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Molecular and Cellular Biology, March 2003, p. 1922-1934, Vol. 23, No. 6
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.6.1922-1934.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Molecular Determinants of Glucocorticoid Receptor Mobility in Living Cells: the Importance of Ligand Affinity

Marcel J. M. Schaaf and John A. Cidlowski*

Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709

Received 29 August 2002/ Returned for modification 14 October 2002/ Accepted 23 December 2002

The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which is activated upon ligand binding, and can alter the expression of target genes either by transrepression or transactivation. We have applied FRAP (fluorescence recovery after photobleaching) to quantitatively assess the mobility of the yellow fluorescent protein (YFP)-tagged human GR {alpha}-isoform (hGR{alpha}) in the nucleus of transiently transfected COS-1 cells and to elucidate determinants of its mobility. Addition of the high-affinity agonist dexamethasone markedly decreases the mobility of the receptor in a concentration-dependent manner, whereas low-affinity ligands like corticosterone decrease the mobility to a much lesser extent. Analysis of other hGR{alpha} ligands differing in affinity suggests that it is the affinity of the ligand that is a major determinant of the decrease in mobility. Similar results were observed for two hGR{alpha} antagonists, the low-affinity antagonist ZK98299 and the high-affinity antagonist RU486. The effect of ligand affinity on mobility was confirmed with the hGR{alpha} mutant Q642V, which has an altered affinity for triamcinolone acetonide, dexamethasone, and corticosterone. Analysis of hGR{alpha} deletion mutants indicates that both the DNA-binding domain and the ligand-binding domain of the receptor are required for a maximal ligand-induced decrease in receptor mobility. Interestingly, the mobility of transfected hGR{alpha} differs among cell types. Finally, the proteasome inhibitor MG132 immobilizes a subpopulation of unliganded receptors, via a mechanism requiring the DNA-binding domain and the N-terminal part of the ligand-binding domain. Ligand binding makes the GR resistant to the immobilizing effect of MG132, and this effect depends on the affinity of the ligand. Our data suggest that ligand binding induces a conformational change of the receptor which is dependent on the affinity of the ligand. This altered conformation decreases the mobility of the receptor, probably by targeting the receptor to relatively immobile nuclear domains with which it transiently associates. In addition, this conformational change blocks immobilization of the receptor by MG132.

* Corresponding author. Mailing address: Laboratory of Signal Transduction, NIEHS, 111 Alexander Dr., P.O. Box 12233, Research Triangle Park, NC 27709. Phone: (919) 541-1564. Fax: (919) 541-1367. E-mail: cidlowski{at}niehs.nih.gov.

Molecular and Cellular Biology, March 2003, p. 1922-1934, Vol. 23, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.6.1922-1934.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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