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. 1999 Sep 6;146(5):929-40.
doi: 10.1083/jcb.146.5.929.

A nuclear action of the eukaryotic cochaperone RAP46 in downregulation of glucocorticoid receptor activity

J Schneikert  1 , S Hübner, E Martin, A C Cato
Affiliations

A nuclear action of the eukaryotic cochaperone RAP46 in downregulation of glucocorticoid receptor activity

J Schneikert et al. J Cell Biol. .

Abstract

RAP46 is a eukaryotic cochaperone that associates with several proteins, including the heat shock protein hsp70/hsc70 and the glucocorticoid receptor (GR). Here we show a downregulation of GR-mediated transactivation by RAP46 via a mechanism independent of a cytoplasmic action of this cochaperone. We demonstrate a specific cytoplasmic-nuclear recruitment of RAP46 by the liganded GR that results in inhibition of the transactivation function of the receptor. A repeated sequence motif [EEX(4)](8) at the NH(2) terminus of RAP46 or BAG-1L, a larger isoform of RAP46, is responsible for this downregulation of GR activity. BAG-1, a shorter isoform with only a duplication of the [EEX(4)] sequence, does not inhibit GR activity. The [EEX(4)](8) motif, when linked to an otherwise unrelated protein, abrogated the inhibitory action of endogenous RAP46 on GR-mediated transactivation. The nuclear effects of RAP46 and BAG-1L are specific since GR-mediated inhibition of AP-1 activity was not affected. These studies identify the [EEX(4)](8) sequence as a signature motif for inhibition of GR-mediated transactivation and demonstrate a specific nuclear action of a eukaryotic cochaperone in the regulation of GR activity.

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Figures

Figure 1
Figure 1
Confocal immunofluorescence analysis of intracellular localization of GR, MR, and RAP46. COS-7 cells were transiently transfected with expression vectors encoding either a GR–GFP (A and B) or GFP–MR (C and D) together with a plasmid expressing HA-tagged RAP46 (A–D). 36 h after transfection the cells were treated for 1 h with vehicle (0.1% ethanol) alone (−) or vehicle containing either 0.1 μM dexamethasone (+ Dex) or 0.1 μM aldosterone (+ Ald) before harvesting, processing, and visualization with a laser confocal microscope. The green fluorescence arises from the GFP-tagged receptors, whereas the red fluorescence comes from staining of RAP46 with the anti-HA mAb 12CA5 (Boehringer Mannheim Corp.), followed by an anti-mouse antibody labeled with rhodamine. The yellow to orange colors indicate areas of colocalization of the two proteins.
Figure 4
Figure 4
Confocal immunofluorescence analysis of intracellular localization of mutant GR and RAP46. COS-7 cells were transiently transfected with expression vectors encoding either a GR1-515-GFP (A) or GRΔ491-515-GFP (B), together with a plasmid expressing HA-tagged RAP46. 36 h after transfection, the cells were treated for 1 h with vehicle (0.1% ethanol) alone (−) or vehicle containing 0.1 μM dexamethasone (+ Dex) before harvesting, processing, and visualization with a laser confocal microscope. The green fluorescence arises from the GFP-tagged receptors, whereas the red fluorescence comes from staining of RAP46 with the anti-HA mAb 12CA5 (Boehringer Mannheim Corp.), followed by an anti-mouse antibody labeled with rhodamine. The yellow to orange colors indicate areas of colocalization of the two proteins.
Figure 4
Figure 4
Confocal immunofluorescence analysis of intracellular localization of mutant GR and RAP46. COS-7 cells were transiently transfected with expression vectors encoding either a GR1-515-GFP (A) or GRΔ491-515-GFP (B), together with a plasmid expressing HA-tagged RAP46. 36 h after transfection, the cells were treated for 1 h with vehicle (0.1% ethanol) alone (−) or vehicle containing 0.1 μM dexamethasone (+ Dex) before harvesting, processing, and visualization with a laser confocal microscope. The green fluorescence arises from the GFP-tagged receptors, whereas the red fluorescence comes from staining of RAP46 with the anti-HA mAb 12CA5 (Boehringer Mannheim Corp.), followed by an anti-mouse antibody labeled with rhodamine. The yellow to orange colors indicate areas of colocalization of the two proteins.
