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Inositol 1,4,5-trisphosphate (IP^sub 3^) is a second messenger that elicits complex spatiotemporal patterns of calcium ion (Ca^sup 2+^) mobilization and has essential roles in the regulation of many cellular functions. In Madin-Darby canine kidney epithelial cells, green fluorescent protein-tagged pleckstrin homology domain translocated from the plasma membrane to the cytoplasm in response to increased concentration of IP^sub 3^. The detection of transLocation enabled monitoring of IP^sub 3^ concentration changes within single cells and revealed spatiotemporal dynamics in the concentration of IP^sub 3^ synchronous with Ca^sup 2+^ oscillations and intracellular and intercellular IP^sub 3^ waves that accompanied Ca^sup 2+^ waves. Such changes in IP, concentration may be fundamental to Ca^sup 2+^ signaling.
IP^sub 3^ production by phospholipase C (PLC)mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP^sub 2^) is an early intracellular event after stimulation by hormones, autacoids, and neurotransmitters. IP^sub 3^ mobilizes Ca^sup 2+^ from intracellular stores through the IP3 receptor, resulting in activation of Ca^sup 2+^-dependent cellular events such as contraction, secretion, gene expression, and synaptic plasticity (1, 2). Ca^sup 2+^ mobilization occurs in complex temporal and spatial patterns, including Ca^sup 2+^ oscillations (3) and Ca^sup 2+^ waves (4). However, the mechanism underlying the generation of the complex patterns has not been fully elucidated, partly because of lack of knowledge regarding IP^sub 3^ dynamics in single cells.
Green fluorescent protein (GFP)-based probes have been used to analyze cellular signaling because they have the advantage that they can be DNA encoded (5). Fusion proteins consisting of GFP and a functional protein domain can function as molecular probes when their intracellular translocation pattern can be visualized (6). The GFP-tagged pleckstrin homology (PH) domain of PLC-delta^sub 1^ (GFP-PHD) is one such probe because it binds to PIP^sub 2^ within the plasma membrane and translocates to the cytoplasm after receptor stimulation (7). Although the translocation was thought to reflect a decrease in the PIP^sub 2^ concentration (7), we obtained evidence that an increase in the cytoplasmic IP^sub 3^ concentration ([IP^sub 3^]^sub i^) causes the translocation of GFP-PHD, and therefore we used GFP-PHD to monitor spatiotemporal changes in [IP^sub 3^]^sub i^ that underlie the complex Ca^sup 2+^ mobilization patterns within single living cells.
We analyzed PIP^sub 2^ binding of the PH domain of PLC-delta^sub 1^ (8) by a surface plasmon assay...