BINDING OF THE RECEPTOR FOR 1,25-DIHYDROXYVITAMIN-D3 TO THE 5'-FLANKING REGION OF THE BOVINE PARATHYROID-HORMONE GENE

Receptors for 1,25-dihydroxyvitamin D3 (1,25-OH)2D3) were prepared from bovine parathyroid glands and incubated with fragments of DNA of the 5'-flanking region of the bovine parathyroid hormone (PTH) gene covering 1700 base pairs (bp) upstream of the initiation site. In filter binding assays, incubation of the DNA fragment spanning -700 to +50 bp with 200 μg cytosolic protein gave 288 ± 63% (mean ± S.D.) of binding in the absence of protein. In contrast, there was no significant reaction with the -1350 to -700 bp fragment, nor was there binding of the receptor to a fragment of DNA covering the coding region of the PTH gene. Substitution of bovine serum albumin for the receptor preparation did not induce binding to the -700 to +50 bp fragment. The receptor-binding site was further defined to -700 to -100 bp as deletion of the -100 to +50 bp did not reduce receptor binding. Reaction of receptors further purified by sucrose density ultracentrifugation with a monoclonal antibody in immunoblots revealed a single species with a molecular mass of approximately 50,000 Da which was absent in preparations of cos-1 cells. Autoradiography following incubation of receptors immobilized on nitrocellulose filters with the -700 to +50 bp fragment indicated a single reactive band coincident with the band in the immunoblot. The DNA fragment did not bind to filters containing preparations of cos-1 cells. Extraction of the receptors in the presence or absence of 1,25-(OH)2D3 (4 nmol/l) or the presence of KC1 (150 mmol/l) in the incubation medium had no significant effect on DNA binding to the protein in this assay. Autoradiography following incubation of immobilized receptors with the -700 to +50 bp, -485 to -50 bp, -700 to -100 bp and -700 to -485 bp fragments revealed a binding site for the receptor for 1,25-(OH)2D3 between -485 and -100 bp upstream of the initiation site in the bovine PTH gene.