Figure 2
Figure 2
The effects of RAP46 and RAP46Δ70 on dexamethasone binding properties of the GR. 200,000 COS-7 cells were transiently transfected with an empty expression vector or expression vectors encoding GR, RAP46, and RAP46Δ70. Thereafter, the cells were incubated with the indicated amounts of [3H]dexamethasone in the presence or absence of 1,000-fold competitor unlabeled dexamethasone and the bound radioactivity was determined. This was done by subtracting the amount of radioactivity incorporated in the presence of competitor from the radioactivity incorporated in the absence of the competitor. Left, GR-bound [3H]dexamethasone is plotted as a function of hormone concentration in cells transfected with only the GR expression vector (open circles), the GR and RAP46 (filled circles), the GR and RAP46Δ70 (open squares), or with an empty expression vector (open triangles). Presented are the mean values and error bars of two different experiments. In each experiment, the individual measurements were performed in triplicate. Right, The Scatchard plots of the experiments indicating the Kd of GR for dexamethasone in the presence or absence of RAP46 and RAP46Δ70.
Figure 3
Figure 3
Effect of RAP46 on ligand dependent transactivation by GR and MR. A, Schematic diagram of the GR–MR chimeras showing the GR in gray and the MR in stripes. The RAP46 binding site in between amino acids 491 and 515 of the GR (Kullmann et al. 1998) is presented as an unshaded region. The receptor chimeras described have been reported previously by Arriza 1991. DBD, DNA binding domains. B and C, COS-7 cells were transiently cotransfected with an MMTV firefly luciferase indicator gene and a renilla luciferase construct as an internal control, together with the indicated MR and GR expression vectors in the presence of a RAP46 expressing plasmid (filled bars) or of the corresponding control vector (open bars). Hormone treatment (0.1 μM dexamethasone, cortisol, or aldosterone) was performed immediately after the transfection and the cells were harvested 36 h later for luciferase activity measurements. The results are expressed as the level of hormone-induced expression of the MMTV construct after correcting for the transfection efficiency by renilla luciferase measurements. This was presented relative to the cortisol induced expression of the MMTV reporter plasmid by the GR, which was 213-fold, but was arbitrarily set to unity. The bar chart represents the mean value (± SD) of three independent experiments. In B, the hormone used after transfection was 0.1 μM cortisol and the level of induction of MMTV activity in this case was 201-fold, but was also set arbitrarily to 1.0. In these experiments, GGG and MMM stand for the GR and MR.
Figure 3
Figure 3
Effect of RAP46 on ligand dependent transactivation by GR and MR. A, Schematic diagram of the GR–MR chimeras showing the GR in gray and the MR in stripes. The RAP46 binding site in between amino acids 491 and 515 of the GR (Kullmann et al. 1998) is presented as an unshaded region. The receptor chimeras described have been reported previously by Arriza 1991. DBD, DNA binding domains. B and C, COS-7 cells were transiently cotransfected with an MMTV firefly luciferase indicator gene and a renilla luciferase construct as an internal control, together with the indicated MR and GR expression vectors in the presence of a RAP46 expressing plasmid (filled bars) or of the corresponding control vector (open bars). Hormone treatment (0.1 μM dexamethasone, cortisol, or aldosterone) was performed immediately after the transfection and the cells were harvested 36 h later for luciferase activity measurements. The results are expressed as the level of hormone-induced expression of the MMTV construct after correcting for the transfection efficiency by renilla luciferase measurements. This was presented relative to the cortisol induced expression of the MMTV reporter plasmid by the GR, which was 213-fold, but was arbitrarily set to unity. The bar chart represents the mean value (± SD) of three independent experiments. In B, the hormone used after transfection was 0.1 μM cortisol and the level of induction of MMTV activity in this case was 201-fold, but was also set arbitrarily to 1.0. In these experiments, GGG and MMM stand for the GR and MR.
