Evidence that annexin II is not a putative membrane receptor for 1α,25(O-H)2-vitamin D3

The seco-steroid hormone 1alpha,25(OH)(2)-vitamin D-3 (1,25-D-3) is known to generate biological responses via both genomic and non-genomic rapid signal transduction pathways. The calcium regulated annexin II/p11 heterotetramer (AII(2)/P11(2)) was proposed by Baran and co-authors to be the membrane receptor responsible for mediating non-genomic, rapid actions of 1,25-D-3, based on ligand affinity labeling, competition, and saturation analysis experiments. Given the cytosolic presence of both the monomeric and heterotetrameric form of All and their functional regulation by intracellular calcium concentrations, which are known to be affected by 1,25-D3 rapid, non-genomic activities, we investigated in vitro the affinity of [H-3]-1,25-D-3 for the All monomer and AII(2)/P11(2) in the absence and presence of calcium using saturation analysis and gel-filtration chromatography. Using two different techniques for separating bound from free ligand (perchlorate and hydroxylapatite (HAP)) over a series of 30 experiments, no evidence for specific binding of [H-3]1,25-D-3 was obtained with or without the presence of 700 nM exogenous calcium, using either the All monomer or AII(2)/P11(2). However saturable binding of [H-3] 1,25-D-3 to the lipid raft/caveolae enriched rat intestinal fraction was consistently observed (K-d = 3.0 nM; B-max = 45 fmols/mg total protein). All was detected in lipid raft/caveolae enriched fractions from rat and mouse intestine and ROS 17/2.8 and NB4 cells by Western blot, but incubation in the presence of exogenous calcium did not ablate 1,25-D-3 binding as reported by Baran et al. Our results suggest that All does not bind 1,25-D-3 in a physiologically relevant manner; however, recent studies linking AII(2)/P112 phosphorylation to vesicle fusion and its calcium regulated localization may make All a possible down-stream substrate for 1,25-D-3 induced rapid cellular effects. (C) 2004 Wiley-Liss, Inc.