The activation of T lymphocytes is the result of ligand-receptor interaction occurring at the interface of an antigen specific T cell and antigen presenting cell. Central to this interaction is the triggering of the T cell antigen receptor to initiate biochemical events leading to new gene transcription. The T cell antigen receptor consists of an antigen specific disulfide linked heterodimer (Ti). Ti is associated with an invariant molecular complex, termed CD3, which is composed of three to seven chains. Agonists binding to the CD3/Ti complex initiate activation by inducing the hydrolysis of phosphatidyl inositol trisphosphate with consequent second messenger generation, inositol trisphosphate and diacylglycerol. At least one result of the generation of inositol trisphosphate is the increase of cytoplasmic free calcium which has been shown to be a critical event involved in the initiation of IL-2 gene transcription. The goal of the proposed studies is to understand how the structure of the CD3/Ti complex relates to its functional role in signal transduction. In these studies a panel of mutants derived from the human T cell leukemic line Jurkat will be isolated which fail to increase cytoplasmic free calcium in response to agonists. The functional defect in these cells will be localized to the CD3 or Ti chain or more distal molecules which may play a role in signal transduction. Through the identification of chains and residues which have been altered in such mutants, an understanding of the regions of Ti or CD3 involved in signal transduction will be furthered. The information gained in such studies will be built upon by using site-directed mutagenesis of the involved chains of Ti in receptor reconstitution studies utilizing a panel of Jurkat mutants which are deficient in these chains. Such an analysis will help pinpoint critical residues which may be involved in conformational changes between Ti and T3. The signalling mutants generated will also be utilized in studies to assess the role of CD3 phosphorylation in T cell receptor activation and signal transduction.
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