Binding of phospholipase C-related but catalytically inactive protein to phosphatidylinositol 4,5-bisphosphate via the PH domain.Cell Signal 2009; 21(7):1180-6CS
A well-known protein module regulating molecular interactions is the pleckstrin homology (PH) domain whose best-characterised ligand is phosphoinositide. In the present study, we analysed the PH domain from PRIP (phospholipase C-related but catalytically inactive protein, comprising types 1 and 2) regarding phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] binding employing a variety of binding assays. The PH domains prepared from PRIP-1 and -2 showed similar binding profiles to soluble ligands in vitro and showed similar plasma membrane localisation to that of PLC-delta1; however, the PH domain with the N-terminal extension of PRIP-1 but not PRIP-2 showed even distribution throughout the cytoplasm, indicating that the N-terminal extension of PRIP-1 inhibited binding to PtdIns(4,5)P(2) present in the plasma membrane. A chimeric molecule of PLC-delta1 PH domain with the N-terminal extension of PRIP-1 exhibited similar localisation to PRIP-1 PH domain with the N-terminal extension. Binding assay to liposomes containing various concentrations of PtdIns(4,5)P(2) revealed that the PH domain of PLC-delta1 bound steeply to the maximum, even at a concentration of 1.2 mol%, whereas the PH domains from PRIP-1 and -2 bound depending on the concentration up to 5 mol%. We also performed binding experiments using saponin-permeabilised PC12 cells. PH domains from PRIP increased the binding to cells preincubated with the brain cytosol extract in the presence of ATP, during which PtdIns(4,5)P(2) were probably synthesised. The binding of PH domain with the following EF hand motifs showed Ca(2+)-dependent binding. These results indicate that the PH domain of PRIP binds to PtdIns(4,5)P(2) present in the plasma membrane, depending on the concentrations of the lipid ligand and Ca(2+), suggesting that PRIP might play physiological roles in events involved in the changes of these parameters, probably including Ins(1,4,5)P(3).