Figure 5
Figure 5
Regions of RAP46 required for nuclear transport and inhibition of GR activity. A, Schematic representation of different isoforms and deletion mutants of RAP46. Indicated are the isoforms BAG-1L, RAP46, and BAG-1, as well as the deletion mutants RAP46Δ40 and RAP46Δ70. The black vertical bars indicate the positions of the motif [EEX4]. The gray and hatched areas represent the location of a proline rich region (Pro) and of a putative nuclear localization signal (NLS). The sequence of the first 71 NH2-terminal amino acids of RAP46 is also shown with the arrows pointing to the first amino acid in RAP46, RAP46Δ40, BAG-1, and RAP46Δ70. Each [EEX4] motif is underlined. B, Lack of effect of a NH2-terminal deletion mutant of RAP46 on DNA binding by the GR and transactivation by the receptor at the MMTV promoter. COS-7 cells were transiently transfected with a control vector (lanes 1 and 2) or an expression vector for GR (lanes 3–10). In addition, they were cotransfected with either a control vector (lanes 1–4) or a plasmid encoding the wild-type HA-tagged RAP46 (HA-RAP46 wt, lanes 5 and 6) or deletion mutants lacking either the first 40 amino acids (HA-RAP46Δ40, lanes 7 and 8) or the first 70 amino acids (HA-RAP46Δ70, lanes 9 and 10). After treatment with vehicle (0.1% ethanol) alone (− Dex) or with 0.1 μM dexamethasone (+ Dex) for 36 h, whole cell extracts were prepared for EMSA and for GR or RAP46 immunoblot analysis. The cells were also transfected with the MMTV firefly luciferase indicator construct and an internal control encoding the renilla luciferase. Hormone treatment was performed with 0.1 μM dexamethasone. The results are expressed as dexamethasone-induced activity of the MMTV indicator gene presented relative to the induced level in the absence of RAP46 that was set at unity. The bar chart represents the mean value (± SD) of three independent experiments. C and D, Confocal immunofluorescence analysis of intracellular localization of GR and HA-RAP46Δ70 or HA-RAP46ΔC47 constructs. COS-7 cells were transiently transfected with expression vectors encoding a GR–GFP and HA-tagged RAP46Δ70 (C) or GR–GFP and HA-tagged RAP46ΔC47 (D). 36 h after transfection, the cells were treated for 1 h with vehicle (0.1% ethanol) alone (−) or vehicle containing 0.1 μM dexamethasone (+ Dex) before harvesting, processing, and visualization with a laser confocal microscope. The green fluorescence arises from the GFP-tagged receptor, whereas the red fluorescence comes from staining of RAP46Δ70 and RAP46ΔC47 with the anti-HA mAb 12CA5, followed by an anti-mouse antibody labeled with rhodamine. The yellow to orange colors indicate areas of colocalization of the two proteins.
