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Within the human glucocorticoid receptor (GR) steroid binding pocket, tyrosine 735 makes hydrophobic contact with the steroid D ring. Substitution of tyrosine735 selectively impairs glucocorticoid transactivation but not transrepression. We now show, using both mammalian two-hybrid and glutathione-S-transferase pull downs, that such substitutions reduce interaction with steroid receptor coactivator 1, both basally and in response to agonist binding. Using a yeast two-hybrid screen we identified one of the three nuclear receptor interacting domains (NCoR-N1) of nuclear receptor corepressor (NCoR) as interacting with the GR C terminus in an RU486-specific manner. This was confirmed in mammalian two-hybrid experiments, and so we used the NCoR-N1 peptide to probe the GR C-terminal conformation. Substitution of Tyr735phe, Tyr735val, and Tyr735 ser, which impaired steroid receptor coactivator 1 (SRC1) interaction, enhanced NCoR-N1 recruitment, basally and after RU486. RU486 did not direct SRC1 recruitment to any of the GR constructs, and dexamethasone did not allow NCoR-N1 recruitment. Using a glutathione-S-transferase pull-down approach, the NCoR-N1 peptide was found to bind the full-length GR constitutively, and no further induction was seen with RU486, but it was reduced by dexamethasone. As both SRC1 and NCoR are predicted to recognize a common hydrophobic cleft in the GR, it seems that changes favorable to one interaction are detrimental to the other, thus identifying a molecular switch.

Original publication

DOI

10.1210/me.2002-0320

Type

Journal article

Journal

Mol Endocrinol

Publication Date

05/2003

Volume

17

Pages

845 - 859

Keywords

Amino Acid Sequence, Animals, Binding Sites, COS Cells, Dexamethasone, Glutathione Transferase, Histone Acetyltransferases, Hormone Antagonists, Humans, Ligands, Mifepristone, Molecular Sequence Data, Nuclear Proteins, Nuclear Receptor Co-Repressor 1, Nuclear Receptor Coactivator 1, Point Mutation, Receptors, Glucocorticoid, Receptors, Steroid, Repressor Proteins, Structure-Activity Relationship, Transcription Factors, Two-Hybrid System Techniques, Tyrosine