The receptor for antigen and MHC (T3-Ti) on the vast majority of human T lymphocytes is a molecular complex comprised of an immunoglobulin-like Ti alpha-beta disulfide-linked heterodimer and three non-covalently associated monomorphic subunits (T3 gamma, T3 delta and T3 epsilon). Although much is known about the molecular nature of Ti diversity encoded by V, D and J-like elements and the primary structures of individual T3 subunits, the signal transduction process is ill-defined. Its molecular basis will be the subject of the present proposal. Firstly, monoclonal antibodies will be produced to the native external domain of individual T3 subunits expressed in the baculoviral system. These reagents will allow us to determine whether there are unique functional effects attributable to a given subunit. Alterations in (Ca2+)i, phosphoinositide turnover, phosphorylation events and gene transcription will be examined after external domain perturbation. Furthermore, novel strategies for developing antibodies to other putative T3-Ti associated surface structures will be devised and their primary structure analyzed by microsequencing and cDNA cloning. Secondly, through the use of gene transfer techniques and truncation mutants, the signal transduction function of individual cytoplasmic domains of T3 gamma, T3 delta and T3 epsilon will be defined. The role of potential phosphorylation sites in the cytoplasmic domain will be examined via site directed mutagenesis. The significance of the single acidic amino acid residue in the transmembrane segment of each T3 subunit with respect to T3-Ti stability and function will be investigated. Expression of T3-Ti subunit RNAs in xenopus oocytes will be attempted with the view of coupling T3-Ti complex signalling to the endogenous oocyte IP3 triggered C1- channel as a rapid assay for receptor mediated activation events. Thirdly, the role of the T3-Ti subunits in signalling growth inhibition will be characterized. It will be determined whether anti-T3 modulation functionally uncouples the T3-Ti complex from a G protein, inhibits protein kinase C or induces an arrestin- like protein to bind the T3-Ti complex. This desensitization response will be studied in detail with Jurkat mutants which have cytoplasmic deletions of individual T3 subunits. Finally, as the binding of the LFA-3 molecule to the CD2 external domain appears to amplify triggering through the T3-Ti complex, we will determine whether the T3 subunits are modified by phosphorylation or new protein associations after such an interaction.

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National Institute of Allergy and Infectious Diseases (NIAID)
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Immunobiology Study Section (IMB)
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Dana-Farber Cancer Institute
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