Work from a number of laboratories has confirmed the central role played by the CD45 tyrosine phosphatase in the regulation of signal transduction via the T cell and B cell antigen receptors. Our laboratory has generated a CD45-deficient variant of the Jurkat T cell leukemic line. Unlike the wild type parent, stimulation of the T cell receptor (TCR) on the CD45-deficient cells fails to result in the appearance of second messengers. Transfection of wild type CD45 or a chimeric molecule containing CD45 cytoplasmic sequences restores TCR-mediated signaling. One major focus of the current work is to understand better the structural features of CD45 responsible for its ability to regulate TCR signaling. Experiments are proposed which will alter the CD45 cDNA prior to transfection into the deficient cells in an effort to understand better the structural features of CD45 important in its ability to bind to other cellular proteins and support TCR signaling. These studies will also examine the structure-function relationships of lymphocyte phosphatase associated protein (LPAP) in the regulation of T cell activation. The next major aim of this proposal is to utilize CD45-deficient variants of two B cell leukemia lines which represent different stages of B cell maturation. Our goal will be to compare and contrast the requirement for CD45 and LPAP in the activation of these two B cell lines. For the final specific aim of this project, we propose to follow up on an observation that surface expression of hemapoietic cell-specific phosphatase (HCP) fails to rescue signal transduction via the TCR on our CD45-deficient cells and in fact inhibits TCR signaling in wild-type Jurkat cells. We propose studies to examine the structure-function relationships of HCP in regards to its dominant interfering effect on TCR signaling and to search for proteins with which it may interact. It is hoped that these studies will provide insight into the events associated with normal lymphocyte activation and thus be helpful in our attempts to unravel the events associates with dysregulation of signal transduction leading to pathologic states.
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