Identification of natural ligands of retinoic acid receptor-related orphan receptor alpha ligand-binding domain expressed in Sf9 cells--a mass spectrometry approach

Anal Biochem. 2003 Dec 1;323(1):139-49. doi: 10.1016/j.ab.2003.08.029.

Abstract

The ligand-binding domain (LBD) of the human retinoic acid receptor-related orphan receptor (RORalpha-LBD), expressed in Sf9 cells, was purified and analyzed by electrospray ionization-mass spectrometry (ESI-MS). ESI-MS operated under native conditions showed the presence of a fortuitous ligand with molecular weight 386. Further analysis by gas chromatography-mass spectrometry (GC-MS) allowed the identification of the ligands bound to the LBD. Cholesterol (77%) and 7-dehydrocholesterol (provitamin D(3); 18%) were shown to be the major ligands. A monohydroxylated cholesterol derivative was identified as a minor ligand. In addition, ligand exchange experiments monitored by ESI-MS showed that cholesterol sulfate has a higher affinity for RORalpha-LBD than cholesterol and 25-hydroxycholesterol. Binding of coactivator (CoA) peptide GRIP1P was shown to occur in a stoichiometric manner. Therefore, monitoring of binding of CoAs by mass spectrometry could be used for classification of the ligands as agonist or antagonist molecules.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Carrier Proteins / metabolism
  • Cell Line
  • Cholesterol / analysis
  • Cholesterol / metabolism
  • Cloning, Molecular
  • Gas Chromatography-Mass Spectrometry
  • Humans
  • Ligands
  • Nerve Tissue Proteins / metabolism
  • Nuclear Receptor Subfamily 1, Group F, Member 1
  • Protein Structure, Tertiary
  • Receptors, AMPA / metabolism
  • Receptors, Cytoplasmic and Nuclear / chemistry*
  • Receptors, Cytoplasmic and Nuclear / metabolism
  • Spectrometry, Mass, Electrospray Ionization
  • Trans-Activators / chemistry*
  • Trans-Activators / metabolism

Substances

  • Carrier Proteins
  • GRIP1 protein, human
  • Ligands
  • Nerve Tissue Proteins
  • Nuclear Receptor Subfamily 1, Group F, Member 1
  • RORA protein, human
  • Receptors, AMPA
  • Receptors, Cytoplasmic and Nuclear
  • Trans-Activators
  • Cholesterol