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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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Differential extraction of GR from nuclei of  hormone-treated vs hormonewithdrawn cells. GrH2 cells  grown in hormone-free medium were treated with 10−6 M  corticosterone (Cort) for 1 h,  and then either incubated  with hormone for an additional 30 min (a, A–C; b,  lanes 1–3) or withdrawn from  hormone for 30 min (a, D–F;  b, lanes 4–6). Cells were either  permeabilized using digitonin (a, A and D; b, lanes 1  and 4), or permeabilized and  then subjected to extractions  with either Hypo buffer (a, B  and E; b, lanes 2 and 5) or  CK buffer (a, C and F; b,  lanes 3 and 6). (a) In situ extraction of cells grown on  coverslips. GR was visualized  by IIF using BuGR2. (b) Differential extraction of GR  from cells in suspension. After permeabilization and extraction in suspension, nuclear proteins were resolved  by SDS-PAGE. GR, NuMA,  and hnRNP A1 were detected by Western blot analysis and ECL using BuGR2,  the Ab-1 anti-NuMA, and the  9H10 anti–hnRNP A1 mAbs,  respectively. (c) Quantification of results in b by densitometry (mean ± SD of four  experiments). The relative  ratio of GR to NuMA in intact nuclei (NE) was set at  100. NE, intact GrH2 nuclei  after permeabilization, not  extracted; Hypo and H, nuclei after permeabilization  and Hypo buffer extraction;  CK and C, nuclei after permeabilization and CK buffer  extraction.
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Figure 1: Differential extraction of GR from nuclei of hormone-treated vs hormonewithdrawn cells. GrH2 cells grown in hormone-free medium were treated with 10−6 M corticosterone (Cort) for 1 h, and then either incubated with hormone for an additional 30 min (a, A–C; b, lanes 1–3) or withdrawn from hormone for 30 min (a, D–F; b, lanes 4–6). Cells were either permeabilized using digitonin (a, A and D; b, lanes 1 and 4), or permeabilized and then subjected to extractions with either Hypo buffer (a, B and E; b, lanes 2 and 5) or CK buffer (a, C and F; b, lanes 3 and 6). (a) In situ extraction of cells grown on coverslips. GR was visualized by IIF using BuGR2. (b) Differential extraction of GR from cells in suspension. After permeabilization and extraction in suspension, nuclear proteins were resolved by SDS-PAGE. GR, NuMA, and hnRNP A1 were detected by Western blot analysis and ECL using BuGR2, the Ab-1 anti-NuMA, and the 9H10 anti–hnRNP A1 mAbs, respectively. (c) Quantification of results in b by densitometry (mean ± SD of four experiments). The relative ratio of GR to NuMA in intact nuclei (NE) was set at 100. NE, intact GrH2 nuclei after permeabilization, not extracted; Hypo and H, nuclei after permeabilization and Hypo buffer extraction; CK and C, nuclei after permeabilization and CK buffer extraction.

Mentions: Fig. 1 shows the differential extraction of GR from nuclei of hormone-treated vs hormone-withdrawn cells. GRs accumulated in nuclei after a 1 h of corticosterone treatment (Fig. 1 a, A, and Fig. 1 b, lane 1) and remained nuclear after 30 min of hormone withdrawal (Fig. 1 a, D, and Fig. 1 b, lane 4). While Hypo buffer extraction of permeabilized cells removed ∼20% of the nuclear GR from hormone-treated cells (Fig. 1 a, B; Figs. 1 b and c, lane 2), 80% of nuclear GRs were extracted by Hypo buffer from hormone-withdrawn cells (Fig. 1 a, E; Fig. 1, b and c, lane 5). Thus, although a brief hormone withdrawal does not apparently alter the nuclear localization of GRs, unliganded and liganded nuclear receptors differ dramatically in their nuclear affinity. Importantly, these results also establish that GR nuclear export is not merely restricted by high affinity binding of GR to nuclei. GRs in hormonetreated cells are not artificially trapped within nuclei by our permeabilization conditions, as a high salt, detergent wash (i.e., CK buffer) efficiently extracts 80% of nuclear GR (Fig. 1 a, C and F; Fig. 1, b and c, lanes 3 and 6). DAPI staining confirmed that nuclei remained intact after this extraction (not shown). The residual amount of GR that resists CK extraction (Fig. 1, b and c, lane 3) may represent nuclear matrix–associated receptors (Tang and DeFranco, 1996).


