Thyroid Hormone Receptor Nuclear Export

Abstract

Most shuttling proteins exit the nucleus via a CRM-1 mediated process. A role has been proposed for calreticulin (CRT) in mediating steroid receptor nuclear export, although this is debated. We recently showed that the thyroid hormone receptor (TR), shuttles rapidly between the nucleus and cytoplasm. Nuclear export of TR is not blocked by leptomycin B, a specific inhibitor of CRM1, indicating that TR export is CRM1-independent. In contrast to TR, its oncogenic homolog v-ErbA follows a CRM1-mediated pathway. To determine whether nuclear export of TR occurs by a CRT-mediated pathway, in vitro digitonin permeabilization assays were used to assess export requirements. HeLa cells were transfected with a GFP-TR expression construct. Twenty-four hours after transfection, the cells were permeabilized with digitonin, and incubated in either recombinant GST-CRT or His-CRM-1 in export buffer. Cells were fixed at time points between 0 to 40 min and the distribution of TR was visualized by fluorescence microscopy. Our data show that GFP-TR remains localized in the permeabilized cell nucleus over time in the presence of rabbit reticulocyte lysate, export buffer alone, or CRM-1. However, in the presence of CRT, after 10 min GFP-TR no longer localizes in the nucleus and undergoes nuclear export. These findings suggest that TR follows a CRT-mediated export pathway. Understanding the mechanism for TR nuclear export contributes to understanding the normal cellular response to thyroid hormone, and also provides important insight into the mode of action of oncogenic variants of TR. This research was funded in part by NIH #DK058028-02 and by a HHMI grant through the Undergraduate Biological Sciences Education Program to William & Mary.