Thyroid Hormone Receptor α1 Follows a Cooperative CRM1/Calreticulin-mediated Nuclear Export Pathway

Abstract

The thyroid hormone receptor ␣1 (TR␣) 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 TR␣, 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 TR␣ is its ability to exit the nucleus through the nuclear pore complex. TR␣ 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 TR␣. We show that, in addition to shuttling in heterokaryons, TR␣ shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TR␣ directly interacts with calreticulin, and point to the intriguing possibility that TR␣ follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TR␣ from the nucleus to cytoplasm. The thyroid hormone receptor ␣1 (TR␣)4 is a member of the nuclear receptor superfamily of transcription factors. TR␣ acts as an intracellular receptor for thyroid hormone (T 3 ), thereby regulating expression of T 3 -responsive genes associated with many aspects of development, growth, and metabolism. Among the nuclear receptors, TR␣ is particularly intriguing in that it can modulate transcription whether or not it is bound to T 3 . Consistent with this dual role as an activator or repressor of transcription, at steady state TR␣ appears to be almost exclusively localized in the nucleus. However, we have shown that the receptor, in fact, shuttles rapidly between the nucleus and cytoplasm (1). Whereas the significance of this nucleocytoplasmic shuttling remains to be precisely characterized, this activity may be related directly to regulation of TR␣ target genes as well as to yet unknown non-genomic functions (2).Nucleocytoplasmic shuttling occurs as a result of a dynamic balance between the recognition of nuclear localization signals (NLS) and nuclear export signals (NES) by particular import and export factors termed importins and exportins, respectively (3). Most nuclear receptors appear to enter the nucleus via importin ␣/␤ recognition and subsequent translocation through the nuclear pore complex (3, 4). Unlike nuclear import, however, the export pathways followed by nuclear receptors remain more elusive.The most thoroughly studied and well characterized nuclear export pathway involves the exportin CRM1. Many shuttling transcription factors outside of the nuclear receptor superfamily follow a CRM1-dependent mechanism (5, 6). Like other nuclear receptors, however, TR␣ lacks the leucine-rich NES associated with classical CRM1-mediated nuclear export (1, 7). Concordantly, we ha...

Multiple exportins influence thyroid hormone receptor localization

“…After normalization, convergence of the representative curves for bleached and unbleached nuclei toward one another represents the degree of fluorescence equilibration between these compartments. When one bleached and one unbleached nucleus are present, complete equilibration occurs at 0.5 fluorescence units (Grespin et al, 2008). …”

“…Encoded by two different genes, thyroid hormone receptors TRα1 and TRβ1 respond to T 3 levels by activating or repressing target gene expression. Although primarily found in the nucleus at steady-state, TRα1 and TRβ1 can rapidly shuttle between the nucleus and cytoplasm (Baumann et al, 2001; Bunn et al, 2001; Grespin et al, 2008). Nuclear import and export of proteins occurs through the nuclear pore complexes, mediated by members of the karyopherin β family called importins and exportins, respectively (Kimura and Imamoto, 2014; Pemberton and Paschal, 2005).…”

“…Previously, we showed that TR can exit the nucleus by a pathway mediated by the export factor CRM1 (chromosome region maintenance 1), also known as exportin 1, in cooperation with calreticulin (Grespin et al, 2008); however, the exact interaction and mechanism remain unclear. Two main lines of evidence suggested that TR might also follow a CRM1/calreticulin-independent nuclear export pathway.…”

“…First, under conditions in which TR still shuttles, shuttling of p53 and the oncoprotein v-ErbA is completely blocked in the presence of the CRM1-specific inhibitor leptomycin B (LMB) (Bunn et al, 2001; DeLong et al, 2004). Also, during fluorescence recovery after photobleaching (FRAP) experiments, when one nucleus in a multinucleate HeLa cell was photobleached, recovery of fluorescence in the bleached nucleus in the presence of LMB was reduced by only 60%, relative to recovery in the absence of LMB (Grespin et al, 2008). Second, no CRM1-dependent NES in TR has yet been characterized (Mavinakere et al, 2012).…”

“…Second, no CRM1-dependent NES in TR has yet been characterized (Mavinakere et al, 2012). Indeed, our studies showed that TR interacts directly with calreticulin but complex formation with CRM1 was not detectable in pull-down assays (Grespin et al, 2008). In an effort to identify and clarify the mode of TR nuclear export, we previously carried out a comprehensive analysis of TR to screen for NES motifs.…”