Binding of PLCδ1PH-GFP to PtdIns(4,5)P2 prevents inhibition of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 by neomycin

Aim: To investigate the effects of the pleckstrin homology (PH) domain of phospholipase C-delta 1 (PLC delta 1PH) on inhibition of phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2] by neomycin. Methods: A fusion construct of green fluorescent protein (GFP) and PLC delta 1PH (PLC delta 1PH-GFP), which is known to bind PtdIns(4,5)P-2 specifically, together with laser-scanning confocal microscopy, was used to trace PtdIns(4,5)P-2 translocation. Results: Stimulation of the type 1 muscarinic receptor and the bradykinin 2 receptor induced a reversible PLC delta 1PH-GFP translocation from the membrane to the cytosol in COS-7 cells. PLC inhibitor U73122 blocked the translocation. Wortmannin, a known PtdIns kinase inhibitor, did not affect the translocation induced by ACh, but blocked recovery after translocation, indicating that PtdIns(4,5)P-2 hydrolysis occurs through receptor-mediated PLC activation. Neomycin, a commonly used phospholipase C blocker, failed to block the receptor-induced PLC delta 1PH-GFP translocation, indicating that neomycin is unable to block PLC-mediated PtdIns(4,5)P-2 hydrolysis. However, in the absence of PLC(delta 1)PHGFP expression, neomycin abolished the receptor-induced hydrolysis of PtdIns(4,5)P-2 by PLC. Conclusion: Although PLC delta 1PH and neomycin bind to PtdIns(4,5)P-2 in a similar way, they have distinct effects on receptor-mediated activation of PLC and PtdIns(4,5)P-2 hydrolysis.