SIGNAL TRANSDUCTION IN T CELLS

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The purpose of this proposal is to investigate the role of GTP binding proteins in human T cell activitation. GTP binding proteins, composed of three subunits (Alpha,Beta,Gamma), have been shown to couple ligand- receptor interactions to intracellular effectors. No such proteins have been identified as yet in cells of the immune system. We have accumulated preliminary evidence which strongly suggests that such proteins do exist in human T lymphocytes. We have demonstrated the presence of GTP-binding protein Alpha subunits in membranes of human T cells by using 32p-NAD and by taking advantage of the ability of bacterial toxins (cholera and pertussis toxin) to specifically ADP- ribosylate the Alpha subunits in vitro using the radioactive NAD as a donor of the ADP-ribose. Furthermore, these toxins can modulate activation of T cells induced via the TCR/CD3 complex. For example, cholera toxin at picomolar concentrations inhibits anti-CD3+IL2 mediated proliferation of human T cells. In contrast, this toxin does not inhibit ionomycin+PMA proliferation even at 10-fold higher concentration. Additional data suggest that the cholera toxin mediated inhibition is probably at the step induced by anti-CD3 and not at the step controlled by IL2. We have also identified and isolated GTP-binding protein alpha subunit cDNA clones by screening a Gamma gt11 cDNA library from the human leukemic T cell line Jurkat with synthetic oligodeoxynucleotides. A partial nucleotide sequence of one of the cDNA clones has been also obtained. Finally, antibodies to synthetic peptides that represent conserved regions of Alpha subunits have been prepared as probes for characterizing these proteins in T cells. We proposed to characterize the isolated a subunit cDNA clones and compare their sequences to those of already cloned Alpha subunits. Using DNA-RNA hybridization analysis we shall determine whether or not the isolated cDNA clones are uniquely expressed in cells of the lymphoid lineage. Furthermore, we shall compare the expression of these genes in resting and activated T cells, as well as, in T cells at various stages of their differentiation (thymocytes). Furthermore, we'll determine the topographic association of GTP-binding proteins to specific T cell surface receptors by using bifunctional crosslinking chemistry. Finally, we'll utilize synthetic peptides that represent conserved portions of the alpha subunits and their reactive antibodies in order to determine the involvement of GTP-binding proteins in the triggering of T cells via the TCR/CD3 molecular complex. We'll measure the effects of these agents on IP3 and Ca++ accumulation using a permeabilized cell system and electroporation.