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

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

Differential extraction of GR from nuclei of  hormone-treated vs hormonewithdrawn cells. GrH2 cells  grown in hormone-free medium were treated with 10−6 M  corticosterone (Cort) for 1 h,  and then either incubated  with hormone for an additional 30 min (a, A–C; b,  lanes 1–3) or withdrawn from  hormone for 30 min (a, D–F;  b, lanes 4–6). Cells were either  permeabilized using digitonin (a, A and D; b, lanes 1  and 4), or permeabilized and  then subjected to extractions  with either Hypo buffer (a, B  and E; b, lanes 2 and 5) or  CK buffer (a, C and F; b,  lanes 3 and 6). (a) In situ extraction of cells grown on  coverslips. GR was visualized  by IIF using BuGR2. (b) Differential extraction of GR  from cells in suspension. After permeabilization and extraction in suspension, nuclear proteins were resolved  by SDS-PAGE. GR, NuMA,  and hnRNP A1 were detected by Western blot analysis and ECL using BuGR2,  the Ab-1 anti-NuMA, and the  9H10 anti–hnRNP A1 mAbs,  respectively. (c) Quantification of results in b by densitometry (mean ± SD of four  experiments). The relative  ratio of GR to NuMA in intact nuclei (NE) was set at  100. NE, intact GrH2 nuclei  after permeabilization, not  extracted; Hypo and H, nuclei after permeabilization  and Hypo buffer extraction;  CK and C, nuclei after permeabilization and CK buffer  extraction.
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Figure 1: Differential extraction of GR from nuclei of hormone-treated vs hormonewithdrawn cells. GrH2 cells grown in hormone-free medium were treated with 10−6 M corticosterone (Cort) for 1 h, and then either incubated with hormone for an additional 30 min (a, A–C; b, lanes 1–3) or withdrawn from hormone for 30 min (a, D–F; b, lanes 4–6). Cells were either permeabilized using digitonin (a, A and D; b, lanes 1 and 4), or permeabilized and then subjected to extractions with either Hypo buffer (a, B and E; b, lanes 2 and 5) or CK buffer (a, C and F; b, lanes 3 and 6). (a) In situ extraction of cells grown on coverslips. GR was visualized by IIF using BuGR2. (b) Differential extraction of GR from cells in suspension. After permeabilization and extraction in suspension, nuclear proteins were resolved by SDS-PAGE. GR, NuMA, and hnRNP A1 were detected by Western blot analysis and ECL using BuGR2, the Ab-1 anti-NuMA, and the 9H10 anti–hnRNP A1 mAbs, respectively. (c) Quantification of results in b by densitometry (mean ± SD of four experiments). The relative ratio of GR to NuMA in intact nuclei (NE) was set at 100. NE, intact GrH2 nuclei after permeabilization, not extracted; Hypo and H, nuclei after permeabilization and Hypo buffer extraction; CK and C, nuclei after permeabilization and CK buffer extraction.
Mentions: Fig. 1 shows the differential extraction of GR from nuclei of hormone-treated vs hormone-withdrawn cells. GRs accumulated in nuclei after a 1 h of corticosterone treatment (Fig. 1 a, A, and Fig. 1 b, lane 1) and remained nuclear after 30 min of hormone withdrawal (Fig. 1 a, D, and Fig. 1 b, lane 4). While Hypo buffer extraction of permeabilized cells removed ∼20% of the nuclear GR from hormone-treated cells (Fig. 1 a, B; Figs. 1 b and c, lane 2), 80% of nuclear GRs were extracted by Hypo buffer from hormone-withdrawn cells (Fig. 1 a, E; Fig. 1, b and c, lane 5). Thus, although a brief hormone withdrawal does not apparently alter the nuclear localization of GRs, unliganded and liganded nuclear receptors differ dramatically in their nuclear affinity. Importantly, these results also establish that GR nuclear export is not merely restricted by high affinity binding of GR to nuclei. GRs in hormonetreated cells are not artificially trapped within nuclei by our permeabilization conditions, as a high salt, detergent wash (i.e., CK buffer) efficiently extracts 80% of nuclear GR (Fig. 1 a, C and F; Fig. 1, b and c, lanes 3 and 6). DAPI staining confirmed that nuclei remained intact after this extraction (not shown). The residual amount of GR that resists CK extraction (Fig. 1, b and c, lane 3) may represent nuclear matrix–associated receptors (Tang and DeFranco, 1996).

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