Abstract
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 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 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.
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Project supported by the National Natural Science Foundation of China (No 30270361), Ministry of Science and Technology of China (No 2003CCA00300), HL Zhang is a beneficiary of the National Science Fund for Distinguished Young Scholars of China (No 30325038).
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Wang, C., Du, Xn., Jia, Qz. et al. Binding of PLCδ1PH-GFP to PtdIns(4,5)P2 prevents inhibition of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 by neomycin. Acta Pharmacol Sin 26, 1485–1491 (2005). https://doi.org/10.1111/j.1745-7254.2005.00223.x
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DOI: https://doi.org/10.1111/j.1745-7254.2005.00223.x
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