blot analysis (Fig. 3, right panel). The transactivation function of each of the deletion mutants was shown as percentage ratio to that of MR A/B-C. Both the truncation of 169 a.a. from the N-terminal region (M1) and a deletion from a.a.450 to 595 in the C terminus of the A/B domain (M5) resulted in marked reductions of the AF-1 activities. The mutant lacking both regions (M4) completely lost the AF-1 activity, suggesting that the N-terminal (designated as AF-1a hereafter) and C-terminal (AF-1b) regions of the MR A/B domain constitute the full activity of the AF-1.

Fig. 3.

Mapping of Core Regions for the MR AF-1 Activity Scheme of deletion mutants of the A/B domain (M1∼M9) are shown in the left panel along with the full-length of MR A/B-C (WT). Each receptor expression vector (1 μg) was transfected to COS-1 cells, together with 3 μg of GRE2-tk-CAT reporter plasmid, and the transactivation function of each mutant receptor was measured by CAT assay as described in Materials and Methods. Each value represents the mean ± se of three individual transfections and is expressed as a percentage ratio to the full-length A/B-C activity (the background activity of the reporter plasmid was expressed as 0). Deduced core regions of MR AF-1 are shown in the top column and designated as AF-1a and AF-1b. Expression levels of the A/B domain deletion mutants estimated by a Western blot analysis as described in Fig. 1 are shown in the right panel.

Enhancement of the MR AF-1 Activity by p300 and TIF2, but Not by SRC-1 and AIB1

The SRC-1/TIF2 family and CBP/p300 family proteins are shown to potentiate the AF-2 activity by directly binding to the E/F domains in a ligand-dependent way (7, 8, 15). More recently, the AF-1 activities of several nuclear receptors were shown to be potentiated by the presence of these coactivators (29–33). We therefore examined whether these reported coactivators potentiate or not the MR AF-1 activity by coexpression of these coactivators. In COS-1 cells, with GRE2-TATA-CAT as a reporter gene, SRC-1e (a short but more potent isoform of SRC-1a, see Ref. 34), TIF2, and p300 enhanced the ligand-induced transactivation of the full-length MR approximately 2- to 3-fold, respectively (Fig. 4A), as well as for the MR AF-2 (data not shown), suggesting that they act as coactivators for the MR AF-2. In contrast, no significant enhancement by SRC-1a or AIB1 was detected (Fig. 4A). Under these conditions, TIF2 and p300, but not SRC-1e, were potent also for the MR AF-1 (MR A/B-C), enhancing its transactivation 1.7- and 2.7-fold, respectively (Fig. 4B). p300 enhanced the activities of both AF-1a and AF-1b, but TIF2 was effective only for the AF-1b (Fig. 4C). From these results, it was likely that for the transactivation function of MR AF-1, p300 and TIF2 act as coactivators. To further test the possible interactions of these coactivators with the MR A/B domain, a mammalian two-hybrid assay with chimeric p300 and TIF2 proteins fused to the VP16 activation domain were performed in COS-1 cell with the MR A/B-C as a bait. TIF2 interacted strongly with the MR E/F domain in a ligand-dependent manner (32-fold), but weakly with the MR A/B domain (3.2-fold) in the mammalian two-hybrid system. The p300 N-terminal region (596 a.a. residues) encompassing the nuclear receptor-interacting region interacted poorly with both the MR A/B and E/F domains (1.7-fold), and the other regions tested in p300 did not exhibit any significant interaction with the MR A/B and E/F domains (data not shown). Interaction with p300 was detected in both of the A/B core domains, AF-1a and AF-1b, whereas only AF-1b strongly interacted with TIF2 (Fig. 5B).

Fig. 4.

Potentiation of MR AF-1 Activity by TIF2 and p300 Effects of coactivators on transactivation functions of the full-length MR in the presence of 10 nm aldosterone (A), MR AF-1 (B), and each AF-1 core region (C) were examined in COS-1 cells. SRC-1a, SRC-1e, TIF2, AIB1, p300, and parent empty expression vectors were cotransfected in the indicated amount (1–6 μg) along with 1 μg of receptor expression vector of full-length MR (A), MR A/B-C (B), M3 mutant (AF-1a in Fig. 3, left in panel C) or M8 mutant (AF-1b in Fig. 3, right in panel C) and 5 μg of GRE2-TATA-CAT reporter plasmid, and CAT assay was performed as described in Materials and Methods. Each value represents the mean ± se of three individual transfections and is shown as the fold induction by coactivators.

