Cell-cell interactions are important in many aspects of immune function. Contact between T lymphocytes and their cognate partners is mediated in large part by the interaction of the T cell restricted molecule CD2 with its ubiquitous ligand LFA-3. Abrogation of CD2-LFA-3 interaction severely impairs T cell mediated immune function including that of helper T cells, cytolytic T cells and natural killer cells. Furthermore, the expression of CD2 on thymocytes and LFA-3 on thymic stromal elements suggests a role for CD2-LFA-3 interaction during T lineage development. During the last funding interval, cDNA cloning of human and murine CD2 has been completed, the genomic organization and chromosome assignment of the gene loci determined, recombinant CD2 expressed, physicochemical properties of the extracellular segment and N-terminal adhesion domain investigated, and signal transduction function of the cytoplasmic tail characterized. The importance of both CD2 mediated adhesion and signal transduction function was defined for the process of T cell receptor (TCR) mediated activation. In turn, the dependency of CD2 signalling on TCR function was also uncovered. The present proposal will provide detailed structure-function analysis of the CD2 intracellular and extracellular segments. First, coupling of CD2 signalling to CD3zeta will be investigated and any physical association between CD3zeta and the CD2 cytoplasmic region determined. Milligram quantities of the CD2 cytoplasmic domain will be produced by E. coli expression for use as an affinity ligand to detect CD2 associated cytoplasmic protein and to resolve the structure of the CD2 cytoplasmic domain using 4-D NMR. Potential associations of CD2 with cytoskeletal components, particularly following ligation of the CD2 extracellular segment by LFA-3 will be investigated. Second, detailed mapping of the LFA-3 binding region on the CD2 extracellular segment will be performed by site-directed mutagenesis in conjunction with a solution structure of human CD2. The suggestion that there may be additional extracellular ligands of CD2 will be directly addressed utilizing recombinant CD2 extracellular segment. Such ligands will be tested for their ability to bind directly to CD2 ex vivo as well as by immunohistochemical analysis utilizing labeled CD2 extracellular segment. If ligands are identified, they will be cloned and their binding site on the CD2 extracellular segment defined relative to LFA-3. Finally, homologous recombination technologies will be used to make CD2-/- mice or mice expressing altered CD2 forms. The role of CD2 in thymocyte development with emphasis on repertoire formation including positive and negative selection will be determined.
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