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PMID:12902338

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Citation

Li, G, Wang, S and Gelehrter, TD (2003) Identification of glucocorticoid receptor domains involved in transrepression of transforming growth factor-beta action. J. Biol. Chem. 278:41779-88

Abstract

The transforming growth factor-beta (TGF-beta) and glucocorticoid signaling pathways interact both positively and negatively in regulating a variety of physiological and pathologic processes. We previously reported that liganded glucocorticoid receptor (GR) repressed TGF-beta induction of human plasminogen activator inhibitor-1 gene transcription by directly targeting the transcriptional activation function of Smad3. To identify the domain(s) in the glucocorticoid receptor involved in this repression, we have examined the ability of various GR truncation, deletion, and substitution mutants to repress TGF-beta transactivation in Hep3B human hepatoma cells that lack functional endogenous GR. Partial deletions in the ligand-binding domain (LBD), including the tau2 and tauc regions, greatly reduced or eliminated GR repression, whereas deletion of the N-terminal AF1 (tau1) domain and substitution mutations in the DNA-binding domain had little or no effect. Liganded androgen receptor repressed TGF-beta transactivation, whereas mineralocorticoid receptor did not, and studies with rat GR-mineralocorticoid receptor chimeras confirmed that the GR C-terminal domains were required for repression. RU486, a strong antagonist of transactivation by GR, partially reversed repression by wild type GR. Co-immunoprecipitation experiments in Hep3B cells indicated that physical interaction between GR and Smad3 is necessary but not sufficient for repression. Physical interaction required activation of Smad3 by TGF-beta but not dexamethasone binding to GR. Glutathione S-transferase pull-down assays demonstrated that several regions of the LBD could mediate GR-Smad3 physical interaction. We conclude that the LBD of GR, but not the DNA-binding domain or the N-terminal activation domain, is required for GR-mediated transrepression of TGF-beta transactivation.

Links

PubMed Online version:10.1074/jbc.M305350200

Keywords

Binding Sites/genetics; Cell Line, Tumor; DNA-Binding Proteins/metabolism; Down-Regulation; Humans; Mutation; Plasminogen Activator Inhibitor 1/genetics; Promoter Regions, Genetic; Protein Structure, Tertiary; Receptors, Glucocorticoid/chemistry; Receptors, Glucocorticoid/genetics; Receptors, Glucocorticoid/physiology; Smad3 Protein; Trans-Activators/metabolism; Transcriptional Activation; Transforming Growth Factor beta/physiology

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:GCR

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P84022

F

Seeded From UniProt

complete

HUMAN:GCR

involved_in

GO:0000122: negative regulation of transcription by RNA polymerase II

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:GCR

involved_in

GO:0071560: cellular response to transforming growth factor beta stimulus

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:GCR

involved_in

GO:0071549: cellular response to dexamethasone stimulus

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

RAT:MCR

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P84022

F

Seeded From UniProt

complete

HUMAN:SMAD3

involved_in

GO:0071560: cellular response to transforming growth factor beta stimulus

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:SMAD3

enables

GO:0031962: mineralocorticoid receptor binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P22199

F

Seeded From UniProt

complete

HUMAN:SMAD3

enables

GO:0035259: glucocorticoid receptor binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P04150

F

Seeded From UniProt

complete

HUMAN:ANDR

involved_in

GO:0071383: cellular response to steroid hormone stimulus

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:ANDR

involved_in

GO:0000122: negative regulation of transcription by RNA polymerase II

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

Notes

See also

References

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