Fig. 5.

In Vivo Interactions of MR Deletion Mutants with the AF-2 Coactivators Interactions of TIF2 and p300 with MR AF-1 and AF-2 (A), and each AF-1 core region (B) were tested by modified mammalian two-hybrid assay in COS-1 cells. Five micrograms of VP16 activation domain fusion vectors bearing either TIF2 or p300 (pVP-TIF2, pVP-p300) and vacant parent vector (pVP16 parent) were transfected along with 2 μg of MR A/B-C (panel A, left 3 lanes), MR C-DE/F (panel A, right 6 lanes), M3 mutant (AF-1a in Fig. 3; left 3 lanes in panel B) or M8 mutant (AF-1b in Fig. 3; right 3 lanes in panel B) and 3 μg of GRE2-tk-CAT reporter plasmid. CAT assay was performed as described in Materials and Methods. In the case of MR C-DE/F, interactions were tested in the absence (−) or presence (+) of 10 nm aldosterone. Each value represents the mean ± se of three individual transfections and is expressed as the fold induction from the activity of the pVP16 parent vector-transfected cell, respectively.

Indirect Interactions of TIF2 and p300 with the MR A/B Domain

To clarify whether the functional interactions of TIF2 and p300 with the MR A/B domains are direct or mediated through some unknown nuclear factor(s) in mammalian cells, we further tested these interactions in a yeast two-hybrid assay and a glutathione-_S_-transferase (GST) pull-down assay. Using chimeric MR proteins fused to GAL4-DBD as a bait, interactions of TIF2 and p300 with the A/B and E/F domains were examined. We observed ligand-dependent interactions of the coactivators with the MR E/F domain, as observed in mammalian cells (Fig. 6B); however, we could not find any interaction of these coactivators with MR A/B domain (Fig. 6A), even with either AF-1a or AF-1b (data not shown). Similar results were also seen when these coactivators were used as a bait (data not shown). In a GST pull-down assay with chimeric MR proteins fused to GST and 35S-labeled coactivators, ligand-dependent interactions with TIF2 and p300 were detected in the MR E/F domain (lane 3 in Fig. 6, C and D). However, no such physical interaction was found in the A/B domain, even in the AF-1a or AF-1b (lanes 4–6 in Fig. 6, C and D). Taken all together, these results suggest that interactions of TIF2 and p300 with the MR AF-1 are indirect or too weak to be detectable due to improper modification of the expressed A/B domain proteins in this assay. However, we tempt to speculate that some unknown nuclear factor(s) is mediated to directly associate with the A/B domain, possibly recruiting TIF2 and p300 for the full activity of the MR AF-1.

Fig. 6.

No Interaction of the MR A/B Domain with TIF2 and p300 in a Yeast Two-Hybrid System and in a GST Pull-Down Assay Physical interactions of TIF2 and p300 with MR AF-1 were tested by yeast two-hybrid assay (A and B) and in vitro GST pull-down assay (C and D). GAL4-AD fusion vector bearing either TIF2, p300 (pGAD-TIF2, pGAD-p300), or vacant parent vector (pGAD10 parent) was transfected to yeast along with chimeric MR mutants fused to GAL4-DBD, GAL4BD-MR A/B (A), and GAL4BD-MR DE/F (B). Transformed yeast clones were grown in liquid medium in the absence (−) or presence (+) of 10 μm aldosterone. Interactions between MR deletion mutants and TIF2 and p300 were measured as β-galactosidase activities normalized with the optical density of yeast culture (Miller Unit). Each value represents the mean ± se of three experiments and is expressed as fold induction from the activity of the pGAD10 parent vector transfected yeast, respectively. Chimeric MR proteins fused to GST were expressed in E. coli as shown in panel E and immobilized on glutathione-Sepharose beads. In vitro translated TIF2 (C) or p300 (D) labeled with[ 35S]methionine was incubated with the beads. Bound protein was analyzed by SDS-PAGE (7.5%) and visualized by autoradiography. Note that aldosterone (10 nm) induced interactions of the E/F domain with TIF2 and p300, as expected from previous reports (7, 15 ).

