Pharmaceutical Sciences Faculty Publications

Analysis of Transcription Complexes and Effects of Ligands by Microelectrospray Ionization Mass Spectrometry

Document Type

Article

Publication Date

12-1-1999

Journal Title

Nature Biotechnology

ISSN

1087-0156

Volume

17

Issue

12

First Page

1214

Last Page

1218

DOI

10.1038/70767

PubMed ID

10585721

Abstract

The human vitamin D receptor (VDR) and retinoid X receptor-alpha (RXRalpha) modulate gene activity by forming homodimeric or heterodimeric complexes with specific DNA sequences and interaction with other elements of the transcriptional apparatus in the presence of their known endogenous ligands 1alpha,25-dihydroxyvitamin D3 (1, 25-[OH]2D3) and 9-cis-retinoic acid (9-c-RA). We used rapid buffer exchange gel filtration in conjunction with microelectrospray ionization mass spectrometry (microESI-MS) to study the binding of these receptors to the osteopontin vitamin D response element (OP VDRE). In the absence of DNA, both VDR and RXRalpha existed primarily as monomers, but in the presence of OP VDRE, homodimeric RXRalpha and heterodimeric RXRalpha-VDR complexes were shown to bind OP VDRE. Addition of 9-c-RA increased RXRalpha homodimer-OP VDRE complexes, and addition of 1,25-(OH) 2D3 resulted in formation of 1, 25-(OH)2D 3-VDR-RXRalpha-OP VDRE complexes. Addition of low-affinity binding ligands had no detectable effect on the VDR-RXRalpha-OP VDRE transcription complex. These results demonstrate the utility of microESI-MS in analyzing multimeric, high-molecular-weight protein-protein and protein-DNA complexes, and the effects of ligands on these transcriptional complexes.

Keywords

Base sequence, DNA primers, humans, ligands, mass spectrometry, receptors, calcitriol, retinoic acid, transcription factors, genetic

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