title: 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.
abstract: 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.
authors.affiliation: Endocrine Sciences Research Group, Faculty of Medicine, University of Manchester, United Kingdom. fras@fs1.ser.man.ac.uk
authors.forename: Adam
authors.initials: A
authors.lastname: Stevens
authors.forename: Helen
authors.initials: H
authors.lastname: Garside
authors.forename: Andrew
authors.initials: A
authors.lastname: Berry
authors.forename: Charlotte
authors.initials: C
authors.lastname: Waters
authors.forename: Anne
authors.initials: A
authors.lastname: White
authors.forename: David
authors.initials: D
authors.lastname: Ray
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D051548:Histone Acetyltransferases; D006727:Hormone Antagonists / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D015735:Mifepristone / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D056971:Nuclear Receptor Co-Repressor 1; D056920:Nuclear Receptor Coactivator 1; D017354:Point Mutation; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D006727:Hormone Antagonists; D008024:Ligands; C539127:NCOR1 protein, human; D009687:Nuclear Proteins; D056971:Nuclear Receptor Co-Repressor 1; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D012097:Repressor Proteins; D014157:Transcription Factors; D015735:Mifepristone; D014443:Tyrosine; D003907:Dexamethasone; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1; D005982:Glutathione Transferase