Affinity labeling of the 1 alpha,25-dihydroxyvitamin D3 receptor.
Genomic actions of the calciotropic hormone 1 alpha, 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 1 alpha, 25-dihydroxyvitamin D3 receptor, VDR. In order to study this key step in the cascade, we synthesized 1 alpha,25-dihydroxy[26(27)-3H]vitamin D3-3-deoxy-3 beta-bromoacetate (1,25(OH)2[3H]D3-BE) and 1 alpha,25-dihydroxyvitaminD3-3 beta-[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 1 alpha,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.
Ray
R
R
Vitamin D Laboratory, Boston University School of Medicine, Massachusetts 02118, USA.
Swamy
N
N
MacDonald
P N
PN
Ray
S
S
Haussler
M R
MR
Holick
M F
MF
D000345:Affinity Labels; D000818:Animals; D016367:Baculoviridae; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D006801:Humans; D008970:Molecular Weight; D051381:Rats; D018167:Receptors, Calcitriol / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins
D000345:Affinity Labels; D018167:Receptors, Calcitriol; D011994:Recombinant Proteins; D002117:Calcitriol