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 both the PI and PTK second messenger pathways. 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-deficient mutants of the human T cell leukemic lines, HPB-ALL and Jurkat have been developed.
The specific aims of this project are to study how the structure of CD45 may relate to its function as a tyrosine phosphatase and as a regulator of TCR mediated signal transduction. This will be accomplished by site- directed mutagenesis of murine CD45 and the creation of chimeric molecules before reconstitution by transfection into the CD45-deficient cells. The ability of the various mutants to restore TCR-mediated signal transduction will be evaluated. More detailed studies addressing how CD45 regulates TCR-mediated signal transduction are proposed focusing on the role of CD45 in regulating the activity of the src-like kinases, fyn and lck. Additionally, experiments are described which will attempt to identify physiological substrates of CD45 by co-immunoprecipitation in our T cell leukemic lines and normal human T lymphocytes. A genetic approach, utilizing mutagenesis and cell-sorting techniques to isolate pseudo- revertants, to attempt to identify potential CD45 substrates is also proposed. Finally, we propose to evaluate the role of CD45 in the regulation of non TCR-associated PTK's by examining the function of the insulin receptor in our CD45-positive, CD45-deficient, and CD45-transfected clones.
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