DISCUSSION

As the A/B domain is poorly conserved among the members of the nuclear receptor superfamily, the function of the A/B domain in terms of ligand-induced transactivation and actions of coactivators has not been fully studied. Here we characterized the A/B domain and identified the AF-1 function. Deletion of the A/B domain caused an evident reduction in the ligand-induced transactivation, and the truncation of the C-terminal E/F domain produced an intrinsic activity in the transactivation of the MR A/B domain. A point mutant of the full-length MR in helix 12 of the E/F domain to destroy the AF-2 still retained the ligand-induced transactivation function (Fig. 2B), clearly indicating a significant role for AF-1 in the ligand-induced transactivation function of MR and a ligand-induced functional interaction between AF-1 and AF-2 of MR. By our preliminary experiments, the activity of the MR AF-1 appeared cell type specific (data not shown), as expected from the results concerning the AF-1 activities in steroid hormone receptors. If the MR AF-1 is cell type specific, in some cell lines it may be at negligible levels as compared with that of AF-2. This may explain why in previous reports, no AF-1 activity was detected in the A/B domain (19, 21). We further mapped two core regions essential for the AF-1 activity [amino acids (a.a.) 1–169 and a.a. 451–603 of the A/B domain], which are not overlapped with the reported MR AF-1 core region (a.a. 328–382, Ref. 20). This discrepancy may be due to the MR mutants tested, since in that study, the deletion mutants retain the AF-2 domain when the AF-1 core domain was mapped. Although in overall amino acid sequence, MR exhibits greatest homology to the glucocorticoid receptor in the nuclear receptor superfamily, no significant homology is found between their core regions in AF-1 (22).

Molecular dissection of ERα suggested that the AF-1 function is critical to the tissue-specific actions of estrogen-partial antagonists such as tamoxifen and raloxifen, since such compounds block the function of AF-2, but not AF-1 (35, 36). Furthermore, ligand-induced and functional synergism was detected between the AF-1 and AF-2 in many nuclear receptors (29, 37), supporting the idea that the AF-1 significantly contributes to the ligand-induced transactivation function of nuclear receptors. Therefore, one may speculate that the MR AF-1 significantly contributes to tissue-specific actions of mineralocorticoid and synthetic ligands for MR, since the length of the A/B domain is twice that of the E/F domain. In this respect, as the functional and physical interactions between the N-terminal and the C-terminal domains are described in other steroid receptors (33, 37–40), MR may be used to study the role of the A/B domain in the ligand-induced transactivation.

For the ligand-induced transactivation of nuclear receptors, ligand-dependent interactions with coactivator complexes are essential. To date, two classes of coactivator complexes have been identified. One of them is considered to include p300/CBP, the SRC-1/TIF2 family proteins, and SRA (14), possibly with many unknown components, and the other is DRIP/TRAP complex (16, 17), which is composed of at least 9 factors. Since direct and ligand-dependent interaction occurs in p300/CBP and the SRC-1/TIF2 family proteins acting as a coactivator for AF-2s of various nuclear receptors including MR, we examined whether these coactivators act also for the MR AF-1 to bridge with the AF-2 for forming a stable MR complex with the coactivator complex. p300 and TIF2 (but neither SRC-1 nor AIB1) potentiated the activity of the MR AF-1, and functional interactions were detected in a mammalian two-hybrid system. However, no direct interaction with both coactivators was detected in a GST-pull down assay and a yeast two-hybrid system, although we cannot exclude a possibility that the expressed MR deletion mutants are not functionally produced and/or modified to recruit coactivators. However, we are speculating that unknown nuclear factors other than p300 and TIF2 associate with the A/B domain for the full activity of the AF-1. This hypothesis is supported by our recent findings that a newly identified coactivator, p68 (38), serves as a specific coactivator for the AF-1 of hERα, by binding directly to the hERα A/B domain, but not to the E/F domain nor to the other nuclear receptors tested including ERβ. In light of these findings, it should be possible to identify proteins associating with the MR A/B domain. Such proteins may be a component(s) of the two known coactivator complexes, with which nuclear receptors are shown to interact in a ligand-dependent way through their E/F domains. Otherwise, the MR AF-1 may recruit a coactivator complex distinct from the coactivator complexes for AF-2.

