T lymphocytes play an essential role in the body's defense against pathogens. Stimulation of T cells by specific antigens involves activation of the T cell antigen receptor complex (TCR) resulting in the generation of phosphatidylinositol (PI)-derived second messengers and the activation of a protein tyrosine kinase (PTK). Recent evidence from our laboratory and others has indicated that another T cell surface antigen, CD45, is important in T cell activation. The surface expression of CD45, a protein tyrosine phosphatase, appears to be critical in order for the TCR to couple with the PI second messenger pathway. The overall goal of this proposal is to understand the mechanism by which CD45 regulates signal transduction via the TCR. To pursue this question, CD45-negative mutants of the human T cell leukemic lines, HPB-ALL and Jurkat have been developed.
The specific aims of the project are to evaluate the status of the PTK second messenger pathway by analysis of the appearance of new phosphotyrosine containing proteins in wild type human T cells, the CD45-negative mutants, and negative cells reconstituted with murine CD45 cDNA before and after stimulation via the TCR. The function of the PTK and PI pathways and their relative importance in the generation of distal biologic events will then be assessed. Additionally, the ability of the CD45-negative cells to be stimulated by """"""""accessory molecules"""""""" will be evaluated, studying both proximal (second messenger generation) and distal (production of lymphokines, expression of activation antigens) signalling events. Experiments also designed to identify CD45 substrates utilizing reagents specific for several candidate proteins are presented. The final specific aim of the proposal is the creation of mutant CD45 molecules (by site-directed mutagenesis and the formation of chimeric molecules) in order to study some of the function/structure relationships of the CD45 tyrosine phosphatase. Specifically, the role of the phosphatase domain(s) of CD45 and the location of this molecule within the cell in permitting TCR mediated signal transduction will be evaluated.
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