IN VIVO STRUCTURE FUNCTION RELATIONSHIP OF SLP-76

University Of Pennsylvania, Philadelphia PA

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Abstract

DESCRIPTION (provided by applicant): Recently, it has become clear that adapters, proteins that 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 76dDa (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 three 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, breeding SLP-76 transgenic animals onto the SLP-76-/- background, and generating SLP-76 mutations in the endogenous locus by "knock-in" methodology. We additionally will make use of fetal thymic organ culture to examine the ability of other proteins to complement developmental deficiencies associated with expression of SLP-76 mutants. Our second specific aim will investigate the relationship between SLP-76 and PLCy1 and ITK, two proteins that play important roles in TCR signaling. These studies will make use of biochemical approaches, transfections into cell lines, and studies in intact animals. Our third 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. These studies will also evaluate how SLP-76 localization is modulated by T cell activation and by the association of SLP-76 with other proteins. Our proposed experiments will make use of both scientific cores associated with this Program and will involve collaborations with other P01 investigators. It is hope that collectively our work will increase our understanding of the role of SLP-76 (and adapter proteins more generally) in the regulation of cellular activation.