MATERIALS AND METHODS

Construction of MR Expression Vectors

MR expression vectors were constructed to express chimeric MR mutants fused to FLAG in its N terminus. The cDNA coding the full length of rMR (a.a.1–981, Ref. 41) fused to FLAG was obtained by RT-PCR using rat kidney mRNA with an upstream primer, 5′-GGG-GTA-CCA-CCA-TGG-ATT-ACA-AGG-ACG-ACG-ATG-ACA-AGA-TGG-AAA-CCA-AAG-GCT-AC-3′, and a downstream primer, 5′-GCT-CTA-GAT-CAC-TTT-CTG-TGA-AAG-TAA-AGG-G-3′. The PCR product was digested with _Kpn_I/_Xba_I and subcloned into pcDNA3 mammalian expression vector (pcDNA3-MR; Invitrogen, San Diego, CA), and the sequence was verified by dideoxy-sequencing (ABI 377. Perkin-Elmer Corp., Norwalk, CT). A cDNA fragment coding for the A/B-C region (a.a.1–680) of MR fused to FLAG was amplified with pcDNA3-MR by PCR using the upstream primer used for pcDNA3-MR and a downstream primer, 5′-GCT-CTA-GAT-CAC-CCC-AGC-TTCTTT-GAC-3′, and subcloned into pcDNA3 to generate pcDNA3-MR A/B-C. A cDNA fragment coding the C–DE/F region (a.a. 604–981) of MR fused to FLAG was obtained with a pair of primers; an upstream primer, 5′-GGG-GTA-CCA-CCA-TGG-ATT-ACA-AGG-ACG-ACG-ATG-ACA-AGT-GTT-TGG-TGT-GTG-GAG-ATG-3′, and the downstream primer used for pcDNA3-MR and subcloned into pcDNA3 to generate pcDNA3-MR C–DE/F. The N-terminal (M1, M2, and M3) and the C-terminal (M4∼M9) deletion mutants were generated in the same way by PCR with a particular set of primers. A human MR A/B-C cDNA fragment coding a.a.1–679 (42) fused to FLAG was amplified by PCR using pRShMR (American Type Culture Collection, Manassas, VA) as template, with a proper set of primer and subcloned into pcDNA3 to generate pcDNA3-hMR A/B-C. The MRE959Q and MR C-DE/FE959Q mutant, in which Glu959 was substituted with Gln, were constructed with a site-directed mutagenesis kit (Quick Change, Stratagene, La Jolla, CA) with sense primer, 5′-CCC-GCC-ATG-CTG-GTG-CAG-ATC-ATC-ACC-GAC-C-3′, and antisense primer, 5′-GGT-CGG-TGA-TGA-TCT-GCA- CCA-GCA-TGG-CGG-G-3′.

Other Plasmids

Expression vectors of human SRC-1a, TIF2 and AIB1, were previously described (43). pcDNA3-SRC-1e (44) was constructed by RT-PCR using HeLa cell mRNA, and pcDNA3-p300 was made from CMVβ-p300 (a gift from Dr. A. Fukamizu, University of Tsukuba). GRE2-tk-CAT contains two GREs (45) found in the tyrosine aminotransferase gene promoter and thymidine kinase promoter in front of the CAT reporter gene in pBL-CAT. pGAD-TIF2 (43) contains cDNA coding a.a. 669-1465 of hTIF2. This hTIF2 cDNA fragment was also subcloned into pVP16 mammalian two-hybrid activation domain vector (CLONTECH Laboratories, Inc., Palo Alto, CA; pVP-TIF2). pGAD-p300 and pVP-p300 were constructed by subcloning the cDNA fragment coding a.a.1–596 of p300 described above into each two-hybrid activation domain fusion vector.

Cell Culture and Transfections

COS-1 cells were maintained in DMEM (without phenol red) supplemented with 5% charcoal-stripped FCS. The cells were transfected at approximately 20–30% confluency in 10-cm culture dishes by the calcium phosphate precipitation method with the vectors described in the figure legends, along with 3 μg of pCH110 (Amersham Pharmacia Biotech, Piscataway, NJ) expressing β-galactosidase as an inner control for transfection efficiency. The culture medium was renewed 24 h after transfection, and after a further 24 h, the cells were harvested. The cells were treated for 24 h after transfection in the presence and absence of cognate ligands. d-Aldosterone (Sigma, St. Louis, MO) dissolved in ethanol was added in 0.1% volume to the culture medium to obtain appropriate concentrations. Cell extracts were prepared by freezing and thawing, normalized with β-galactosidase activities, and measured for chloramphenicol acetyl transferase activities (46, 47).

