title: Binding characteristics of a membrane receptor that recognizes 1 alpha,25-dihydroxyvitamin D3 and its epimer, 1 beta,25-dihydroxyvitamin D3.
abstract: The steroid hormone 1 alpha,25-dihydroxyvitamin D3 has been shown to exert rapid effects (15 s to 5 min) in osteoblasts. These effects occur in osteoblast-like cells lacking the nuclear vitamin D receptor, ROS 24/1, suggesting that a separate signalling system mediates the rapid actions. These non-genomic actions include rapid activation of phospholipase C and opening of calcium channels, pointing to a membrane localization of this signalling system. Previous studies have shown that the 1 beta epimer of 1 alpha,25-dihydroxyvitamin D3 can block these rapid actions, indicating that the 1 beta epimer may bind to the receptor responsible for the rapid actions in a competitive manner. We have assessed the displacement of 3H-1 alpha,25-dihydroxyvitamin D3 by vitamin D compounds, as well as the apparent dissociation constant of 1 alpha,25-dihydroxyvitamin D3 and its 1 beta epimer for the membrane receptor in membrane preparations from ROS 24/1 cells. Increasing concentrations of 1 alpha,25-dihydroxyvitamin D3, 7.25 nM to 725 nM, displaced 3H-1 alpha,25-dihydroxyvitamin D3 from the membranes with 725 nM of the hormone displacing 40-49% of the radioactivity. Similarly, 1 beta,25-dihydroxyvitamin D3, 7.25 nM and 72.5 nM, displaced 1 alpha,25-dihydroxyvitamin D3 binding while 25-hydroxyvitamin D3, 72.5 nM and 725 nM, did not. The apparent dissociation constant (KD) for 1 alpha,25-dihydroxyvitamin D3 was determined from displacement of 3H-1 alpha,25-dihydroxyvitamin D3 yielding a value of 8.1 x 10(-7) M by Scatchard analysis. The KD for the 1 beta epimer determined from displacement of 3H-1 beta,25-dihydroxyvitamin D3 was 4.8 x 10(-7) M.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Orthopedics, University of Massachusetts Medical Center, Worcester 01655.
authors.forename: D T
authors.initials: DT
authors.lastname: Baran
authors.forename: R
authors.initials: R
authors.lastname: Ray
authors.forename: A M
authors.initials: AM
authors.lastname: Sorensen
authors.forename: T
authors.initials: T
authors.lastname: Honeyman
authors.forename: M F
authors.initials: MF
authors.lastname: Holick
mesh_terms: D000818:Animals; D001665:Binding Sites; D002117:Calcitriol / Q000378:metabolism* / Q000493:pharmacokinetics*; D002462:Cell Membrane; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D012516:Osteosarcoma / Q000473:pathology; D051381:Rats; D013237:Stereoisomerism; D014407:Tumor Cells, Cultured
chemical_list: D008565:Membrane Proteins; D002117:Calcitriol