The T cell receptor (TCR) is a multi-subunit complex that mediates recognition of peptide antigens in the context of MHC molecules and is central for initiating signal transduction events including those initiated through accessory structures. During the last funding period, we utilized Xenopus oocyte reconstitution as well as lymphoid cell line transfection studies to show that the dimeric TCR component CD3zelu is structurally and functionally similar to FcepsilonRIgamma, a signaling subunit of the high affinity IgE receptor. We also found that FcepsilonRIgamma substitutes for CD3zelu to form the TCR in naturally occurring LGL. In addition, we isolated the 23KD murine CD3eta protein, microsequenced CNBr fragments and cloned CD3eta at both cDNA and genomic levels. CD3eta was shown to be an alternatively spliced product of the same genetic locus encoding CD3zelu on murine chromosome 1 where the FcepsilonRIgamma gene is also found. The different functional roles of CD3eta vs. CD3zelu are not yet apparent, although CD3eta is thought to be involved in thymic development. During the next granting period, we will definitively characterize the role of CD3eta in thymic selection. B6D2 transgenic (tg) mice overexpressing CD3eta protein by >100 fold have already been created and will be backcrossed onto C57BL/6 (H-2b) and DBA/2 (H-2d) mice. Differential Vbeta usage and susceptibility to anti- CD3eta mAb induced DNA fragmentation in vivo will be assessed in comparison to non-tg littermates. Disruption of the CDeta locus by targeted recombination of ES cells will also be exploited. Second, the constitutive or induced association of novel intracellular proteins with the TCR complex will be systematically investigated using a battery of detergents and ionic conditions with and without chemical crosslinkers. 31-13 TCR-Jurkat or other variant cells transfected with CD8alpha/CD3zelu/eta chimeric constructs, or as control, CD8alpha will serve as incisive tools to detect TCR associations since such a chimera mediates all the signals of the native TCR. Microsequencing and cDNA cloning of candidate proteins will be performed and their CD3zelu/eta association verified by immunoprecipitation and western blotting analysis with specific antibodies. In parallel, direct screening of lambdagt11 T cell cDNA expression libraries will be performed with DC3zelu/eta derived peptides or fusion proteins containing the HMK phosphorylation site for 32P labeling. Finally, reconstitution studies in Xenopus oocytes will allow us to determine the minimal components of the TCR complex required for signaling phosphoinositide hydrolysis and.or protein tyrosine kinase activity. Oocyte microinjections will be performed with synthetic RNAs encoding CD8alpha/CD3zelu+/-p56lck+/-CD45 and signaling after anti-CD8alpha mAb crosslinking assessed. Any new cDNAs derived from aim 2 will be tested in this system as will others obtained by complementation methodologies.
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