NK cells mediate important anti-tumor and anti-viral innate immune responses and can contribute to potent graft-versus-leukemia responses in allogeneic stem cell transplants. Unlike CTLs, where the crucial recognition event driving activation is mediated by a single receptor, the unique aB TCR expressed on that particular cell, NK cells must function by integrating a complex set of signals from the diverse array of activating and inhibitory NK receptors engaged upon interrogation of target cells. The ultimate goal of these studies is to understand the mechanism of NK cell signal integration. To achieve this long-term goal, we have begun with the C-type lectin-like NK receptor NKG2D, first determining receptor, ligand (MICs, ULBPs, RAE-ls) and complex crystal structures, characterizing interaction affinities, kinetics and thermodynamics by SPR and analyzing the interfaces computationally - all leading to an understanding of recognition and the determinants underlying structure and function. In Aim 1, we propose to complete these studies with specific mutations to confirm our recognition model, crystallographic and SPR analyses of the uniquely divergent ligands MIC-A*004 and ULBP4, and by determining the structural constraints on NKG2D signal transduction through the ectodomain, using mutagenesis and cell-based activation assays. We next propose to extend these studies to the rest of the NKG2x-CD94 receptor family, having already analyzed additional structures of their ligand (HLA-E) and the correlation between its thermal stability, expression and affinity. In Aim 2, we propose crystallographic and continuing SPR analyses of the structures, recognition machinery, affinities and kinetics of NKG2x-CD94-HLA-E interactions, concurrent with cell-based studies of the relative strengths of the signals delivered by the various activating and inhibitory CTLD NK receptors in driving activation. Completing Aims 1 and 2 will result in fully characterizing the extracellular components of the CTLD arm of the NK cell signal integration mechanism, and allow us to rationalize those parameters with measurements of relative signal strengths. Finally, in Aim 3, we propose to study other receptors that interact with MICs: V81 y8 TCRs. Having expressed soluble forms of three different MIC-responsive y8 TCRs, we propose SPR interaction studies (including whether NKG2D-TCR-MIC interactions are cooperative, anti-cooperative or independent) and crystallographic analyses to characterize the complexes.