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

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

Research output: Contribution to journalArticle

33 Citations (Scopus)

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
StatePublished - Jan 26 1996

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Thymus
Calcitriol Receptors
Baculoviridae
Osteosarcoma
Labeling
Thymus Gland
Rats
Binding Sites
Calcitriol
Cholecalciferol
Cytosol
Hormones
bromoacetate
25-hydroxyvitamin D3-bromoacetate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Affinity labeling of the 1α,25-dihydroxyvitamin D3 receptor. / Ray, Rahul; Swamy, Narasimha; MacDonald, Paul N.; Ray, Swapna; Haussler, Mark R; Holick, Michael F.

In: Journal of Biological Chemistry, Vol. 271, No. 4, 26.01.1996, p. 2012-2017.

Research output: Contribution to journalArticle

Ray, R, Swamy, N, MacDonald, PN, Ray, S, Haussler, MR & Holick, MF 1996, 'Affinity labeling of the 1α,25-dihydroxyvitamin D3 receptor', Journal of Biological Chemistry, vol. 271, no. 4, pp. 2012-2017. https://doi.org/10.1074/jbc.271.4.2012
Ray, Rahul ; Swamy, Narasimha ; MacDonald, Paul N. ; Ray, Swapna ; Haussler, Mark R ; Holick, Michael F. / Affinity labeling of the 1α,25-dihydroxyvitamin D3 receptor. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 4. pp. 2012-2017.
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