Antibodies against the PH domain of phospholipase C-δ1 inhibit Ins(1,4,5)P₃-Mediated Ca²⁺ release from the endoplasmic reticulum

The pleckstrin homology domain (PH domain) is now well known as a structural module for the binding of inositol compounds. In the present study, polyclonal antibodies against the peptide KVKSSSWRRERFYK, derived from the N-terminal of the PH domain of phospholipase C-δ1 (PLC-δ1), were raised in rabbits. These were then tested for their ability to inhibit the binding of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] to the binding proteins including the receptor molecule. The Fab fragment of the antibodies but not the whole molecule inhibited the binding of Ins(1,4,5)P₃ not only to PLC-δ1 but also to the Ins(1,4,5)P₃ receptor, indicating that the antibodies raised recognized the binding site for Ins(1,4,5)P₃ in the receptor. Rat basophilic leukemic cells were permeabilized with saponin and assayed for Ins(1,4,5)P₃mediated Ca²⁺ release. Pretreatment of permeabilized RBL cells with the Fab fragment of the antibodies diminished the release of Ca²⁺ caused by Ins(1,4,5)P₃, and further absorption experiments using a variety of synthetic peptides suggested that the tripeptide KVK is the epitope of the antibodies. Structural information about KVK will help in screening for Ins(1,4,5)P₃ antagonists.