Involvement of EF hand motifs in the Ca2+-dependent binding of the pleckstrin homology domain to phosphoinositides

The pleckstrin homology (PH) domains of phospholipase C (PLC)-delta 1 and a related catalytically inactive protein, p130, both bind inositol phosphates and inositol lipids. The binding to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2] by PLC-delta 1 is proposed to be the critical interaction required for membrane localization to where the substrate resides; it is also required for the Ca2+-depsndent activation of PLC-delta 1 observed in the permeabilized cells. In the proximity of the PH domain, both PLC-delta 1 and p130 possess the EF-hand domain, containing classical motifs implicated in calcium binding. Therefore, in the present study we examined whether the binding of the PH domain to PtdIns(4,5)P-2 is regulated by changes in free Ca2+ concentration within the physiological range. A Ca2+ dependent increase in the binding to PtdIns(4,5)P-2 was observed with a full-length PLC-delta 1, while the isolated PH domain did not show any Ca2+ dependence. However, the connection of the EF-hand motifs to the PH domain restored the Ca2+ dependent increase in binding, even in the absence of the C2 domain. The p130 protein showed similar properties to PLC-delta 1, and the EF-hand motifs were again required for the PH domain to exhibit a Ca2+ dependent increase in the binding to PtdIns(4,5)P-2. The isolated PH domains from several other proteins which have been demonstrated to bind PtdIns(4,5)P-2 showed no Ca2+ dependent enhancement of binding. However, when present within a chimera also containing PLC-delta 1 EF-hand motifs, the Ca2+ dependent binding was again observed. These results suggest th;it the binding of Ca2+ to the EF-hand motifs can modulate binding to PtdIns(4,5)P-2 mediated by the PH domain.