Binding of PLCδ1PH-GFP to Ptdlns(4,5)P2 prevents inhibition of phospholipase C-mediated hydrolysis of Ptdlns(4,5)P 2 by neomycin

Abstract

Aim: To investigate the effects of the pleckstrin homology (PH) domain of phospholipase C81 (PLC61PH) on inhibition of phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by neomycin. Methods: A fusion construct of green fluorescent protein (GFP) and PLC81PH (PLC61PH-GFP), which is known to bind PtdIns(4,5)P2 specifically, together with laser-scanning confocal microscopy, was used to trace PtdIns(4,5)P2 translocation. Results: Stimulation of the type 1 muscarinic receptor and the bradykinin 2 receptor induced a reversible PLC81PH-GFP translocation from the membrane to the cytosol in COS-7 cells. PLC inhibitor U73122 blocked the translocation. Wortmannin, a known Ptdlns kinase inhibitor, did not affect the translocation induced by ACh1 but blocked recovery after translocation, indicating that PtdIns(4,5)P2 hydrolysis occurs through receptor-mediated PLC activation. Neomycin, a commonly used phospholipase C blocker, failed to block the receptor-induced PLC 81PH-GFP translocation, indicating that neomycin is unable to block PLC-mediated PtdIns(4,5)P2 hydrolysis. However, in the absence Of PLC61PH-GFP expression, neomycin abolished the receptor-induced hydrolysis of PtdIns(4,5)P2 by PLC. Conclusion: Although PLC 81PH and neomycin bind to PtdIns(4,5)P2 in a similar way, they have distinct effects on receptor-mediated activation of PLC and PtdIns(4,5)P2 hydrolysis. ©2005 CPS and SIMM.