Abstract
Natural mutations of the human glucocorticoid receptor (GR) isoform alpha 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 (t 1/2) after photobleaching and, hence, significantly increased intranuclear motility and decreased "chromatin retention." The t 1/2 values of the mutants correlated positively with their transcriptional activities and depended on the GR domain affected. GRbeta, a natural splice variant of the GR gene, also demonstrated a shorter t 1/2 than GRalpha. The motility responsiveness of the natural and artificial mutant receptors examined, and of GRbeta, 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.
Highlights
Glucocorticoid resistance is a rare, familial or sporadic condition characterized by generalized, partial end-organ insensitivity to glucocorticoids caused by mutations in the glucocorticoid receptor (GR) gene [1,2]
The activated GRα attracts nuclear receptor coactivators, through its 2 activation functions AF-1 and -2, which are respectively located in the NH2-terminal domain and the ligand-binding domain (LBD), as well as other chromatinremodeling complexes and transcriptional components, including general transcription factors and the RNA polymerase II, to the gene promoter regions, modulating the transcriptional rates of glucocorticoid-responsive genes [14]
We recently examined the molecular defects of several GRα mutant receptors that cause familial or sporadic glucocorticoid resistance by examining their ligand-binding activity, cytoplasmic-to-nuclear translocation, and interactions with glucocorticoid response elements (GREs) and p160 type nuclear receptor coactivators and by determining their transcriptional activities [3,5,6,8,9,12,13]
Summary
Glucocorticoid resistance is a rare, familial or sporadic condition characterized by generalized, partial end-organ insensitivity to glucocorticoids caused by mutations in the GR gene [1,2]. The activated GRα attracts nuclear receptor coactivators, through its 2 activation functions AF-1 and -2, which are respectively located in the NH2-terminal domain and the ligand-binding domain (LBD), as well as other chromatinremodeling complexes and transcriptional components, including general transcription factors and the RNA polymerase II, to the gene promoter regions, modulating the transcriptional rates of glucocorticoid-responsive genes [14]. We found that the pathologic and synthetic GRα mutants with changes in specific functional domains of the receptor generally demonstrated a defect in their intranuclear motility in vivo and responded differentially to treatment with the proteasomal inhibitor MG-132, one of the known determinants of such motility These results indicate that pathologic mutant glucocorticoid receptors have a dynamic defect in their “chromatin retention” possibly because of loss of attractive interactions with key nuclear partner molecules necessary for full transcriptional activity
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