Detection of nucleic acid-nuclear hormone receptor complexes with mass spectrometry.
Nuclear receptors, such as the retinoic acid receptor (RAR) or the 9-cis retinoic acid receptor (RXR), interact not only with their ligands but also with other types of receptors and with DNA. Here, two complementary mass spectrometry (MS) methods were used to study the interactions between retinoic receptors (RXR/RAR) and DNA: non-denaturing nano-electrospray (nanoESI MS), and high-mass matrix-assisted laser desorption ionization (MALDI MS) combined with chemical cross-linking. The RAR x RXR heterodimer was studied in the presence of a specific DNA sequence (DR5), and a specific RAR x RXR x DNA complex was detected with both MS techniques. RAR by itself showed no significant homodimerization. A complex between RAR and the double stranded DR5 was detected with nanoESI. After cross-linking, high-mass MALDI mass spectra showed that the RAR binds the single stranded DR5, and the RAR dimer binds both single and double stranded DR5. Moreover, the MALDI mass spectrum shows a larger RAR dimer signal in the presence of DNA. These results suggest that a gene-regulatory site on DNA can induce quaternary structural changes in a transcription factor such as RAR.
Bich
Claudia
C
Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich, Switzerland.
Bovet
Cédric
C
Rochel
Natacha
N
Peluso-Iltis
Carole
C
Panagiotidis
Andreas
A
Nazabal
Alexis
A
Moras
Dino
D
Zenobi
Renato
R
D001665:Binding Sites; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D011485:Protein Binding; D018168:Receptors, Retinoic Acid / Q000032:analysis / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D018168:Receptors, Retinoic Acid; D004247:DNA