Stimulation of many receptors, including the T cell receptor (TCR), leads to an increase in the activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC catalyzes the hydrolysis of phosphatidylinositol bisphosphate to inositol-1,4,5-triphosphate and diacylglycerol, leading to an increase in intracellular calcium concentration, and activation of protein kinase C. This pathway is thought to play a central role in the complex process of T cell activation, and yet the expression, and regulation of the PI-PLC molecule itself have not been examined in T cells. Recently, at least 5 structurally divergent isoforms of PI-PLC have been identified, and many cells may simultaneously express multiple isoforms. This finding has raised the possibility that different isoforms subserve distinct functions in relation to cellular activation. Initial studies have shown that the human T cell line, Jurkat expresses at least 2 isoforms of PI-PLC: PLCalpha and PLCgamma, and that stimulation of Jurkat cells with phytohemagglutinin and phorbol myristate acetate, leads to differential regulation of transcripts encoding PLCalpha and PLCgamma. Thus, Jurkat cells provide a useful system in which to examine the functions and regulation of PI-PLC isoforms, and their roles in T cell activation. The proposed studies will examine the following hypotheses: 1) That there is differential expression of PI-PLC isoforms among different functional subsets of T cells, and during T cell ontogeny; 2) That more than 1 isoform can couple to the specific isoforms after T cell activation, and 3) That expression of one or more isoforms of PI-PLC is required for TCR-mediated production of IL-2 and induction of IL-2 receptor expression. In the first part of the studies, northern and western blotting techniques will be used to examine the expression and regulation of PI-PLC isoforms in functional subsets of T cells, T cell clones and in thymocytes. The effects of TCR-mediated activation of Jurkat and other T cell populations of PI-PLC isoform expression will be assessed in parallel experiments. The second phase of the project will involve transfection of Jurkat with expression vectors carrying cDNA sequences of individual PI-PLC isoforms in reverse orientation. The resulting antisense RNA inhibition should ablate the expression of specific PI-PLC isoforms. These Jurkat transfectants will be used in functional studies to define the contribution of each expressed PI-PLC isoform to both the receptor-mediated hydrolysis of inositolphospholipids, and to subsequent stages of T cell activation, such as production of IL-2 and expression of IL-2 receptors.
