Recently, it has become clear that adapters, proteins which function to mediate intermolecular interactions, play as crucial a role in signal transduction as do receptors, enzymes, and downstream effectors. Two hematopoietic cell specific adapters, SH2 domain containing leukocyte phosphoprotein of 76 kDa (SLP-76) and Linker of Activation of T cells (LAT), have been shown to be essential for T cell development and for signal transduction mediated by the mature T cell antigen receptor (TCR). We reason that since SLP-76 possesses no enzymatic function, it is through the intermolecular interactions it coordinates that it serves its critical functions. To investigate this hypothesis, we propose in our first specific aim to perform an in vivo structure/function analysis of SLP-76 using two complementary approaches; reconstitution of lethally irradiated host mice with SLP-76 deficient bone marrow, which has been transduced with wild type or mutant SLP-76 transgenes and breeding SLP-76 transgenic animals onto the SLP-76 -/- background. We propose further to examine the intermolecular interactions mediated by SLP-76 in intact cells focusing on the regions of the molecule identified to be most critical for function. Our second specific aim will address the subcellular localization of SLP-76 and determine if the multimolecular complexes coordinated by SLP-76 differ based on location within the cell. Our third specific aim will investigate the importance of the interaction between SLP-76 and LAT in the regulation of TCR signals. These studies will make use of mutant cell lines to analyze the biochemistry of the signaling events as well as genetically altered mice to address the in vivo relevance of the findings obtained in the cell lines. Collectively, we hope these studies will provide insights into the complex regulation of signaling by SLP-76 and LAT in T cell development and activation.
