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 micrograms 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 KCl (150 mmol/l) in the incubation medium had no significant effect on DNA binding to the protein in this assay. (ABSTRACT TRUNCATED AT 250 WORDS)
Farrow
S M
SM
Department of Medicine, Middlesex Hospital, London.
Hawa
N S
NS
Karmali
R
R
Hewison
M
M
Walters
J C
JC
O'Riordan
J L
JL
D000818:Animals; D002117:Calcitriol / Q000378:metabolism*; D002417:Cattle; D004247:DNA / Q000378:metabolism; D008722:Methods; D010281:Parathyroid Hormone / Q000235:genetics*; D011485:Protein Binding; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000378:metabolism*
D010281:Parathyroid Hormone; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D004247:DNA; D002117:Calcitriol