Binding of PLCdelta1PH-GFP to PtdIns(4,5)P2 prevents inhibition of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 by neomycin.
OBJECTIVE
To investigate the effects of the pleckstrin homology (PH) domain of phospholipase C(delta1) (PLC(delta1)PH) 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 PLC (delta1)PH (PLC(delta1)PH-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 PLC(delta1)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)P2 hydrolysis occurs through receptor-mediated PLC activation. Neomycin, a commonly used phospholipase C blocker, failed to block the receptor-induced PLCd1PH-GFP translocation, indicating that neomycin is unable to block PLC-mediated PtdIns(4,5)P2 hydrolysis. However, in the absence of PLCd1PH-GFP expression, neomycin abolished the receptor-induced hydrolysis of PtdIns(4,5)P2 by PLC.


CONCLUSIONS
Although PLCd1PH 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.
Wang
Chuan
C
Department of Pharmacology, Hebei Medical University, Shijiazhuang 050017, China.
Du
Xiao-Na
XN
Jia
Qing-Zhong
QZ
Zhang
Hai-Lin
HL
D000109:Acetylcholine / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D019556:COS Cells / Q000166:cytology; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D009355:Neomycin / Q000494:pharmacology*; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D054803:Phospholipase C delta; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D011760:Pyrrolidinones / Q000494:pharmacology; D043782:Receptor, Bradykinin B2 / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D004963:Estrenes; D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011760:Pyrrolidinones; D043782:Receptor, Bradykinin B2; D011976:Receptors, Muscarinic; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D049452:Green Fluorescent Proteins; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D009355:Neomycin; D000109:Acetylcholine; D001920:Bradykinin; D000077191:Wortmannin