Open AccessBinding of PLCδ 1 PH‐GFP to PtdIns(4,5)P 2 prevents inhibition of phospholipase C‐mediated hydrolysis of PtdIns(4,5)P 2 by neomycin 1
Author(s) -
WANG Chuan,
DU Xiaona,
JIA Qingzhong,
ZHANG Hailin
Publication year - 2005
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2005.00223.x
Subject(s) - phospholipase c , chemistry , hydrolysis , phospholipase , biochemistry , microbiology and biotechnology , enzyme , green fluorescent protein , biology , gene
Aim: To investigate the effects of the pleckstrin homology (PH) domain of phospholipase C δ1 (PLC δ1 PH) 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 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 receptor 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 hydrolysis occurs through receptor‐mediated PLC activation. Neomycin, a commonly used phospholipase C blocker, failed to block the receptor‐induced PLC δ1 PH‐GFP translocation, indicating that neomycin is unable to block PLC‐mediated PtdIns(4,5)P 2 hydrolysis. However, in the absence of PLC δ1 PHGFP expression, neomycin abolished the receptor‐induced hydrolysis of PtdIns(4,5)P 2 by PLC. Conclusion: Although PLC δ1 PH 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.