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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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TRα requires CRT for nuclear export. crt-/- cells were transfected with a GFP-TRα expression plasmid and nucleocytoplasmic shuttling was monitored through FRAP (n = 8). White arrowheads indicate bleached nuclei. DIC images were taken to delineate cell borders and a fluorescence recovery graph indicating relative shuttling of GFP-TRα was generated. Bar, 10 μm.
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fig2: TRα requires CRT for nuclear export. crt-/- cells were transfected with a GFP-TRα expression plasmid and nucleocytoplasmic shuttling was monitored through FRAP (n = 8). White arrowheads indicate bleached nuclei. DIC images were taken to delineate cell borders and a fluorescence recovery graph indicating relative shuttling of GFP-TRα was generated. Bar, 10 μm.

Mentions: TRα Shuttling Is Inhibited in Living Cells Deficient in CRT Expression—CRT has previously been shown to function as an exportin for nuclear receptors related to TRα (8, 11, 12, 31). To address the question of whether TRα export is also mediated by CRT, we transiently transfected mouse embryonic fibroblast cells isolated from CRT knock-out mouse embryos (crt-/- cells) (23) with GFP-TRα and monitored FRAP in bleached nuclei of transfected CRT-deficient monokaryons (Fig. 2).


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)

TRα requires CRT for nuclear export. crt-/- cells were transfected with a GFP-TRα expression plasmid and nucleocytoplasmic shuttling was monitored through FRAP (n = 8). White arrowheads indicate bleached nuclei. DIC images were taken to delineate cell borders and a fluorescence recovery graph indicating relative shuttling of GFP-TRα was generated. Bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: TRα requires CRT for nuclear export. crt-/- cells were transfected with a GFP-TRα expression plasmid and nucleocytoplasmic shuttling was monitored through FRAP (n = 8). White arrowheads indicate bleached nuclei. DIC images were taken to delineate cell borders and a fluorescence recovery graph indicating relative shuttling of GFP-TRα was generated. Bar, 10 μm.
Mentions: TRα Shuttling Is Inhibited in Living Cells Deficient in CRT Expression—CRT has previously been shown to function as an exportin for nuclear receptors related to TRα (8, 11, 12, 31). To address the question of whether TRα export is also mediated by CRT, we transiently transfected mouse embryonic fibroblast cells isolated from CRT knock-out mouse embryos (crt-/- cells) (23) with GFP-TRα and monitored FRAP in bleached nuclei of transfected CRT-deficient monokaryons (Fig. 2).

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
Related in: MedlinePlus