Hormone-induced translocation of thyroid hormone receptors in living cells visualized using a receptor green fluorescent protein chimera.
Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcription factors that regulate growth, differentiation, and development. To understand the role of the hormone, 3,3', 5-triiodo-L-thyronine (T3), in the nuclear translocation and targeting of TRs to the regulatory sites in chromatin, we appended green fluorescent protein (GFP) to the human TR subtype beta1 (TRbeta1). The fusion of GFP to the amino terminus of TRbeta1 protein did not alter T3 binding or transcriptional activities of the receptor. The subcellular localization of GFP-TRbeta1 in living cells was visualized by laser-scanning confocal microscopy. In the presence of T3, the expressed GFP-TRbeta1 was predominately localized in the nucleus, exhibiting a nuclear/cytoplasmic ratio of approximately 5.5. No GFP-TRbeta1 was detected in the nucleolus. In the absence of T3, more GFP-TRbeta1 was present in the cytoplasm, exhibiting a nuclear/cytoplasmic ratio of approximately 1.5. In these cells, cytoplasmic GFP-TRbeta1 could be induced to enter the nucleus by T3. The T3-induced translocation was blocked when Lys184-Arg185 in domain D of TRbeta1 was mutated to Ala184-Ala185. Furthermore, the inability of the mutant TR to translocate to the nucleus correlated with the loss of most of its transcriptional activity. These results suggest that TR functions may, in part, be regulated by T3-induced nuclear entry.
Laboratory of Molecular Biology, National Institutes of Health, Bethesda, Maryland 20892, USA.
X G
XG
Zhu
J A
JA
Hanover
G L
GL
Hager
S Y
SY
Cheng
D001692:Biological Transport; D002451:Cell Compartmentation; D003584:Cytological Techniques; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014284:Triiodothyronine / Q000378:metabolism*
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014284:Triiodothyronine; D049452:Green Fluorescent Proteins