Affinity Labeling of the 1,25-Dihydroxyvitamin D Receptor (*)

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

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