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Analysis of Vitamin D Analog-Induced Heterodimerization of Vitamin D Receptor with Retinoid X Receptor Using the Yeast Two-Hybrid System

Department of Medicine Stanford University School of Medicine Stanford, California 94305

Several synthetic analogs of 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] are potent inducers of cellular differentiation and inhibitors of cell growth, yet they are less calcemic than 1,25-(OH)₂D₃ itself. The mechanisms by which these vitamin D analogs elicit a different profile of cellular activities than 1,25-(OH)₂D₃ are not fully understood. We propose that the analogs bind to the vitamin D receptor (VDR) to produce a conformational change that is more or less constrained than that induced by 1,25-(OH)₂D₃. This conformational change determines the extent of the VDR-retinoid X receptor (RXR) heterodimerization which, in turn, determines the interaction with other factors that specify the selectivity and magnitude of gene transactivation. We used the yeast two-hybrid system to evaluate a series of six vitamin D analogs for their ability to induce VDR-RXR heterodimerization. The VDR-RXR interaction was elicited by the analogs in a concentration-dependent manner. To evaluate how this activity compared with other known steps in 1,25-(OH)₂D₃ action, we also measured the ability of the same six analogs to bind to VDR, to enhance the binding of VDR-RXR to DNA, to transactivate a vitamin D-response element-reporter construct, and to inhibit proliferation in mammalian cells. Our results indicate that, for most analogs, the level of transcriptional activation correlates well with the strength of VDR-RXR heterodimerization in intact cells. We conclude that the yeast two-hybrid system provides a useful means to investigate heterodimerization potency and that this property contributes significantly to the overall pattern of analog activity. The yeast two-hybrid system, being an intact cell assay and easy to perform, may be a useful supplement to the conventional assays employed to screen vitamin D analogs.

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