Figure 5
Figure 5
Regions of RAP46 required for nuclear transport and inhibition of GR activity. A, Schematic representation of different isoforms and deletion mutants of RAP46. Indicated are the isoforms BAG-1L, RAP46, and BAG-1, as well as the deletion mutants RAP46Δ40 and RAP46Δ70. The black vertical bars indicate the positions of the motif [EEX4]. The gray and hatched areas represent the location of a proline rich region (Pro) and of a putative nuclear localization signal (NLS). The sequence of the first 71 NH2-terminal amino acids of RAP46 is also shown with the arrows pointing to the first amino acid in RAP46, RAP46Δ40, BAG-1, and RAP46Δ70. Each [EEX4] motif is underlined. B, Lack of effect of a NH2-terminal deletion mutant of RAP46 on DNA binding by the GR and transactivation by the receptor at the MMTV promoter. COS-7 cells were transiently transfected with a control vector (lanes 1 and 2) or an expression vector for GR (lanes 3–10). In addition, they were cotransfected with either a control vector (lanes 1–4) or a plasmid encoding the wild-type HA-tagged RAP46 (HA-RAP46 wt, lanes 5 and 6) or deletion mutants lacking either the first 40 amino acids (HA-RAP46Δ40, lanes 7 and 8) or the first 70 amino acids (HA-RAP46Δ70, lanes 9 and 10). After treatment with vehicle (0.1% ethanol) alone (− Dex) or with 0.1 μM dexamethasone (+ Dex) for 36 h, whole cell extracts were prepared for EMSA and for GR or RAP46 immunoblot analysis. The cells were also transfected with the MMTV firefly luciferase indicator construct and an internal control encoding the renilla luciferase. Hormone treatment was performed with 0.1 μM dexamethasone. The results are expressed as dexamethasone-induced activity of the MMTV indicator gene presented relative to the induced level in the absence of RAP46 that was set at unity. The bar chart represents the mean value (± SD) of three independent experiments. C and D, Confocal immunofluorescence analysis of intracellular localization of GR and HA-RAP46Δ70 or HA-RAP46ΔC47 constructs. COS-7 cells were transiently transfected with expression vectors encoding a GR–GFP and HA-tagged RAP46Δ70 (C) or GR–GFP and HA-tagged RAP46ΔC47 (D). 36 h after transfection, the cells were treated for 1 h with vehicle (0.1% ethanol) alone (−) or vehicle containing 0.1 μM dexamethasone (+ Dex) before harvesting, processing, and visualization with a laser confocal microscope. The green fluorescence arises from the GFP-tagged receptor, whereas the red fluorescence comes from staining of RAP46Δ70 and RAP46ΔC47 with the anti-HA mAb 12CA5, followed by an anti-mouse antibody labeled with rhodamine. The yellow to orange colors indicate areas of colocalization of the two proteins.
Figure 5
Figure 5
Regions of RAP46 required for nuclear transport and inhibition of GR activity. A, Schematic representation of different isoforms and deletion mutants of RAP46. Indicated are the isoforms BAG-1L, RAP46, and BAG-1, as well as the deletion mutants RAP46Δ40 and RAP46Δ70. The black vertical bars indicate the positions of the motif [EEX4]. The gray and hatched areas represent the location of a proline rich region (Pro) and of a putative nuclear localization signal (NLS). The sequence of the first 71 NH2-terminal amino acids of RAP46 is also shown with the arrows pointing to the first amino acid in RAP46, RAP46Δ40, BAG-1, and RAP46Δ70. Each [EEX4] motif is underlined. B, Lack of effect of a NH2-terminal deletion mutant of RAP46 on DNA binding by the GR and transactivation by the receptor at the MMTV promoter. COS-7 cells were transiently transfected with a control vector (lanes 1 and 2) or an expression vector for GR (lanes 3–10). In addition, they were cotransfected with either a control vector (lanes 1–4) or a plasmid encoding the wild-type HA-tagged RAP46 (HA-RAP46 wt, lanes 5 and 6) or deletion mutants lacking either the first 40 amino acids (HA-RAP46Δ40, lanes 7 and 8) or the first 70 amino acids (HA-RAP46Δ70, lanes 9 and 10). After treatment with vehicle (0.1% ethanol) alone (− Dex) or with 0.1 μM dexamethasone (+ Dex) for 36 h, whole cell extracts were prepared for EMSA and for GR or RAP46 immunoblot analysis. The cells were also transfected with the MMTV firefly luciferase indicator construct and an internal control encoding the renilla luciferase. Hormone treatment was performed with 0.1 μM dexamethasone. The results are expressed as dexamethasone-induced activity of the MMTV indicator gene presented relative to the induced level in the absence of RAP46 that was set at unity. The bar chart represents the mean value (± SD) of three independent experiments. C and D, Confocal immunofluorescence analysis of intracellular localization of GR and HA-RAP46Δ70 or HA-RAP46ΔC47 constructs. COS-7 cells were transiently transfected with expression vectors encoding a GR–GFP and HA-tagged RAP46Δ70 (C) or GR–GFP and HA-tagged RAP46ΔC47 (D). 36 h after transfection, the cells were treated for 1 h with vehicle (0.1% ethanol) alone (−) or vehicle containing 0.1 μM dexamethasone (+ Dex) before harvesting, processing, and visualization with a laser confocal microscope. The green fluorescence arises from the GFP-tagged receptor, whereas the red fluorescence comes from staining of RAP46Δ70 and RAP46ΔC47 with the anti-HA mAb 12CA5, followed by an anti-mouse antibody labeled with rhodamine. The yellow to orange colors indicate areas of colocalization of the two proteins.
