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Aim: To investigate the effects of the pleckstrin homology (PH) domain of phospholipase Cδ1 (PLCδ1PH) 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 PLCδ1PH (PLCδ1PH-GFP), which is known to bind Ptdlns(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 PLCδ1PH-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)P2 hydrolysis occurs through receptor-mediated PLC activation.Neomycin, a commonly used phospholipase C blocker, failed to block the receptor-induced PLCδ1PH-GFP translocation, indicating that neomycin is unable to block PLC-mediated PtdIns(4,5)P2 hydrolysis. However, in the absence of PLCδ1PH-GFP expression, neomycin abolished the receptor-induced hydrolysis of PtdIns(4,5)P2 by PLC. Conclusion: Although PLCδ1PH 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.