Dissociation of Steroid Receptor Coactivator 1 and Nuclear Receptor Corepressor Recruitment to the Human Glucocorticoid Receptor by Modification of the Ligand-Receptor Interface: The Role of Tyrosine 735

• Published in: Molecular endocrinology (Baltimore, Md.) Md.), 2003-05, Vol.17 (5), p.845-859
• Main Authors: Stevens, Adam, Garside, Helen, Berry, Andrew, Waters, Charlotte, White, Anne, Ray, David
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; COS Cells; Dexamethasone - metabolism; Dexamethasone - pharmacology; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Histone Acetyltransferases; Hormone Antagonists - metabolism; Hormone Antagonists - pharmacology; Humans; Ligands; Mifepristone - metabolism; Mifepristone - pharmacology; Molecular Sequence Data; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Coactivator 1; Point Mutation; Receptors, Glucocorticoid - drug effects; Receptors, Glucocorticoid - genetics; Receptors, Glucocorticoid - metabolism; Receptors, Steroid - genetics; Receptors, Steroid - metabolism; Repressor Proteins - genetics; Repressor Proteins - metabolism; Structure-Activity Relationship; Transcription Factors - genetics; Transcription Factors - metabolism; Two-Hybrid System Techniques; Tyrosine - chemistry; Tyrosine - genetics; Tyrosine - metabolism
• ISSN: 0888-8809; 1944-9917
• DOI: 10.1210/me.2002-0320

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.