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Subnuclear trafficking of glucocorticoid receptors in vitro: chromatin recycling and nuclear export.

Yang J, Liu J, DeFranco DB - J. Cell Biol. (1997)

Bottom Line: Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality.If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited.Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

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In vivo recycling of nuclear GRs. GrH2 cells were either treated with corticosterone (Cort) for 1 h (a, lanes 1 and 2; b,  A and B), withdrawn from hormone for 20 min after the 1-h hormone treatment (a, lanes 3 and 4; b, C and D), or treated with  hormone for an additional 10 min after the 20-min withdrawal (a,  lanes 5 and 6; b, E and F). (a) Suspension assay: cells were harvested and either permeabilized and not extracted (NE) or permeabilized and then extracted with Hypo buffer (H) in suspension. Nuclear GRs were detected by Western blot analysis.  NuMA and hnRNP A1 proteins were also visualized on the same  blot. (b) Coverslip assay: cells grown on coverslips were directly  permeabilized and either not extracted (NE; A, C, and E) or extracted with Hypo buffer after permeabilization (Hypo; B, D, and  F). Cells were fixed and GRs were detected by IIF.
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Figure 4: In vivo recycling of nuclear GRs. GrH2 cells were either treated with corticosterone (Cort) for 1 h (a, lanes 1 and 2; b, A and B), withdrawn from hormone for 20 min after the 1-h hormone treatment (a, lanes 3 and 4; b, C and D), or treated with hormone for an additional 10 min after the 20-min withdrawal (a, lanes 5 and 6; b, E and F). (a) Suspension assay: cells were harvested and either permeabilized and not extracted (NE) or permeabilized and then extracted with Hypo buffer (H) in suspension. Nuclear GRs were detected by Western blot analysis. NuMA and hnRNP A1 proteins were also visualized on the same blot. (b) Coverslip assay: cells grown on coverslips were directly permeabilized and either not extracted (NE; A, C, and E) or extracted with Hypo buffer after permeabilization (Hypo; B, D, and F). Cells were fixed and GRs were detected by IIF.

Mentions: Since the rapid release of GR from chromatin upon hormone withdrawal is not coupled to its rapid nuclear export, receptors appear to be held within a distinct, low affinity subnuclear compartment. Upon prolonged hormone withdrawal, nuclear GRs redistribute to the cytoplasm where they eventually regain competence to respond to a secondary hormone challenge (Qi et al., 1989). Is cytoplasmic reentry obligatory for recycled receptors to regain functionality? Given the effectiveness of the hypotonic extraction in distinguishing between chromatin-bound and -released receptors, we asked whether GRs could regain high affinity chromatin binding if charged with hormone while still resident within nuclei. We therefore applied a secondary hormone treatment to GrH2 cells, which had previously been treated with corticosterone for 1 h but briefly withdrawn from hormone (i.e., 20 min). Nuclear GRs were detected by either Western blot analysis (Fig. 4 a) or IIF (Fig. 4 b) after either permeabilization and no extraction (NE), or permeabilization and Hypo buffer extraction (Hypo; H). As shown previously, a 1-h hormone treatment led to the accumulation of nuclear GRs that were not extracted by Hypo buffer (Fig. 4 a, lanes 1 and 2; Fig. 4 b, A and B). In addition, 20 min after hormone withdrawal, GRs remained nuclear (Fig. 4 a, lane 3; Fig. 4 b, C) but were extracted by Hypo buffer (Fig. 4 a, lane 4; Fig. 4 b, D), indicating that most GRs were released from chromatin. Interestingly, when hormone-withdrawn cells were briefly exposed to a 10-min secondary hormone treatment, GRs regained their resistance to hypotonic extraction (Fig. 4 a, lanes 5 and 6; Fig. 4 b, E and F). Thus, nuclear GRs that have been released from chromatin during hormone withdrawal can rebind chromatin upon a secondary hormone exposure.


