Affinity labeling of the 1 alpha,25-dihydroxyvitamin D-3 receptor

Genomic actions of the calciotropic hormone 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) involves a multistep process that is triggered by the highly specific binding of 1,25(OH)(2)D-3 to 1 alpha,25-dihydroxyvitamin D-3 receptor, VDR. In order to study this key step in the cascade, we synthesized 1 alpha,25-dihydroxy[26(27)-H-3]vitamin D-3-3-deoxy-3 beta-bromoacetate (1,25(OH)(2)[H-3]D-3-BE) and 1 alpha,25-dihydroxyvitaminD(3)-3 beta-[1-C-14]bromoacetate(1,25(OH)(2)D-3-[C-14]BE), binding-site directed analogs of 1,25(OH)(2)D-3, and affinity-labeled baculovirus-expressed recombinant human VDR (with 1,25(OH)(2)[H-3]D-3-BE), and naturally occurring VDRs in cytosols from calf thymus homogenate and rat osteosarcoma (ROS 17/2.8) cells (with 1, 25(OH)(2)D-3-[C-14]BE). In each case, specificity of labeling was demonstrated by the drastic reduction in labeling when the incubation was carried out in the presence of an excess of nonradioactive 1 alpha,25(OH)(2)D-3. These results strongly suggested that 1,25(OH)(2)[H-3]D-3-BE and 1,25(OH)(2)D-3-[C-14]BE covalently modified the 1,25(OH)(2)D-3-binding sites in baculovirus-expressed recombinant human VDR and naturally occurring calf thymus VDR and rat osteosarcoma VDR, respectively.