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

Schneikert J, Hübner S, Martin E, Cato AC - J. Cell Biol. (1999)

Bottom Line: We demonstrate a specific cytoplasmic-nuclear recruitment of RAP46 by the liganded GR that results in inhibition of the transactivation function of the receptor.BAG-1, a shorter isoform with only a duplication of the [EEX(4)] sequence, does not inhibit GR activity.The nuclear effects of RAP46 and BAG-1L are specific since GR-mediated inhibition of AP-1 activity was not affected.

View Article: PubMed Central - PubMed

Affiliation: Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, D-76021 Karlsruhe, Germany.

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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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.
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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.

Mentions: Through its association with the GR and MR in the cytoplasm, RAP46 may alter the ligand binding properties of these receptors in line with its cochaperone activity (Stuart et al. 1998). We therefore analyzed the hormone binding properties of these receptors in the presence of RAP46 in whole cells and in cytosol preparations. These studies revealed only a slight change in the ligand binding activity of the receptors in the presence of RAP46. For example, Scatchard plot analysis showed an insignificant change in the dissociation constant (Kd) of the receptor for dexamethasone from 20 to 17 nM in the presence of RAP46 (Fig. 2, right). A slight (20%) reduction in the maximum hormone binding capacity (Bmax) of the receptor was also detected (Fig. 2, left). This downregulation of the Bmax is minimal compared with our previous report of a strong RAP46-mediated inhibition of transactivation by the GR (Kullmann et al. 1998). Thus, RAP46 must exert its negative regulatory function at other stages in the action of the GR.


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

Schneikert J, Hübner S, Martin E, Cato AC - J. Cell Biol. (1999)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169481&req=5

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.
Mentions: Through its association with the GR and MR in the cytoplasm, RAP46 may alter the ligand binding properties of these receptors in line with its cochaperone activity (Stuart et al. 1998). We therefore analyzed the hormone binding properties of these receptors in the presence of RAP46 in whole cells and in cytosol preparations. These studies revealed only a slight change in the ligand binding activity of the receptors in the presence of RAP46. For example, Scatchard plot analysis showed an insignificant change in the dissociation constant (Kd) of the receptor for dexamethasone from 20 to 17 nM in the presence of RAP46 (Fig. 2, right). A slight (20%) reduction in the maximum hormone binding capacity (Bmax) of the receptor was also detected (Fig. 2, left). This downregulation of the Bmax is minimal compared with our previous report of a strong RAP46-mediated inhibition of transactivation by the GR (Kullmann et al. 1998). Thus, RAP46 must exert its negative regulatory function at other stages in the action of the GR.

Bottom Line: We demonstrate a specific cytoplasmic-nuclear recruitment of RAP46 by the liganded GR that results in inhibition of the transactivation function of the receptor.BAG-1, a shorter isoform with only a duplication of the [EEX(4)] sequence, does not inhibit GR activity.The nuclear effects of RAP46 and BAG-1L are specific since GR-mediated inhibition of AP-1 activity was not affected.

View Article: PubMed Central - PubMed

Affiliation: Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, D-76021 Karlsruhe, Germany.

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.

Show MeSH
Related in: MedlinePlus