Analysis of transcription complexes and effects of ligands by microelectrospray ionization mass spectrometry

The human vitamin D receptor (VDR) and retinoid X receptor-alpha (RXR alpha ) modulate gene activity by forming homodimeric or heterodimeric complexes with specific DNA sequences and interaction with other elements of the tran scriptional apparatus in the presence of their known endogenous ligands 1 a lpha,25-dihydroxyvitamin D-3 (1,25-[OH](2)D-3) and 9-cis-retinoic acid (9-c -RA). We used rapid buffer exchange gel filtration in conjunction with micr oelectrospray ionization mass spectrometry (mu ESI-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 RXR alpha existed primarily as monome rs, but in the presence of OP VDRE, homodimeric RXR alpha and heterodimeric RXR alpha-VDR complexes were shown to bind OP VDRE. Addition of 9-c-RA inc reased RXR alpha homodimer-OP VDRE complexes, and addition of 1,25-(OH)2D3 resulted in formation of 1,25-(OH)(2)D-3-VDR-RXR alpha-OP VDRE complexes. A ddition of low-affinity binding ligands had no detectable effect on the VDR -RXR alpha-OP VDRE transcription complex. These results demonstrate the uti lity of mu ESI-MS in analyzing multimeric, high-molecular-weight protein-pr otein and protein-DNA complexes, and the effects of ligands on these transc riptional complexes.