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Published 27 May 2003. doi:10.1083/jcb.200301070
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© The Rockefeller University Press, 0021-9525/2003/5/779 $5.00
The Journal of Cell Biology, Volume 161, Number 4, 779-791

Article

 Kinetic analysis of receptor-activated phosphoinositide turnover

Chang Xu, James Watras and Leslie M. Loew

Department of Physiology and Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, CT 06030

Address correspondence to Leslie M. Loew, Dept. of Physiology and Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, CT 06030. Tel.: (860) 679-3568. Fax: (860) 679-1039. E-mail: les{at}volt.uchc.edu

We studied the bradykinin-induced changes in phosphoinositide composition of N1E-115 neuroblastoma cells using a combination of biochemistry, microscope imaging, and mathematical modeling. Phosphatidylinositol-4,5-bisphosphate (PIP2) decreased over the first 30 s, and then recovered over the following 2–3 min. However, the rate and amount of inositol-1,4,5-trisphosphate (InsP3) production were much greater than the rate or amount of PIP2 decline. A mathematical model of phosphoinositide turnover based on this data predicted that PIP2 synthesis is also stimulated by bradykinin, causing an early transient increase in its concentration. This was subsequently confirmed experimentally. Then, we used single-cell microscopy to further examine phosphoinositide turnover by following the translocation of the pleckstrin homology domain of PLC{delta}1 fused to green fluorescent protein (PH-GFP). The observed time course could be simulated by incorporating binding of PIP2 and InsP3 to PH-GFP into the model that had been used to analyze the biochemistry. Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP2 on the membrane or an increase in InsP3 in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.

Key Words: lipid signaling; bradykinin; phospholipase C; mathematical modeling; confocal microscopy

* Abbreviations used in this paper: GTP{gamma}S, guanosine 5'-O-(3-thiotriphosphate); InsP3, inositol 1,4,5-trisphosphate; PH, pleckstrin homology; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-bisphosphate.


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