Dynamic Shuttling and Intranuclear Mobility of Nuclear Hormone Receptors*

We expressed green fluorescent protein (GFP) chimeras of estrogen, retinoic acid, and thyroid hormone receptors (ERs, RARs, and TRs, respectively) in HeLa cells to examine nucleocytoplasmic shuttling and intranuclear mobility of nuclear hormone receptors (NRs) by confocal microscopy. These receptors were predominantly in the nucleus and, interestingly, underwent intranuclear reorganization after ligand treatment. Nucleocytoplasmic shuttling was demonstrated by heterokaryon experiments and energy-dependent blockade of nuclear import and leptomycin-dependent blockade of nuclear export. Ligand addition decreased shuttling by GFP-ER, whereas heterodimerization with retinoid X receptor helped maintain TR and RAR within the nucleus. Intranuclear mobility of the GFP-NRs was studied by fluorescence recovery after photo-bleaching ± cognate ligands. Both GFP-TR and GFP-RAR moved rapidly in the nucleus, and ligand binding did not significantly affect their mobility. In contrast, estrogen binding decreased the mobility of GFP-ER and also increased the fraction of GFP-ER that was unable to diffuse. These effects were even more pronounced with tamoxifen. Co-transfection of the co-activator, SRC-1, further slowed the mobility of liganded GFP-ER. Our findings suggest estradiol and tamoxifen exert differential effects on the intranuclear mobility of GFP-ER. They also show that ligand-binding and protein-protein interactions can affect the intracellular mobility of some NRs and thereby may contribute to their biological activity.

[1]  J. Barsony,et al.  Retinoid X receptor dominates the nuclear import and export of the unliganded vitamin D receptor. , 2002, Molecular endocrinology.

[2]  P. Yen,et al.  Physiological and molecular basis of thyroid hormone action. , 2001, Physiological reviews.

[3]  G. Hager,et al.  Nuclear Cytoplasmic Shuttling by Thyroid Hormone Receptors , 2001, The Journal of Biological Chemistry.

[4]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[5]  Grace C. Lin,et al.  Dimerization with Retinoid X Receptors Promotes Nuclear Localization and Subnuclear Targeting of Vitamin D Receptors* , 2000, The Journal of Biological Chemistry.

[6]  B. Spiegelman,et al.  Degradation of the Peroxisome Proliferator-activated Receptor γ Is Linked to Ligand-dependent Activation* , 2000, The Journal of Biological Chemistry.

[7]  B. O’Malley,et al.  The 26S Proteasome Is Required for Estrogen Receptor-α and Coactivator Turnover and for Efficient Estrogen Receptor-α Transactivation , 2000 .

[8]  J. McNally,et al.  The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. , 2000, Science.

[9]  K. Artzt,et al.  The Quaking I-5 Protein (QKI-5) Has a Novel Nuclear Localization Signal and Shuttles between the Nucleus and the Cytoplasm* , 1999, The Journal of Biological Chemistry.

[10]  C. Glass,et al.  Coactivator and corepressor complexes in nuclear receptor function. , 1999, Current opinion in genetics & development.

[11]  J. Davie,et al.  Direct visualization of the human estrogen receptor alpha reveals a role for ligand in the nuclear distribution of the receptor. , 1999, Molecular biology of the cell.

[12]  T. Reich,et al.  Nucleocytoplasmic Trafficking of Steroid-free Glucocorticoid Receptor* , 1999, The Journal of Biological Chemistry.

[13]  S. Cheng,et al.  Hormone-induced Translocation of Thyroid Hormone Receptors in Living Cells Visualized Using a Receptor Green Fluorescent Protein Chimera* , 1998, The Journal of Biological Chemistry.

[14]  Minoru Yoshida,et al.  CRM1 is responsible for intracellular transport mediated by the nuclear export signal , 1997, Nature.

[15]  D. Livingston,et al.  The nuclear hormone receptor coactivator SRC-1 is a specific target of p300. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[16]  G. Hager,et al.  Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Parker,et al.  The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling. , 1993, Journal of cell science.

[18]  E. Milgrom,et al.  Nucleocytoplasmic shuttling of the progesterone receptor. , 1991, The EMBO journal.

[19]  Constantine A. Pappas,et al.  Nuclear location of hormone-free estrogen receptors by monoclonal antibodies could be a tissue-fixation dependent artifact , 1988, Steroids.

[20]  J. Gorski,et al.  Nuclear localization of unoccupied oestrogen receptors , 1984, Nature.

[21]  G. Greene,et al.  Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells , 1984, Nature.

[22]  B. O’Malley,et al.  FRAP reveals that mobility of oestrogen receptor-α is ligand- and proteasome-dependent , 2000, Nature Cell Biology.