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Aim:To investigate the effects of the pleckstrin homology(PH)domain of phos-pholipase C_(δ1)(PLC_(δ1)PH)on inhibition of phospholipase C(PLC)-mediated hy-drolysis of phosphatidylinositol 4,5-bisphosphate[PtdIns(4,5)P2]by neomycin.Methods:A fusion construct of green fluorescent protein(GFP)and PLC_(δ1)PH(PLC_(δ1)PH-GFP),which is known to bind PtdIns(4,5)P_2 specifically,together withlaser-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 recep-tor induced a reversible PLC_(δ1)PH-GFP translocation from the membrane to thecytosol in COS-7 cells.PLC inhibitor U73122 blocked the translocation.Wortmannin,a known PtdIns kinase inhibitor,did not affect the translocationinduced by ACh,but blocked recovery after translocation,indicating thatPtdIns(4,5)P_2 hydrolysis occurs through receptor-mediated PLC activation.Neomycin,a commonly used phospholipase C blocker,failed to block the recep-tor-induced PLC_(δ1)PH-GFP translocation,indicating that neomycin is unable toblock PLC-mediated Ptdlns(4,5)Pe hydrolysis.However,in the absence of PLC_(δ1)PH-GFP expression,neomycin abolished the receptor-induced hydrolysis ofPtdIns(4,5)P2 by PLC.Conclusion:Although PLC_(δ1)PH and neomycin bind toPtdIns(4,5)P2 in a similar way,they have distinct effects on receptor-mediatedactivation of PLC and PtdIns(4,5)P2 hydrolysis.
Aim: To investigate the effects of the pleckstrin homology (PH) domain of phos-pholipase C_ (δ1) (PLC_ (δ1) PH) on inhibition of phospholipase C (PLC) -mediated hy-drolysis of phosphatidylinositol 4,5-bisphosphate [ PtdIns (4,5) P2] by neomycin.Methods: A fusion construct of green fluorescent protein (GFP) and PLC_ (δ1) PH (PLC_ (δ1) PH-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 recep- tor induced a reversible PLC_ (δ1) PH-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 resistance occurs through receptor-mediated PLC activation. Neomycin, a commonly used phospholipase C blocker, failed to block the recep-tor- induced PLC_ (δ1) PH-GFP translocation, indicating that neomycin is unable to block the PLC-mediated Ptdlns (4,5) Pe hydrolysis. However, in the absence of PLC_ (δ1) PH- GFP expression, neomycin abolished the receptor- of PtdIns (4,5) P2 by PLC.Conclusion: Although PLC_ (δ1) PH 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.