Yeast Two-Hybrid Assay

Yeast two-hybrid assay was performed in Saccharomyces cerevisiae strain Y153 (_MAT_a gal4 gal80 his3 trp1–901 ade2–101 ura3–52 leu2–3 leu2–112 URA3::GAL HIS3). The N-terminal A/B domain (a.a.1–596) or C-terminal DE/F domain (a.a.672–981) of rMR was subcloned into pGBT9 GAL4 DNA-binding domain fusion vector (CLONTECH Laboratories, Inc.) in frame and used as bait. Y153 was transformed with bait vector and prey vectors containing co-activator fragment (pGAD-TIF2 or pGAD-p300) by the lithium acetate method. Their interactions were measured byβ -galactosidase liquid assay according to the manufacturer’s instructions.

GST Pull-Down Assay

The cDNAs encoding rMR domains were subcloned into GEX-4T-GST-fusion protein expression vectors (Amersham Pharmacia Biotech). Each GST fusion protein was expressed in Escherichia coli and bound to glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). Its size was predicted by SDS-PAGE. Human pcDNA3-TIF2 and pcDNA3-p300 were used to produce[ 35S] methionine-labeled proteins using an in vitro translation system (TNT-coupled Reticulocyte Lysate System. Promega Corp., Madison, WI). The 35S-labeled TIF2 and p300 were incubated with beads bound either to GST or a GST-fused MR fragment in NET-N buffer[ 0.5% Nonidet P-40, 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA] with 1 mm phenylmethylsulfonylfluoride for 3 h. After the free proteins had been washed away from the beads, bound proteins were extracted into loading buffer and separated by 7.5% SDS-PAGE, and visualized by autoradiography (13).

Western Blot Analysis

COS-1 cells were transfected with the indicated expression plasmids, lysed in TNE buffer [10 mm Tris-HCl (pH 7.8), 1% NP-40, 0.15 m NaCl, 1 mm EDTA]. The chimeric MR mutants fused to FLAG in their N-terminal ends were separated by 10% SDS-PAGE, transferred onto polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Richmond, CA), and then detected by immunoblotting with monoclonal antibody to FLAG[ IBI; Eastman Kodak Co., Rochester, NY (13)].

Acknowledgments

We thank Dr. Y. Arao and T. Hashimoto for advice with the yeast two-hybrid assay, H. Tai for construction of coactivator expression vectors, Dr. J. Yanagisawa for advice on the GST pull-down assay, and Dr. A. Fukamizu for the provision of plasmid.

This work was supported in part by a grant-in-aid for priority areas from the Ministry of Education, Science, Sports and Culture of Japan (to S.K.).

Funder

1998

Aldosterone action: fact, failure and the future

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Characterization of Transactivational Property and Coactivator Mediation of Rat Mineralocorticoid Receptor Activation Function-1 (AF-1) (original) (raw)

Cite

Abstract

INTRODUCTION

RESULTS

Identification of the Transactivation Function (AF-1) in the N-Terminal Domain of MR

Transactivation Function of AF-1 in Full-Length MR

Core Regions Responsible for the MR AF-1 Activity

Enhancement of the MR AF-1 Activity by p300 and TIF2, but Not by SRC-1 and AIB1

Indirect Interactions of TIF2 and p300 with the MR A/B Domain

DISCUSSION

MATERIALS AND METHODS

Construction of MR Expression Vectors

Other Plasmids

Cell Culture and Transfections

Yeast Two-Hybrid Assay

GST Pull-Down Assay

Western Blot Analysis

Acknowledgments

Citations

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Citing articles via

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Characterization of Transactivational Property and Coactivator Mediation of Rat Mineralocorticoid Receptor Activation Function-1 (AF-1) (original) (raw)

Cite

Abstract

INTRODUCTION

RESULTS

Identification of the Transactivation Function (AF-1) in the N-Terminal Domain of MR

Transactivation Function of AF-1 in Full-Length MR

Core Regions Responsible for the MR AF-1 Activity

Enhancement of the MR AF-1 Activity by p300 and TIF2, but Not by SRC-1 and AIB1

Indirect Interactions of TIF2 and p300 with the MR A/B Domain

DISCUSSION

MATERIALS AND METHODS

Construction of MR Expression Vectors

Other Plasmids

Cell Culture and Transfections

Yeast Two-Hybrid Assay

GST Pull-Down Assay

Western Blot Analysis

Acknowledgments

Citations

Views

Altmetric

Email alerts

Citing articles via

Latest

Most Read

Most Cited