THE HIGH-AFFINITY LIGAND-BINDING CONFORMATION OF THE NUCLEAR 1,25-DIHYDROXYVITAMIN-D-3 RECEPTOR IS FUNCTIONALLY LINKED TO THE TRANSACTIVATION DOMAIN-2 (AF-2)

The nuclear receptor for 1,25-dihydroxyvitamin D-3 (VD), VDR, is a tra nscription factor that mediates all genomic actions of the hormone, Th e activation of VDR by ligand induces a conformational change within i ts ligand binding domain (LED), Due to the lack of a crystal structure analysis, biochemical methods have to be applied in order to investig ate the details of this receptor-ligand interaction, The limited prote ase digestion assay can be used as a tool for the determination of a f unctional dissociation constant (K-df) Of VDR with any potential ligan d, This method provided with the natural hormone VD two protease-resis tant fragments of the VDR LED and with the 20-epi conformation of VD, known as MC1288, even an additional fragment of intermediate size. The se fragments were interpreted as different receptor conformations and their decreasing size was found to be associated with decreasing ligan d binding affinity, A critical amino acid for VDR's high ligand bindin g conformation has been identified by C-terminal receptor truncations and point mutations as phenylalanine 422, This amino acid appears to d irectly contact the ligand and belongs to the ligand-inducible activat ion function-2 (AF-2) domain, Moreover, functional assays supported th e observation that high affinity ligand binding is directly linked to transactivation function.