Nucleocytoplasmic Shuttling of the Thyroid Hormone Receptorα

• Published in: Molecular endocrinology (Baltimore, Md.) Md.), 2001-04, Vol.15 (4), p.512-533
• Main Authors: Bunn, Caroline F, Neidig, Jessica A, Freidinger, Kathryn E, Stankiewicz, Tracy A, Weaver, Brian S, McGrew, Julie, Allison, Lizabeth A
• Format: Article
• Language: English
• Subjects: Animals; Apyrase - pharmacology; Carrier Proteins - drug effects; Carrier Proteins - metabolism; Cell Nucleus - metabolism; Cells, Cultured; Cytoplasm - metabolism; Exportin 1 Protein; Fatty Acids, Unsaturated - pharmacology; Female; Genes, Dominant; Green Fluorescent Proteins; Humans; Karyopherins; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Mammals; Mice; Mutation; Nuclear Proteins - metabolism; Oncogene Proteins v-erbA - metabolism; Oocytes - drug effects; Protein Transport - drug effects; Receptors, Cytoplasmic and Nuclear; Receptors, Thyroid Hormone - genetics; Receptors, Thyroid Hormone - metabolism; Ribosomal Proteins - metabolism; Temperature; Xenopus
• ISSN: 0888-8809; 1944-9917
• DOI: 10.1210/mend.15.4.0619

The thyroid hormone receptor α (TRα) exhibits a dual role as an activator or repressor of gene transcription in response to thyroid hormone (T3). Our studies show that TRα, formerly thought to reside solely in the nucleus tightly bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. The finding that TRα shuttles reveals an additional checkpoint in receptor control of gene expression. Using Xenopus oocyte microinjection assays, we show that there are two coexisting mechanisms for nuclear entry of TRα. First, nuclear import of TRα (molecular mass 46 kDa) was not sensitive to general inhibitors of signal-mediated transport, indicating that TRα can enter the oocyte nucleus by passive diffusion. Second, when TRα was tagged with glutathione-S-transferase, import of the fusion protein (molecular mass 73 kDa) was completely blocked by these inhibitors, demonstrating that an alternative, signal-mediated import pathway exists for TRα. Nuclear retention of TRα in oocytes is enhanced in the presence of T3, suggesting that more intranuclear binding sites are available for the ligand-bound receptor. Using mammalian cells, we show that shuttling of green fluorescent protein (GFP)-tagged and untagged TRα is inhibited in both chilled and energy-depleted cells, suggesting that there is an energy-requiring step in the nuclear retention/export process. Nuclear export of TRα is not blocked by leptomycin B, a specific inhibitor of the export receptor CRM1, indicating that TRα does not require the CRM1 pathway to exit the nucleus. Dominant negative mutants of TR with defects in DNA binding and transactivation accumulate in the cytoplasm at steady state, illustrating that even single amino acid changes in functional domains may alter the subcellular distribution of TR. In contrast to TRα, nuclear export of its oncogenic homolog v-ErbA is sensitive to leptomycin B, suggesting that the oncoprotein follows a CRM1-mediated export pathway. Acquisition of altered nuclear export capabilities may contribute to the oncogenic properties of v-ErbA.