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Thyroid hormone receptor alpha1 follows a cooperative CRM1/calreticulin-mediated nuclear export pathway.

Grespin ME, Bonamy GM, Roggero VR, Cameron NG, Adam LE, Atchison AP, Fratto VM, Allison LA - J. Biol. Chem. (2008)

Bottom Line: An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex.We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well.Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of William and Mary, 10675 John Jay Hopkins Drive, Williamsburg, VA 23187, USA.

ABSTRACT
The thyroid hormone receptor alpha1 (TRalpha) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T(3)). Previously, we have shown that TRalpha, formerly thought to reside solely in the nucleus bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex. TRalpha export is not sensitive to treatment with the CRM1-specific inhibitor leptomycin B (LMB) in heterokaryon assays, suggesting a role for an export receptor other than CRM1. Here, we have used a combined approach of in vivo fluorescence recovery after photobleaching experiments, in vitro permeabilized cell nuclear export assays, and glutathione S-transferase pull-down assays to investigate the export pathway used by TRalpha. We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm.

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Nuclear export of thyroid hormone receptor (TRα) in permeabilized cells. A, GFP-TRα remains nuclear at t40 in the presence of 670 nm CRT. HeLa cells transfected with a GFP-TRα expression plasmid were digitonin permeabilized, and incubated in an export reaction containing CRT, energy regeneration system, and export buffer alone. At t40 no GFP-TRα export was observed. B, export reactions containing RRL, energy regeneration system, and export buffer were able to support >80% loss of nuclear fluorescence of GFP-TRα at t40. C, export reactions containing both CRT and RRL were able to support >95% loss of nuclear fluorescence of GFP-TRα at t40. White values were normalized to 2,000 using IPlab 3.55 for A-C. D, enlarged section from panels B, t40 (*), and C, t40 (**). White values for * from panel B and ** from panel C were adjusted to 200 for low intensity visualization of the residual GFP-TRα in export reactions. Bar, 10 μm.
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fig6: Nuclear export of thyroid hormone receptor (TRα) in permeabilized cells. A, GFP-TRα remains nuclear at t40 in the presence of 670 nm CRT. HeLa cells transfected with a GFP-TRα expression plasmid were digitonin permeabilized, and incubated in an export reaction containing CRT, energy regeneration system, and export buffer alone. At t40 no GFP-TRα export was observed. B, export reactions containing RRL, energy regeneration system, and export buffer were able to support >80% loss of nuclear fluorescence of GFP-TRα at t40. C, export reactions containing both CRT and RRL were able to support >95% loss of nuclear fluorescence of GFP-TRα at t40. White values were normalized to 2,000 using IPlab 3.55 for A-C. D, enlarged section from panels B, t40 (*), and C, t40 (**). White values for * from panel B and ** from panel C were adjusted to 200 for low intensity visualization of the residual GFP-TRα in export reactions. Bar, 10 μm.

Mentions: Efficient Nuclear Export of TRα in Permeabilized Cells Requires Cytosol and CRT—To provide further evidence for a role of CRT in the nuclear export of TRα, we performed permeabilized cell in vitro nuclear export assays utilizing purified recombinant GST-CRT. For this assay, HeLa cells were transiently transfected with a GFP-TRα expression plasmid. GFP-TRα displays a complete and strong nuclear fluorescence 16 h post-transfection. At this point the outer cell membrane was permeabilized with digitonin and export reactions were performed. First, we sought to assess whether exogenous CRT was sufficient to induce nuclear export of TRα. Our results showed no observable change in TRα nuclear localization between 0 and 40 min irrespective of varying CRT concentration from 270 nm to 1 μm. During this period, all TRα remained localized to the nucleus (Fig. 6A).


Thyroid hormone receptor alpha1 follows a cooperative CRM1/calreticulin-mediated nuclear export pathway.

Grespin ME, Bonamy GM, Roggero VR, Cameron NG, Adam LE, Atchison AP, Fratto VM, Allison LA - J. Biol. Chem. (2008)

Nuclear export of thyroid hormone receptor (TRα) in permeabilized cells. A, GFP-TRα remains nuclear at t40 in the presence of 670 nm CRT. HeLa cells transfected with a GFP-TRα expression plasmid were digitonin permeabilized, and incubated in an export reaction containing CRT, energy regeneration system, and export buffer alone. At t40 no GFP-TRα export was observed. B, export reactions containing RRL, energy regeneration system, and export buffer were able to support >80% loss of nuclear fluorescence of GFP-TRα at t40. C, export reactions containing both CRT and RRL were able to support >95% loss of nuclear fluorescence of GFP-TRα at t40. White values were normalized to 2,000 using IPlab 3.55 for A-C. D, enlarged section from panels B, t40 (*), and C, t40 (**). White values for * from panel B and ** from panel C were adjusted to 200 for low intensity visualization of the residual GFP-TRα in export reactions. Bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2533095&req=5

fig6: Nuclear export of thyroid hormone receptor (TRα) in permeabilized cells. A, GFP-TRα remains nuclear at t40 in the presence of 670 nm CRT. HeLa cells transfected with a GFP-TRα expression plasmid were digitonin permeabilized, and incubated in an export reaction containing CRT, energy regeneration system, and export buffer alone. At t40 no GFP-TRα export was observed. B, export reactions containing RRL, energy regeneration system, and export buffer were able to support >80% loss of nuclear fluorescence of GFP-TRα at t40. C, export reactions containing both CRT and RRL were able to support >95% loss of nuclear fluorescence of GFP-TRα at t40. White values were normalized to 2,000 using IPlab 3.55 for A-C. D, enlarged section from panels B, t40 (*), and C, t40 (**). White values for * from panel B and ** from panel C were adjusted to 200 for low intensity visualization of the residual GFP-TRα in export reactions. Bar, 10 μm.
Mentions: Efficient Nuclear Export of TRα in Permeabilized Cells Requires Cytosol and CRT—To provide further evidence for a role of CRT in the nuclear export of TRα, we performed permeabilized cell in vitro nuclear export assays utilizing purified recombinant GST-CRT. For this assay, HeLa cells were transiently transfected with a GFP-TRα expression plasmid. GFP-TRα displays a complete and strong nuclear fluorescence 16 h post-transfection. At this point the outer cell membrane was permeabilized with digitonin and export reactions were performed. First, we sought to assess whether exogenous CRT was sufficient to induce nuclear export of TRα. Our results showed no observable change in TRα nuclear localization between 0 and 40 min irrespective of varying CRT concentration from 270 nm to 1 μm. During this period, all TRα remained localized to the nucleus (Fig. 6A).

Bottom Line: An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex.We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well.Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of William and Mary, 10675 John Jay Hopkins Drive, Williamsburg, VA 23187, USA.

ABSTRACT
The thyroid hormone receptor alpha1 (TRalpha) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T(3)). Previously, we have shown that TRalpha, formerly thought to reside solely in the nucleus bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex. TRalpha export is not sensitive to treatment with the CRM1-specific inhibitor leptomycin B (LMB) in heterokaryon assays, suggesting a role for an export receptor other than CRM1. Here, we have used a combined approach of in vivo fluorescence recovery after photobleaching experiments, in vitro permeabilized cell nuclear export assays, and glutathione S-transferase pull-down assays to investigate the export pathway used by TRalpha. We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm.

Show MeSH