The T-cell antigen CD28 provides a co-stimulatory signal that regulates an increasing number of T-cell functions that are central to the immune response. Understanding the intracellular signalling mechanisms that mediate CD28 function is an issue of great importance. Recently, we have shown that CD28 binds to two intracellular signalling molecules, phosphatidylinositol 3-kinase (PI 3-kinase) and GRB-2/SOS. Each mediate distinct and major pathways of signal transduction. Pl3-kinase generates D-3 inositol lipids, and is an essential component in signalling by various receptors. Similarly, GRB-2/SOS regulates the activation of p21 ras, a central part of growth control in various mammalian cells. Site- directed mutagenesis, peptide competition and binding studies demonstrated that PI 3-kinase and GRB-2 bind to the same site within the cytoplasmic tail of CD28 exhibit differences in binding requirements, avidity of binding and dependence on CD28 ligation. Further, they define distinct pathways, thereby providing CD28 with the ability to generate multiple signals in the control of different T-cell functions. In this application, we propose to examine further the nature of CD28-Pl 3-kinase and GRB-2 binding, to identify the kinase responsible for CD8 phosphorylation (Aim #1), to use an in vitro translation system to re-constitute the CD28-Pl 3- kinase and -GRB-2/SOS interaction in order to determine the stoichiometry of binding, size of the complex and the events that regulate binding (Aim #2), to examine the ability of CD80 (B7-1 and B7-2) ligation of CD28 to induce differential binding of Pi 3-kinase and GRB-2/SOS (Aim #3) and to define the connection between the CD28-Pl 3-kinase and -GRB-2/SOS signalling pathways and downstream events mediated by CD28 ligation (activation of the p21ras signalling pathway, IL-2 production, mRNA stability etc.) (Aim #4). This approach will provide a powerful means of dissecting the molecular basis of CD28 function.
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