Kinetic analysis of receptor-activated phosphoinositide turnover

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 (PIP(2)) 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 (InsP(3)) production were much greater than the rate or amount of PIP(2) decline. A mathematical model of phosphoinositide turnover based on this data predicted that PIP(2) 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 PLCδ1 fused to green fluorescent protein (PH-GFP). The observed time course could be simulated by incorporating binding of PIP(2) and InsP(3) 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 PIP(2) on the membrane or an increase in InsP(3) in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.