Identification of natural ligands of retinoic acid receptor-related orphan receptor alpha ligand-binding domain expressed in Sf9 cells--a mass spectrometry approach.
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.
Bitsch
Francis
F
Central Technologies, Novartis Institutes for Biomedical Research, Lichtstrasse 35, CH-4002 Basel, Switzerland. francis.bitsch@pharma.novartis.com
Aichholz
Reiner
R
Kallen
Joerg
J
Geisse
Sabine
S
Fournier
Brigitte
B
Schlaeppi
Jean Marc
JM
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002784:Cholesterol / Q000032:analysis / Q000378:metabolism; D003001:Cloning, Molecular; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008024:Ligands; D009419:Nerve Tissue Proteins / Q000378:metabolism; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1; D017434:Protein Structure, Tertiary; D018091:Receptors, AMPA / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism
D002352:Carrier Proteins; C491310:GRIP1 protein, human; D008024:Ligands; D009419:Nerve Tissue Proteins; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1; C539738:RORA protein, human; D018091:Receptors, AMPA; D018160:Receptors, Cytoplasmic and Nuclear; D015534:Trans-Activators; D002784:Cholesterol