Affinity labeling of the 1α,25-dihydroxyvitamin D₃ receptor

Genomic actions of the calciotropic hormone 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) involves a multistep process that is triggered by the highly specific binding of 1.25(OH)₂D₃ to 1α,25-dihydroxyvitamin D₃ receptor, VDR. In order to study this key step in the cascade, we synthesized 1α,25- dihydroxy[26(27)-³H]vitamin D₃-3-deoxy-3β-bromoacetate (1,25(OH)₂[³H]D₃-BE) and 1α,25-dihydroxyvitaminD₃-3β-[1- ¹⁴C]bromoacetate(1,25(OH)₂D₃[¹⁴C]BE), binding-site directed analogs of 1,25(OH)₂D₃, and affinity-labeled baculovirus-expressed recombinant human VDR (with 1,25(OH)₂[³H]D₃-BE), and naturally occurring VDRs in cytosols from calf thymus homogenate and rat osteosarcoma (ROS 17/2.8) cells (with 1, 25(OH)₂D₃-[¹⁴C]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 1α,25(OH)₂D₃. These results strongly suggested that 1,25(OH)₂[³H]D₃-BE and 1,25(OH)₂D₃-[¹⁴C]BE covalently modified the 1,25(OH)₂D₃-binding sites in baculovirus-expressed recombinant human VDR and naturally occurring calf thymus VDR and rat osteosarcoma VDR, respectively.