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

Mol Endocrinol. 2003 May;17(5):845-59. doi: 10.1210/me.2002-0320. Epub 2003 Jan 30.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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*

Substances

  • Hormone Antagonists
  • Ligands
  • NCOR1 protein, human
  • Nuclear Proteins
  • Nuclear Receptor Co-Repressor 1
  • Receptors, Glucocorticoid
  • Receptors, Steroid
  • Repressor Proteins
  • Transcription Factors
  • Mifepristone
  • Tyrosine
  • Dexamethasone
  • Histone Acetyltransferases
  • NCOA1 protein, human
  • Nuclear Receptor Coactivator 1
  • Glutathione Transferase