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 occu r 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 phosph olipase C and opening of calcium channels, pointing to a membrane loca lization of this signalling system. Previous studies have shown that t he 1 beta epimer of 1 alpha,25-dihydroxyvitmina D-3 can block these ra pid actions, indicating that the Ip epimer may bind to the receptor re sponsible for the rapid actions in a competitive manner. We have asses sed 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 recept or in membrane preparations from ROS 24/1 cells. Increasing concentrat ions 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-dih ydroxyvitamin 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,2 5-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 sug gest the presence of a receptor on the membranes of ROS 24/1 cells tha t recognizes 1 alpha,25-dihydroxyvitamin D-3 and its 1 beta epimer, bu t not 25-hydroxyvitamin D-3. Its ability to recognize the 1 beta epime r which appears to be a specific antagonist of the rapid effects of th e 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.