Binding of 1α,25-dihydroxyvitamin D3 to annexin II:: effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1 alpha ,25-(OH)(2)D-3 and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [C-14]-1 alpha ,25-(OH)(2)D-3 bromoacetate to purified annexin II was inhibited by 1 alpha ,25-(OH)(2)D-3 in a concentration-dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1 alpha ,25-(OH)(2)D-3 at 24 muM, 18 muM, and 12 muM and blunted by 6 muM and 3 muM. At a concentration of 12 muM, 1 beta ,25-(OH)(2)D-3 also diminished the binding of [C-14]-1 alpha ,25-(OH)(2)D-3 bromoacetate to annexin II, but cholecalciferol, 25-(OH)D-3, and 24,25-(OH)(2)D-3 did not. Saturation analyses of the binding of [H-3]-1 alpha ,25-(OH)(2)D-3 to purified annexin II showed a K-D of 5.5 x 10(-9) M, whereas [H-3]-1 beta ,25-(OH)(2)D-3 exhibited a K-D of 6.0 x 10(-9) M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration-dependent effect on [C-14]-1 alpha ,25-(OH)(2)D-3 bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1 alpha ,25(OH)(2)D-3 binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1 beta ,25-(OH)(2)D-3 to inhibit the binding of [C-14]-1 alpha ,25(OH)(2)D-3 bromoacetate to annexin II provides a biochemical explanation for the ability of the 1 beta -epimer to inhibit the rapid actions of the hormone in vitro. J. Cell. Biochem. 80:259-265, 2000. (C) 2000 Wiley-Liss, Inc.