Tandem Affinity Purification and Nano HPLC-ESI-MS/MS Reveal Binding of Vitamin D Receptor to p53 and other New Interaction Partners in HEK 293T Cells

While nuclear cofactors that contribute to vitamin D receptor (VDR)-mediated gene transcription, including retinoid X receptors, nuclear co-activators and co-repressors, have been extensively investigated, little is known about cytoplasmic VDR-binding partners and the physiological relevance of their interaction. To gain new insight into this topic, we isolated whole-cell protein extracts of 1,25-dihydroxyvitamin D₃ stimulated and UV-B-irradiated vs. non-irradiated HEK 293T cells transfected with a plasmid called pURB VDR C-Term TAP tag. VDR complex was purified by tandem affinity purification (TAP). The nuclear tumor-suppressor protein p53 and its negative regulator novel INHAT repressor (NIR), in addition to 43 other nuclear or cytoplasmatic VDR binding partners, were identified using nano high-performance liquid chromatography–electrospray ionization tandem mass spectrometric analysis. VDR binding to p53 was confirmed by western blot analysis. Future studies are required to further elucidate the functional significance of these interactions.

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