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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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Model for nuclear export of thyroid hormone receptor (TRα) involving a cooperative CRT and CRM1-mediated pathway. A, nuclear export complex in cells expressing CRT (HeLa, crt+/+) in which CRT binding promotes a cooperative export pathway involving CRM1. This cooperative interaction is indicated by the double-headed curved arrow. CRM1 binding to TRα may require additional factors (indicated by dashed line). Although CRT levels are low under these conditions, export of TRα is efficient and rapid export is observed. B, inefficient nuclear export of TRα in cells expressing CRT (HeLa, crt+/+) occurs upon treatment with LMB. CRM1 is inactivated but CRT can still support modest export autonomously. C, rapid export of TRα occurs even in the presence of LMB upon PEG-induced heterokaryon fusion. Although the CRM1 pathway is inactivated, transient CRT release from the ER renders sufficient CRT levels to support rapid export of TRα. D, crt-/- cells do not support nuclear export of TRα because CRT is not present to facilitate the CRM1-dependent component of the export pathway.
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fig9: Model for nuclear export of thyroid hormone receptor (TRα) involving a cooperative CRT and CRM1-mediated pathway. A, nuclear export complex in cells expressing CRT (HeLa, crt+/+) in which CRT binding promotes a cooperative export pathway involving CRM1. This cooperative interaction is indicated by the double-headed curved arrow. CRM1 binding to TRα may require additional factors (indicated by dashed line). Although CRT levels are low under these conditions, export of TRα is efficient and rapid export is observed. B, inefficient nuclear export of TRα in cells expressing CRT (HeLa, crt+/+) occurs upon treatment with LMB. CRM1 is inactivated but CRT can still support modest export autonomously. C, rapid export of TRα occurs even in the presence of LMB upon PEG-induced heterokaryon fusion. Although the CRM1 pathway is inactivated, transient CRT release from the ER renders sufficient CRT levels to support rapid export of TRα. D, crt-/- cells do not support nuclear export of TRα because CRT is not present to facilitate the CRM1-dependent component of the export pathway.

Mentions: Here, we present findings that provide evidence for a previously uncharacterized mechanism for the dynamic shuttling of TRα. We have used a combination of in vivo FRAP experiments, in vitro digitonin-permeabilized cell nuclear export assays of transiently transfected cells, and GST pull-down assays to investigate aspects of TRα subcellular trafficking. Taken together, our data suggest a novel export mechanism in which CRT directly binds TRα, and CRT and CRM1 work cooperatively to promote rapid, efficient export of TRα from the nucleus (Fig. 9). Alternatively, when the CRM1 pathway is blocked or CRT levels are increased under cellular stress, CRT can act independently as a less efficient exportin. crt-/- cell lines fail to support nuclear export of TRα, suggesting that CRT is indispensable for TRα nuclear export. Thus, CRT may be the most important component of the TRα nuclear export pathway. These data suggest that CRT deficiency prevents CRM1 interacting either directly or indirectly with TRα, and inhibits both cooperative and autonomous TRα export as a consequence.


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)

Model for nuclear export of thyroid hormone receptor (TRα) involving a cooperative CRT and CRM1-mediated pathway. A, nuclear export complex in cells expressing CRT (HeLa, crt+/+) in which CRT binding promotes a cooperative export pathway involving CRM1. This cooperative interaction is indicated by the double-headed curved arrow. CRM1 binding to TRα may require additional factors (indicated by dashed line). Although CRT levels are low under these conditions, export of TRα is efficient and rapid export is observed. B, inefficient nuclear export of TRα in cells expressing CRT (HeLa, crt+/+) occurs upon treatment with LMB. CRM1 is inactivated but CRT can still support modest export autonomously. C, rapid export of TRα occurs even in the presence of LMB upon PEG-induced heterokaryon fusion. Although the CRM1 pathway is inactivated, transient CRT release from the ER renders sufficient CRT levels to support rapid export of TRα. D, crt-/- cells do not support nuclear export of TRα because CRT is not present to facilitate the CRM1-dependent component of the export pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: Model for nuclear export of thyroid hormone receptor (TRα) involving a cooperative CRT and CRM1-mediated pathway. A, nuclear export complex in cells expressing CRT (HeLa, crt+/+) in which CRT binding promotes a cooperative export pathway involving CRM1. This cooperative interaction is indicated by the double-headed curved arrow. CRM1 binding to TRα may require additional factors (indicated by dashed line). Although CRT levels are low under these conditions, export of TRα is efficient and rapid export is observed. B, inefficient nuclear export of TRα in cells expressing CRT (HeLa, crt+/+) occurs upon treatment with LMB. CRM1 is inactivated but CRT can still support modest export autonomously. C, rapid export of TRα occurs even in the presence of LMB upon PEG-induced heterokaryon fusion. Although the CRM1 pathway is inactivated, transient CRT release from the ER renders sufficient CRT levels to support rapid export of TRα. D, crt-/- cells do not support nuclear export of TRα because CRT is not present to facilitate the CRM1-dependent component of the export pathway.
Mentions: Here, we present findings that provide evidence for a previously uncharacterized mechanism for the dynamic shuttling of TRα. We have used a combination of in vivo FRAP experiments, in vitro digitonin-permeabilized cell nuclear export assays of transiently transfected cells, and GST pull-down assays to investigate aspects of TRα subcellular trafficking. Taken together, our data suggest a novel export mechanism in which CRT directly binds TRα, and CRT and CRM1 work cooperatively to promote rapid, efficient export of TRα from the nucleus (Fig. 9). Alternatively, when the CRM1 pathway is blocked or CRT levels are increased under cellular stress, CRT can act independently as a less efficient exportin. crt-/- cell lines fail to support nuclear export of TRα, suggesting that CRT is indispensable for TRα nuclear export. Thus, CRT may be the most important component of the TRα nuclear export pathway. These data suggest that CRT deficiency prevents CRM1 interacting either directly or indirectly with TRα, and inhibits both cooperative and autonomous TRα export as a consequence.

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