application abstract): The long term objective of the proposed research is to characterize how the potency of the T cell receptor (TCR) signals affects the activity of the Ras family of small GTPases. 'I'CR signals will be viewed by altering the contribution of the CD45 protein tyrosine phosphatase and by using ligands of varying affinities for the TCR. Thee studies will explore the hypothesis that small GTPases Ras and related protein Rapl do not act in isolation from each other but rather remain in close functional relationships which may effectively integrate and modify signal transduction events induced by the TCR complex. The applicant hypothesizes that depending on the isoform of CD 45 and the strength of the T cell receptor ligation, either antagonistic or synergistic arrangements between Ras and Rap1 are included, and that these different relationships which may influence lymphocyte behaviors such as T cell early development, differentiation of matured cells into Th1 or 'I'h2 helper subsets, and the generation of memory T cell pools.
In aim 1, the biochemical and functional experiments will examine the effect s of single CD45 isoforms on Ras and Rapl with respect to their immediate downstream targets and the direct upstream stimulators, the Raf kinases and the guanine nucleotide exchange factors (GEFs) respectively. Because individual.. CD4: isoforms are believed to differentially modify responsiveness of T cells to TCR induction information obtained in these studies will model approaches in aim 2 in which the effects of TCR stimulation with high affinity wild type peptide or low affinity altered peptide ligand, will be analyzed. In particular, following, these stimulations, the applicant will evaluate biochemical activities of Ras and Rapl, and examine functional properties of RAP1 to enhance or suppress Ras- signaling effects on mitogen activated protein kinase (MPK). The applicant plans to eventually deliver the dominant negative or constitutive active mutants of Ras or Rap1 into primary T cells and to test their potential to interfere with the functional polarization processes occurring during immune response. Understanding the molecular mechanisms controlling the activation and differentiation of naive T helper cells is crucial with regard to a variety of immunological conditions including allergic, infectious and autoimmune diseases. In addition, basic studies on the regulation of T lymphocytes by small GTPases have broad applicability to the field of cancer pathogenesis.
