title: Analysis of vitamin D analog-induced heterodimerization of vitamin D receptor with retinoid X receptor using the yeast two-hybrid system.
abstract: Several synthetic analogs of 1 alpha,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.
authors.affiliation: Department of Medicine, Stanford University School of Medicine, California 94305, USA.
authors.forename: X Y
authors.initials: XY
authors.lastname: Zhao
authors.forename: T R
authors.initials: TR
authors.lastname: Eccleshall
authors.forename: A V
authors.initials: AV
authors.lastname: Krishnan
authors.forename: C
authors.initials: C
authors.lastname: Gross
authors.forename: D
authors.initials: D
authors.lastname: Feldman
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter / Q000235:genetics; D018167:Receptors, Calcitriol / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D047488:Retinoid X Receptors; D012441:Saccharomyces cerevisiae; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; D002117:Calcitriol