Biochemical evidence for a 170-kilodalton, AF-2-dependent vitamin D receptor/retinoid X receptor coactivator that is highly expressed in osteoblasts

Human vitamin D receptor (hVDR) fused to glutathione S-transferase was util ized to detect a VDR-interacting protein (VIP) of approximately 170 kDa, VI P170 is expressed in osteoblast-like ROS 17/2.8 cells and, to a lesser exte nt, in COS-7 and HeLa cells. VIP170 may be a coactivator because it interac ts only with 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) ligand-bound hVDR a nd because a mutation (E420A) in the activation function-2 (AF-2) of hVDR a bolishes both receptor-mediated transactivation and VIP170 binding, Unlike L254G hVDR, a heterodimerization mutant with an intact AF-2, the E420A muta nt is only partially attenuated in its association with the retinoid X rece ptor (RXR) DNA-binding partner, Finally, the ability of overexpressed hVDR to squelch glucocorticoid receptor-mediated transactivation is lost in both the L254G and E420A mutants. These results suggest that several protein-pr otein interactions, including VDR association with RXR and VIP170, are requ ired for stabilization of a multimeric complex that transduces the signal f or 1,25(OH)(2)D-3-elicited transactivation. (C) 2000 Academic Press.