The pleckstrin homology domain of phosphoinositide-specific phospholipase Cδ4 is not a critical determinant of the membrane localization of the enzyme

Abstract

The inositol lipid and phosphate binding properties and the cellular localization of phospholipase Cδ4 (PLCδ4) and its isolated pleckstrin homology (PH) domain were analyzed in comparison with the similar features of the PLCδ1 protein. The isolated PH domains of both proteins showed plasma membrane localization when expressed in the form of a green fluorescent protein fusion construct in various cells, although a significantly lower proportion of the PLCδ4 PH domain was membrane-bound than in the case of PLCδ1PH-GFP. Both PH domains selectively recognized phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but a lower binding of PLCδ4PH to lipid vesicles containing PI(4,5)P2 was observed. Also, higher concentrations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) were required to displace the PLCδ4PH from the lipid vesicles, and a lower Ins(1,4,5)P3 affinity of PLCδ4PH was found in direct Ins(1,4,5)P3 binding assays. In sharp contrast to the localization of its PH domain, the full-length PLCδ4 protein localized primarily to intracellular membranes mostly to the endoplasmic reticulum (ER). This ER localization was in striking contrast to the well documented PH domain-dependent plasma membrane localization of PLCδ 1. A truncated PLCδ4 protein lacking the entire PH domain still showed the same ER localization as the full-length protein, indicating that the PH domain is not a critical determinant of the localization of this protein. Most important, the full-length PLCδ4 enzyme still showed binding to PI(4,5)P2-containing micelles, but Ins(1,4,5)P3 was significantly less potent in displacing the enzyme from the lipid than with the PLCδ1 protein. These data suggest that although structurally related, PLCδ1 and PLCδ 4 are probably differentially regulated in distinct cellular compartments by PI(4,5)P2 and that the PH domain of PLCδ 4 does not act as a localization signal.