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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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Time course of GR sensitivity to Hypo buffer extraction. GrH2 cells grown on coverslips were treated with 1 μM corticosterone (Cort) for 1 h. Cells were then withdrawn from hormone for either 0 min (A and B), 10 min (C and D), or 30 min  (E–H). After permeabilization, cells were either fixed directly (G  and H) or subjected to Hypo buffer extraction before fixation  (A–F). (A, C, E, and G) GR staining detected by IIF. (B, D, F,  and H) DNA detected by DAPI staining.
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Figure 3: Time course of GR sensitivity to Hypo buffer extraction. GrH2 cells grown on coverslips were treated with 1 μM corticosterone (Cort) for 1 h. Cells were then withdrawn from hormone for either 0 min (A and B), 10 min (C and D), or 30 min (E–H). After permeabilization, cells were either fixed directly (G and H) or subjected to Hypo buffer extraction before fixation (A–F). (A, C, E, and G) GR staining detected by IIF. (B, D, F, and H) DNA detected by DAPI staining.

Mentions: To examine the kinetics of GR release from chromatin upon hormone withdrawal, we performed a time course study of GR sensitivity to hypotonic extraction. As shown previously, GRs in hormone-treated cells are relatively resistant to Hypo buffer extraction (Fig. 3 A). However, within 10 min of hormone withdrawal, most GRs were extracted by Hypo buffer (Fig. 3 C). Nuclear GRs appeared to be maximally extracted within 30 min of hormone withdrawal (Fig. 3 E). As noted above, in cells permeabilized after a 30-min hormone withdrawal, GRs localized within nuclei (Fig. 3 G). These results suggest that, while GRs are rapidly released from chromatin after dissociation of hormone (i.e., within 30 min), they remain associated with nuclei. Importantly, in hormone-withdrawn cells, the kinetics of bulk GR dissociation from chromatin corresponds precisely with the reversal of glucocorticoid-induced nuclease hypersensitivity within chromatin of the glucocorticoidresponsive TAT gene (Reik et al., 1991). Thus, hormone withdrawal leads to release of both bulk GRs from chromatin, and GRs bound to chromatin at specific target genes. While the possibility that unliganded nuclear GRs remain loosely bound to chromatin cannot be excluded, we will refer to these receptors as localizing within a distinct subnuclear compartment, with the caveat that this has only been defined biochemically and may not represent a unique structural component of the nucleus. As will be revealed below, the subnuclear trafficking of these receptors is distinguished from that of chromatin-bound receptors.


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

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

Time course of GR sensitivity to Hypo buffer extraction. GrH2 cells grown on coverslips were treated with 1 μM corticosterone (Cort) for 1 h. Cells were then withdrawn from hormone for either 0 min (A and B), 10 min (C and D), or 30 min  (E–H). After permeabilization, cells were either fixed directly (G  and H) or subjected to Hypo buffer extraction before fixation  (A–F). (A, C, E, and G) GR staining detected by IIF. (B, D, F,  and H) DNA detected by DAPI staining.
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Related In: Results  -  Collection

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Figure 3: Time course of GR sensitivity to Hypo buffer extraction. GrH2 cells grown on coverslips were treated with 1 μM corticosterone (Cort) for 1 h. Cells were then withdrawn from hormone for either 0 min (A and B), 10 min (C and D), or 30 min (E–H). After permeabilization, cells were either fixed directly (G and H) or subjected to Hypo buffer extraction before fixation (A–F). (A, C, E, and G) GR staining detected by IIF. (B, D, F, and H) DNA detected by DAPI staining.
Mentions: To examine the kinetics of GR release from chromatin upon hormone withdrawal, we performed a time course study of GR sensitivity to hypotonic extraction. As shown previously, GRs in hormone-treated cells are relatively resistant to Hypo buffer extraction (Fig. 3 A). However, within 10 min of hormone withdrawal, most GRs were extracted by Hypo buffer (Fig. 3 C). Nuclear GRs appeared to be maximally extracted within 30 min of hormone withdrawal (Fig. 3 E). As noted above, in cells permeabilized after a 30-min hormone withdrawal, GRs localized within nuclei (Fig. 3 G). These results suggest that, while GRs are rapidly released from chromatin after dissociation of hormone (i.e., within 30 min), they remain associated with nuclei. Importantly, in hormone-withdrawn cells, the kinetics of bulk GR dissociation from chromatin corresponds precisely with the reversal of glucocorticoid-induced nuclease hypersensitivity within chromatin of the glucocorticoidresponsive TAT gene (Reik et al., 1991). Thus, hormone withdrawal leads to release of both bulk GRs from chromatin, and GRs bound to chromatin at specific target genes. While the possibility that unliganded nuclear GRs remain loosely bound to chromatin cannot be excluded, we will refer to these receptors as localizing within a distinct subnuclear compartment, with the caveat that this has only been defined biochemically and may not represent a unique structural component of the nucleus. As will be revealed below, the subnuclear trafficking of these receptors is distinguished from that of chromatin-bound receptors.

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