Figure 5
Figure 5
Regions of RAP46 required for nuclear transport and inhibition of GR activity. A, Schematic representation of different isoforms and deletion mutants of RAP46. Indicated are the isoforms BAG-1L, RAP46, and BAG-1, as well as the deletion mutants RAP46Δ40 and RAP46Δ70. The black vertical bars indicate the positions of the motif [EEX4]. The gray and hatched areas represent the location of a proline rich region (Pro) and of a putative nuclear localization signal (NLS). The sequence of the first 71 NH2-terminal amino acids of RAP46 is also shown with the arrows pointing to the first amino acid in RAP46, RAP46Δ40, BAG-1, and RAP46Δ70. Each [EEX4] motif is underlined. B, Lack of effect of a NH2-terminal deletion mutant of RAP46 on DNA binding by the GR and transactivation by the receptor at the MMTV promoter. COS-7 cells were transiently transfected with a control vector (lanes 1 and 2) or an expression vector for GR (lanes 3–10). In addition, they were cotransfected with either a control vector (lanes 1–4) or a plasmid encoding the wild-type HA-tagged RAP46 (HA-RAP46 wt, lanes 5 and 6) or deletion mutants lacking either the first 40 amino acids (HA-RAP46Δ40, lanes 7 and 8) or the first 70 amino acids (HA-RAP46Δ70, lanes 9 and 10). After treatment with vehicle (0.1% ethanol) alone (− Dex) or with 0.1 μM dexamethasone (+ Dex) for 36 h, whole cell extracts were prepared for EMSA and for GR or RAP46 immunoblot analysis. The cells were also transfected with the MMTV firefly luciferase indicator construct and an internal control encoding the renilla luciferase. Hormone treatment was performed with 0.1 μM dexamethasone. The results are expressed as dexamethasone-induced activity of the MMTV indicator gene presented relative to the induced level in the absence of RAP46 that was set at unity. The bar chart represents the mean value (± SD) of three independent experiments. C and D, Confocal immunofluorescence analysis of intracellular localization of GR and HA-RAP46Δ70 or HA-RAP46ΔC47 constructs. COS-7 cells were transiently transfected with expression vectors encoding a GR–GFP and HA-tagged RAP46Δ70 (C) or GR–GFP and HA-tagged RAP46ΔC47 (D). 36 h after transfection, the cells were treated for 1 h with vehicle (0.1% ethanol) alone (−) or vehicle containing 0.1 μM dexamethasone (+ Dex) before harvesting, processing, and visualization with a laser confocal microscope. The green fluorescence arises from the GFP-tagged receptor, whereas the red fluorescence comes from staining of RAP46Δ70 and RAP46ΔC47 with the anti-HA mAb 12CA5, followed by an anti-mouse antibody labeled with rhodamine. The yellow to orange colors indicate areas of colocalization of the two proteins.
