This proposal is a continuation of our previous studies aimed at analyzing the properties and the function of the CD2/E-receptor pathway of human T cell activation. Our interest is to study the structure-function relationship of CD2 and to define and better understand the molecular and physiological events associated with the triggering of CD2 receptors by mitogens and monoclonal antibodies. We will examine 1) the phosphorylation of CD2 molecules on human T cells in the presence of regimens known to affect cell activation, including the triggering by mitogenic combinations of anti-CD2 monoclonal antibodies. We will analyze the phosphoamino acid composition of 32P CD2. 2) Furthermore, we will pursue the analysis and functional testing of CD2 cDNA clones using DNA transfection techniques. We will attempt to reconstitute an active CD2 receptor by DNA transfer in CD2-negative lymphoid T cell lines. We already have demonstrated that a full-length CD2 cDNA clone can be transiently expressed in Cos-7 cells, using the pcEXV-CD2 vector containing the SV40 promoter. Surface CD2 molecules of transfectant cells expressed the adhesion sites as well as activation sites defined by monoclonal antibodies. We will attempt to isolate stable lymphoid cell line transfectants expressing high levels of cell surface CD2 using the expression vector pBC12/CMV/CD2 containing the human cytomegalovirus immediate-early promoter as well as other expression vectors. Positive selection for stable transfectant will be achieved by co- transfectant plasmids (pcEXV-Neo) containing the mammalian selectable marker neo. The generation of deletion mutants of CD2 cDNA using Exonuclease III digestion or site specific mutagenesis will allow us to examine specific amino acid sequences necessary for the proper functioning of CD2, in particular, the ligand binding site (T111), the activation site (T112/T113), and the role of particular amino acids in the cytoplasmic region, including those modified by phosphorylation, as well as the unusual clusters of basic residues and prolines. Many described roles of CD2 in T cell activation and differentiation make the study of CD2 structure-function extremely important for understanding and potentially manipulating the immune response.
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