Regulation of PLC1a membrane anchoring by its substrate phosphatidylinositol (4,5)-bisphosphate

Basic knowledge as to the subcellular location and dynamics of PLC isozymes is scant. Here, we report on the subcellular location of GFP-PLC1a and the use of total internal reflection fluorescence (TIRF) microscopy to examine the dynamics of GFP-PLC1a at the plasma membrane upon stimulation of Gq-coupled receptors. Using this technique, we observed PLC1a dissociation from the plasma membrane upon addition of agonist. An increase in intracellular calcium and a decrease in PtdIns(4,5)P₂ both coincided with a translocation of PLC1a from the plasma membrane into the cytosol. In order to differentiate between calcium and PtdIns(4,5)P₂, rapamycin-induced heterodimerization of FRB and FKBP12 fused to 5-phosphatase IV was used to instantaneously convert PtdIns(4,5)P₂ into PtdIns(4)P. Addition of rapamycin caused PLC1a to dissociate from the plasma membrane, indicating that removal of PtdIns(4,5)P₂ is sufficient to cause translocation of PLC1a from the plasma membrane. In conclusion, PLC1a localization is regulated by its own substrate.