Conformational change and enhanced stabilization of the vitamin D receptor by the 1,25-dihydroxyvitamin D3 analog KH1060.

The 1,25-dihydroxyvitamin D3 [1,25-(OH)2vitamin D3] analog KH1060 exerts very potent effects on cell proliferation and cell differentiation via the vitamin D receptor (VDR). However, the activities of KH1060 are not associated with an increased affinity for the VDR. We now show that increased stabilization of the VDR-KH1060 complex could be an explanation for its high potencies. VDR half-life studies performed with cycloheximide-translational blocked rat osteoblast-like ROS 17/2.8 cells demonstrated that, in the absence of ligand, VDR levels rapidly decreased. After 2 hr, less than 10% of the initial VDR level could be measured. In the presence of 1,25-(OH)2vitamin D3, the VDR half-life was 15 hr. After 24 hr. less than 20% of the initial VDR content was detectable, whereas, at this time-point, when the cells were incubated with KH1060 80% of the VDR was still present. Differences in 1,25-(OH)2vitamin D3- and KH1060-induced conformational changes of the VDR could underlie the increased VDR stability. As assessed by limited proteolytic digestion analysis, both 1,25-(OH)2vitamin D3 and KH1060 caused a specific conformational change of the VDR. Compared with 1,25-(OH)2vitamin D3, KH1060 induced a conformational change that led to a far more dramatic protection of the VDR against proteolytic degradation. In conclusion, the altered VDR stability and the possibly underlying change in VDR conformation caused by KH1060 could be an explanation for its enhanced bioactivity.