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
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.
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.
- 6.Kino T, Stauber RH, Resau JH, Pavlakis GN, Chrousos GP. (2001) Pathologic human GR mutant has a transdominant negative effect on the wild-type GR by inhibiting its translocation into the nucleus: importance of the ligand-binding domain for intracellular GR trafficking. J. Clin. Endocrinol. Metab. 86: 5600–5608.CrossRefGoogle Scholar
- 12.Charmandari E et al. (2005) A novel point mutation in the ligand-binding domain (LBD) of the human glucocorticoid receptor (hGR) causing generalized glucocorticoid resistance: the importance of the C terminus of hGR LBD in conferring transactivational activity. J. Clin. Endocrinol. Metab. 90: 3696–3705.CrossRefGoogle Scholar
- 13.Charmandari E, Kino T, Souvatzoglou E, Vottero A, Bhattacharyya N, Chrousos GP. (2004) Natural glucocorticoid receptor mutants causing generalized glucocorticoid resistance: molecular genotype, genetic transmission, and clinical phenotype. J. Clin. Endocrinol. Metab. 89: 1939–1949.CrossRefGoogle Scholar
- 14.Kino T, Chrousos GP. (2004) Glucocorticoid Effect on Gene Expression. In: Handbook on Stress and the Brain Part 1. Steckler T, Kalin NH, Reul JMHM (eds.) Elsevier BV, Amsterdam, pp. 295–312.Google Scholar
- 23.Charmandari E, Kino T, Ichijo T, Zachman K, Chrousos GP. (2005) Molecular characterization of the human glucocorticoid receptor (hGR) gene mutations hGRαR477H and hGRαG679S causing generalized glucocorticoid resistance: absence of dominant negative activity despite asymmetric binding to p160 coactivators. The Endocrine Society’s 87th Annual Meeting, San Diego, The Endocrine Society Press, Chevy Chase, MD, p647.Google Scholar
- 42.Kino T, Manoli-Oreopoulos I, Kelkar S, Su AY, Blackman MR, Chrousos GP. (2005) The human glucocorticoid receptor (GR) β isoform, a splicing variant of the classic receptor GRα, exerts intrinsic transcriptional effects in HeLa cells. The Endocrine Society’s 87th Annual Meeting, San Diego, The Endocrine Society Press, Chevy Chase, MD, p.422.Google Scholar