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Human glucocorticoid receptor beta binds RU-486 and is transcriptionally active.

ABSTRACT: Human glucocorticoid receptor (hGR) is expressed as two alternately spliced C-terminal isoforms, alpha and beta. In contrast to the canonical hGRalpha, hGRbeta is a nucleus-localized orphan receptor thought not to bind ligand and not to affect gene transcription other than by acting as a dominant negative to hGRalpha. Here we used confocal microscopy to examine the cellular localization of transiently expressed fluorescent protein-tagged hGRbeta in COS-1 and U-2 OS cells. Surprisingly, yellow fluorescent protein (YFP)-hGRbeta was predominantly located in the cytoplasm and translocated to the nucleus following application of the glucocorticoid antagonist RU-486. This effect of RU-486 was confirmed with transiently expressed wild-type hGRbeta. Confocal microscopy of coexpressed YFP-hGRbeta and cyan fluorescent protein-hGRalpha in COS-1 cells indicated that the receptors move into the nucleus independently. Using a ligand binding assay, we confirmed that hGRbeta bound RU-486 but not the hGRalpha ligand dexamethasone. Examination of the cellular localization of YFP-hGRbeta in response to a series of 57 related compounds indicated that RU-486 is thus far the only identified ligand that interacts with hGRbeta. The selective interaction of RU-486 with hGRbeta was also supported by molecular modeling and computational docking studies. Interestingly, microarray analysis indicates that hGRbeta, expressed in the absence of hGRalpha, can regulate gene expression and furthermore that occupation of hGRbeta with the antagonist RU-486 diminishes that capacity despite the lack of helix 12 in the ligand binding domain.

SUBMITTER: Lewis-Tuffin LJ

PROVIDER: S-EPMC1820503 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Human glucocorticoid receptor beta binds RU-486 and is transcriptionally active.

Lewis-Tuffin Laura J LJ Jewell Christine M CM Bienstock Rachelle J RJ Collins Jennifer B JB Cidlowski John A JA

Molecular and cellular biology 20070122 6

Human glucocorticoid receptor (hGR) is expressed as two alternately spliced C-terminal isoforms, alpha and beta. In contrast to the canonical hGRalpha, hGRbeta is a nucleus-localized orphan receptor thought not to bind ligand and not to affect gene transcription other than by acting as a dominant negative to hGRalpha. Here we used confocal microscopy to examine the cellular localization of transiently expressed fluorescent protein-tagged hGRbeta in COS-1 and U-2 OS cells. Surprisingly, yellow fl ...[more]

PMID: 17242213

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Project description:Alternative splicing of the human glucocorticoid receptor gene generates two isoforms, hGR? and hGR?. hGR? functions as a dominant-negative regulator of hGR? activity and but also has inherent transcriptional activity, collectively altering glucocorticoid sensitivity. Single-nucleotide polymorphisms in the 3' UTR of hGR? have been associated with altered receptor protein expression, glucocorticoid sensitivity, and disease risk. Characterization of the hGR? G3134T polymorphism has been limited to a relatively small, homogenous population. The objective of this study was to determine the prevalence of hGR? G3134T in a diverse population and assess the association of hGR? G3134T in this population with physiological outcomes. In a prospective cohort study, 3730 genetically diverse participants were genotyped for hGR? G3134T and four common GR polymorphisms. A subset of these participants was evaluated for clinical and biochemical measurements. Immortalized human osteosarcoma cells (U-2 OS), stably transfected with wild-type or G3134T hGR?, were evaluated for receptor expression, stability, and genome-wide gene expression. Glucocorticoid-mediated gene expression profiles were investigated in primary macrophages isolated from participants. In a racially diverse population, the minor allele frequency was 74% (50.7% heterozygous carriers and 23.3% homozygous minor allele), with a higher prevalence in Caucasian non-Hispanic participants. After adjusting for confounding variable, carriers of hGR? G3134T were more likely to self-report allergies, have higher serum cortisol levels, and reduced cortisol suppression in response to low-dose dexamethasone. The presence of hGR? G3134T in U-2 OS cells increased hGR mRNA stability and protein expression. Microarray analysis revealed that the presence of the hGR? G3134T polymorphism uniquely altered gene expression profiles in U-2 OS cells and primary macrophages. hGR? G3134T is significantly present in the study population and associated with race, self-reported disease, and serum levels of glucocorticoids. Underlying these health differences may be changes in gene expression driven by altered receptor stability.

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Human glucocorticoid receptor beta binds RU-486 and is transcriptionally active.

Publications

Human glucocorticoid receptor beta binds RU-486 and is transcriptionally active.

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