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Glucocorticoid Receptor Mutants Demonstrate Increased Motility Inside the Nucleus of Living Cells: Time of Fluorescence Recovery After Photobleaching (FRAP) Is an Integrated Measure of Receptor Function

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Abstract

Natural mutations of the human glucocorticoid receptor (GR) isoform α cause the glucocorticoid resistance syndrome. Mutant receptors may have abnormal interactions with the ligand, target DNA sequences, and/or multiple intracellular proteins, as well as aberrant nucleocytoplasmic trafficking. Using fluorescence recovery after photobleaching (FRAP) analysis, all GR pathologic mutant receptors examined, as well as 2 synthetic GR mutants lacking the activation function (AF)-1 or the ligand-binding domain (and hence the AF-2), had defective transcriptional activity and dynamic motility defects inside the nucleus of living cells. In the presence of dexamethasone, these mutants displayed a curtailed 50% recovery time (t1/2) after photobleaching and, hence, significantly increased intranuclear motility and decreased “chromatin retention.” The t1/2 values of the mutants correlated positively with their transcriptional activities and depended on the GR domain affected. GRβ, a natural splice variant of the GR gene, also demonstrated a shorter t1/2 than GRa. The motility responsiveness of the natural and artificial mutant receptors examined, and of GRβ, to the proteasomal inhibitor MG-132 also depended on the mutant domain. Thus, mutant glucocorticoid receptors possess dynamic motility defects in the nucleus, possibly caused by their inability to properly interact with all key partner nuclear molecules necessary for full activation of glucocorticoid-responsive genes.

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Acknowledgments

This study was funded in part by the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA. We thank Drs. R.M. Evans and G.L. Hager for providing their plasmids, and Dr. T. Ichijo, S.S. Rao, and K. Zachman for superb technical assistance.

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Authors and Affiliations

  1. Pediatric Endocrinology Section, Reproductive Biology and Medicine Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 10, CRC, Room 1-3140, 10 Center Drive, MSC 110, Bethesda, MD, 20892-1109, USA

    Tomoshige Kino, Szu-Heng Liou, Evangelia Charmandari & George P Chrousos

  2. First Department of Pediatrics, University of Athens, Athens, Greece

    George P Chrousos

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  1. Tomoshige Kino
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  2. Szu-Heng Liou
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  3. Evangelia Charmandari
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  4. George P Chrousos
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Correspondence to Tomoshige Kino.

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Kino, T., Liou, SH., Charmandari, E. et al. Glucocorticoid Receptor Mutants Demonstrate Increased Motility Inside the Nucleus of Living Cells: Time of Fluorescence Recovery After Photobleaching (FRAP) Is an Integrated Measure of Receptor Function. Mol Med 10, 80–88 (2004). https://doi.org/10.2119/2005-00026.Kino

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