The purpose of this application is to train a physician-scientist specializing in nephrology, with a particular interest in transplantation. This will be accomplished through a two-phase program involving a period of intensive didactic and research training progressing to independent research. The focus of research is on the mechanisms of signal transduction in T lymphocytes. The regulation of the signaling events which lead to the activation of resting T cells and thymocytes is central to the function of the immune system. The goal of this project is to use a combined molecular biological and biochemical approach to study the putative G protein alpha subunit(s) which link the T cell antigen receptor (TCR) to the phosphatidylinositol (PI) signal transduction pathway in T cells. This study is expected to identify a unique G protein or group of G proteins which couple a receptor to the PI pathway. It is therefore concerned with the control of growth and differentiation in general, as well as in T lymphocytes in particular. Phase I The polymerase chain reaction will be used to obtain a series of G protein sequences in Jurkat cells. One or more sequences will be selected as candidates for the G protein which is involved in the PI pathway. Site- directed mutagenesis will then be used to generate sequences encoding constitutively activated forms of these G proteins. In order to study the interaction of the G protein with the PI pathway, the activated G protein will be transiently expressed in Jurkat cells. The inositol phosphate (IP) products of the PI pathway will then be assayed. Phase II Monoclonal antibodies and polyclonal antisera directed against both specific and highly conserved G protein determinants will be developed. Biochemical and immunological methods will be used to examine the tissue distribution of the G protein(s) which activate the PI pathway and to demonstrate that this G protein is physiologically associated with the TCR. The long-term goal is to apply the skills and knowledge gained during this award to the study of T cell-mediated immune mechanisms in renal disease and/or transplantation.
| Graber, M; June, C H; Samelson, L E et al. (1992) The protein tyrosine kinase inhibitor herbimycin A, but not genistein, specifically inhibits signal transduction by the T cell antigen receptor. Int Immunol 4:1201-10 |
