The Unit investigates signal transduction pathways that mediate the actions of hormones and growth factors in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Recent studies have utilized green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains to monitor phosphoinositide changes in single living cells. The PH domain of phospholipase C (PLC) delta1, which binds to the membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], was used to monitor PLC activation based on confocal imaging of fluorescence translocation from the membrane to cytosol that occurs upon phosphoinositide hydrolysis. Current studies have explored the possibility that fluorescence energy transfer (FRET) could be used as a sensitive readout of PLC activation both in cell populations and in single-cells. In cells co-transfected with PH-domains tagged with either the cyan (CFP) or the yellow (YFP) fluorescent protein, it was possible to monitor the dynamics of PI(4,5)P2 hydrolysis (PLC activation) and PI(3,4,5)P3 formation (PI 3-kinase activation) either in single cells or in populations of cells. FRET requires significantly less excitation intensity, enabling prolonged and fast data-acquisition without the bleaching and cell damage that limit confocal experiments. Moreover, by choosing the appropriate protein domains, it is possible to follow changes in specific membrane compartments, such as the Golgi or the plasma membrane.Additional studies investigate the molecular determinants that contribute to the membrane recruitment of inositide binding protein domains. The PH domains of PLC-delta1 and of the recently described PLC-like protein, p130, show a great degree of homology. When expressed in bacteria, both domains bind Ins(1,4,5)P3 with identical affinity. However, PLC-delta1PH-GFP but not p130PH-GFP was localized to the membrane when expressed in COS-7 cells. By using a chimeric approach, it was found that the N-terminal parts of the PH domains containing most of the Ins(1,4,5)P3 binding contacts are interchangeable between the two proteins, while the C-terminal half of the PLC-delta1-(but not of p130) PH domain provides an additional binding force that enables it to localize to the membrane. Thus, although inositide binding is necessary it is not sufficient for effective membrane recruitment of these PH domains. Current studies are aimed at determining the nature of this additional interaction.
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