Subnuclear trafficking of glucocorticoid receptors in vitro: chromatin recycling and nuclear export.

Yang J, Liu J, DeFranco DB - J. Cell Biol. (1997)

In vivo recycling of nuclear GRs. GrH2 cells were either treated with corticosterone (Cort) for 1 h (a, lanes 1 and 2; b,  A and B), withdrawn from hormone for 20 min after the 1-h hormone treatment (a, lanes 3 and 4; b, C and D), or treated with  hormone for an additional 10 min after the 20-min withdrawal (a,  lanes 5 and 6; b, E and F). (a) Suspension assay: cells were harvested and either permeabilized and not extracted (NE) or permeabilized and then extracted with Hypo buffer (H) in suspension. Nuclear GRs were detected by Western blot analysis.  NuMA and hnRNP A1 proteins were also visualized on the same  blot. (b) Coverslip assay: cells grown on coverslips were directly  permeabilized and either not extracted (NE; A, C, and E) or extracted with Hypo buffer after permeabilization (Hypo; B, D, and  F). Cells were fixed and GRs were detected by IIF.
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Related In: Results  -  Collection

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Figure 4: In vivo recycling of nuclear GRs. GrH2 cells were either treated with corticosterone (Cort) for 1 h (a, lanes 1 and 2; b, A and B), withdrawn from hormone for 20 min after the 1-h hormone treatment (a, lanes 3 and 4; b, C and D), or treated with hormone for an additional 10 min after the 20-min withdrawal (a, lanes 5 and 6; b, E and F). (a) Suspension assay: cells were harvested and either permeabilized and not extracted (NE) or permeabilized and then extracted with Hypo buffer (H) in suspension. Nuclear GRs were detected by Western blot analysis. NuMA and hnRNP A1 proteins were also visualized on the same blot. (b) Coverslip assay: cells grown on coverslips were directly permeabilized and either not extracted (NE; A, C, and E) or extracted with Hypo buffer after permeabilization (Hypo; B, D, and F). Cells were fixed and GRs were detected by IIF.
Mentions: Since the rapid release of GR from chromatin upon hormone withdrawal is not coupled to its rapid nuclear export, receptors appear to be held within a distinct, low affinity subnuclear compartment. Upon prolonged hormone withdrawal, nuclear GRs redistribute to the cytoplasm where they eventually regain competence to respond to a secondary hormone challenge (Qi et al., 1989). Is cytoplasmic reentry obligatory for recycled receptors to regain functionality? Given the effectiveness of the hypotonic extraction in distinguishing between chromatin-bound and -released receptors, we asked whether GRs could regain high affinity chromatin binding if charged with hormone while still resident within nuclei. We therefore applied a secondary hormone treatment to GrH2 cells, which had previously been treated with corticosterone for 1 h but briefly withdrawn from hormone (i.e., 20 min). Nuclear GRs were detected by either Western blot analysis (Fig. 4 a) or IIF (Fig. 4 b) after either permeabilization and no extraction (NE), or permeabilization and Hypo buffer extraction (Hypo; H). As shown previously, a 1-h hormone treatment led to the accumulation of nuclear GRs that were not extracted by Hypo buffer (Fig. 4 a, lanes 1 and 2; Fig. 4 b, A and B). In addition, 20 min after hormone withdrawal, GRs remained nuclear (Fig. 4 a, lane 3; Fig. 4 b, C) but were extracted by Hypo buffer (Fig. 4 a, lane 4; Fig. 4 b, D), indicating that most GRs were released from chromatin. Interestingly, when hormone-withdrawn cells were briefly exposed to a 10-min secondary hormone treatment, GRs regained their resistance to hypotonic extraction (Fig. 4 a, lanes 5 and 6; Fig. 4 b, E and F). Thus, nuclear GRs that have been released from chromatin during hormone withdrawal can rebind chromatin upon a secondary hormone exposure.

Bottom Line: Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality.If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited.Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

Show MeSH
Related in: MedlinePlus