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

Rahul Ray, Narasimha Swamy, Paul N. MacDonald, Swapna Ray, Mark R Haussler, Michael F. Holick

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)2012-2017
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number4
DOIs
Publication statusPublished - Jan 26 1996

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ASJC Scopus subject areas

  • Biochemistry

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