BINDING CHARACTERISTICS OF A MEMBRANE-RECEPTOR THAT RECOGNIZES 1ALPHA,25-DIHYDROXYVITAMIN D-3 AND ITS EPIMER, 1-BETA,25-DIHYDROXYVITAMIN D-3

The steroid hormone 1 alpha,25-dihydroxyvitamin D-3 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-dihydroxyvitmina D-3 can block these rapid actions, indicating that the Ip epimer may bind to the receptor responsible for the rapid actions in a competitive manner. We have assessed the displacement of H-3-1 alpha,25-dihydroxyvitamin D-3 by vitamin D compounds, as well as the apparent dissociation constant of 1 alpha,25-dihydroxyvitamin D-3 and its 1 beta epimer for the membrane receptor in membrane preparations from ROS 24/1 cells. Increasing concentrations of 1 alpha,25-dihydroxyvitamin D-3, 7.25 nM to 725 nM, displaced H-3-1 alpha,25-dihydroxyvitamin D-3 from the membranes with 725 nM of the hormone displacing 40-49% of the radioactivity. Similarly, 1 beta,25-dihydroxyvitamin D-3, 7.25 nM and 72.5 nM, displaced 1 alpha,25-dihydroxyvitamin D-3 binding while 25-hydroxyvitamin D-3, 72.5 nM and 725 nM, did not. The apparent dissociation constant (K-D) for 1 alpha,25-dihydroxyvitamin D-3 was determined from displacement of H-3-1 alpha,25-dihydroxyvitamin D-3 yielding a value of 8.1 x 10(-7) M by Scatchard analysis. The K-D for the 1 beta epimer determined from displacement of H-3-1 beta,25-dihydroxyvitamin D-3 was 4.8 x 10(-7) M. The data suggest the presence of a receptor on the membranes of ROS 24/1 cells that recognizes 1 alpha,25-dihydroxyvitamin D-3 and its 1 beta epimer, but not 25-hydroxyvitamin D-3. Its ability to recognize the 1 beta epimer which appears to be a specific antagonist of the rapid effects of the hormone suggests that these studies may be the initial steps in the isolation and characterization of the signalling system mediating the rapid actions of vitamin D. (C) 1994 Wiley-Liss, Inc.