This proposal tests a new hypothesis that phosphoinositide (PI) 3-kinase synergizes with phospholipase C in initiating receptor-mediated calcium signaling via a phosphatidylinositol 3,4,5-trisphosphate PI(3,4,5) P3]-sensitive Ca2+-entry pathway. This Pl(3,4,5)P3-mediated Ca2+ influx mechanism is unique in many types of hematopoietic cells, and represents a crucial element in PI 3-kinase-mediated signaling cascades that lead to cell activation. Consequently, this novel Ca2+-entry system may serve as a potential target for diseases related to, but not limited to, blood clotting, type I allergy, and T-cell immunity. This hypothesis will be tested in Jurkat T cells, an exquisite model to study PI 3-kinase signal transduction and Ca2+-entry mechanisms.
Specific aim 1 is to establish the obligatory role of PI 3-kinase in receptor-mediated Ca2+ signaling. This will be accomplished by a combined strategy using pharmacological inhibitors and a molecular genetic approach in which Jurkat T cells are stably transfected with a dominant-negative form of the p85 regulatory subunit to suppress the activity of endogenous PI 3-kinase. This strategy allows the assessment of the functional role of PI 3-kinase in Ca2+ regulation following receptor activation. The second specific aim will be to study interactions between PI(3,4,5)P3-mediated Ca2+ entry and other Ca2+ signaling components in the regulation of T-cell Ca2+ homeostasis. Receptor-mediated Ca2+ signaling is a highly complicated event that involves the concerted action of multiple Ca2+-transporting mechanisms in different cellular compartments of T cells. One plausible factor that links these multiple mechanisms is Ca2+ itself. We propose an integrated approach to study the communication of Pl(3,4,5)P3-mediated Ca2+ entry with lP3-mediated Ca2+ release and with store-operated (capacitative) Ca2+ influx. The third specific aim will be to purify and characterize the PI(3,4,5)P3-binding protein in the T-cell plasma membrane. High affinity PI(3,4,5)P3-binding proteins have been demonstrated in T cell plasma membranes using biotinylated PI(3,4,5)P3 as an affinity probe. This affinity ligand in conjunction with a photoaffinity probe permits rapid purification of the PI(3,4,5)P3-binding protein for sequencing and characterization. Sequence homology analysis will provide useful information concerning the identity of the PI(3,4,5)P3-binding protein. Functional characterization will be carried out by examining the effect of affinity-purified proteins on PI(3,4,5)P3- and anti-CD3 mAb-activated Ca2+ influx in Jurkat T cells. These studies will help understand how the PI(3,4,5)P3-binding protein mediates Ca2+ entry across the plasma membrane.
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