Figure 6
Figure 6
Dominant–negative effect of the first 69 NH2-terminal amino acids of RAP46 on transactivation and DNA binding by the GR. A, GR-mediated enhancement of transactivation by the first 69 NH2-terminal amino acids of RAP46 fused to GFP. MCF-7 cells were transiently transfected with the MMTV firefly luciferase indicator gene and the internal control pTKRenilla-Luc construct, as well as plasmids encoding either RAP46, GFP, or N69GFP. Dexamethasone treatment (0.1 μM) was performed immediately after the transfection. Shown are results of the dexamethasone-induced MMTV activity expressed relative to unity, which is the value given to the hormone induced activity of the indicator plasmid in the presence of the empty expression vector. The bar charts represent the mean value (± SEM) of two independent experiments. B, The N69GFP fusion overcomes the inhibitory effect of RAP46 on DNA binding by the GR. Shown are the EMSA and immunoblots with extracts of COS-7 cells transiently transfected with either a control vector (lane 1) or a plasmid encoding GR (lanes 2–7). In addition, the cells were transfected with constructs encoding HA-RAP46 (lanes 5–7), the empty expression vector pcDNA3 (lanes 1, 2, and 5), GFP (lanes 3 and 6), or the N69GFP fusion (lanes 4 and 7). After transfection, the cells were treated with 0.1 μM dexamethasone for 36 h and cellular extracts were prepared for the EMSA and immunoblot assay.
Figure 6
Figure 6
Dominant–negative effect of the first 69 NH2-terminal amino acids of RAP46 on transactivation and DNA binding by the GR. A, GR-mediated enhancement of transactivation by the first 69 NH2-terminal amino acids of RAP46 fused to GFP. MCF-7 cells were transiently transfected with the MMTV firefly luciferase indicator gene and the internal control pTKRenilla-Luc construct, as well as plasmids encoding either RAP46, GFP, or N69GFP. Dexamethasone treatment (0.1 μM) was performed immediately after the transfection. Shown are results of the dexamethasone-induced MMTV activity expressed relative to unity, which is the value given to the hormone induced activity of the indicator plasmid in the presence of the empty expression vector. The bar charts represent the mean value (± SEM) of two independent experiments. B, The N69GFP fusion overcomes the inhibitory effect of RAP46 on DNA binding by the GR. Shown are the EMSA and immunoblots with extracts of COS-7 cells transiently transfected with either a control vector (lane 1) or a plasmid encoding GR (lanes 2–7). In addition, the cells were transfected with constructs encoding HA-RAP46 (lanes 5–7), the empty expression vector pcDNA3 (lanes 1, 2, and 5), GFP (lanes 3 and 6), or the N69GFP fusion (lanes 4 and 7). After transfection, the cells were treated with 0.1 μM dexamethasone for 36 h and cellular extracts were prepared for the EMSA and immunoblot assay.
Figure 7
Figure 7
Effect of RAP46 and BAG-1L on DNA-binding, transactivation, and transrepression by the GR. COS-7 cells were transiently transfected with either a control vector, a plasmid encoding the GR, RAP46, or BAG-1L. After transfection, the cells were treated with 0.1 μM dexamethasone for 36 h and whole cell extracts were prepared for EMSA. In addition, the effect of RAP46 and BAG-1L on transactivation at the MMTV promoter and GR-mediated repression at the human collagenase I promoter were analyzed. MCF-7 cells were transiently transfected with a control vector or a plasmid encoding RAP46 or BAG-1L, in addition to the MMTV firefly luciferase indicator or collagenase luciferase constructs and a plasmid encoding the renilla luciferase as an internal control. The activity of the collagenase promoter was induced with 80 ng/ml TPA and the cells were treated immediately after transfection with vehicle alone (0.1% ethanol; −) or 0.1 μM dexamethasone (+) for 36 h. The bar chart shows the normalized luciferase activity (firefly/renilla) of the MMTV and collagenase gene constructs. The results represent the mean value (± SD) of three independent experiments.
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