Apoptotic calcium release from inositol 1,4,5-trisphosphate receptors (IP3Rs) regulates cell death in response to multiple stimuli, including engagement of the Fas receptor. We have found that the Fas receptor (FasR) directly engages and activates components of the T cell receptor complex (TCR) to elicit apoptotic calcium release from IP3R channels. However, the underlying molecular mechanisms remain to be established. Our preliminary data suggests that Fas and TCR components are recruited into detergent-resistant membrane microdomains enriched in cholesterol and glycosphingolipids (lipid rafts) following Fas stimulation. Furthermore, our data indicates that this is associated wih the rapid stimulus-dependent palmitoylation of the Src family kinase Lck. The central hypothesis of this proposal is that apoptotic calcium release is mediated by rapid palmitoylation of components of the Fas receptor complex, recruitment into lipid rafts, and subsequent activation of TCR components. We will investigate this hypothesis in two Specific Aims. In Specific Aim 1, we will determine if recruitment of the FasR/TCR supramolecular signaling complex into lipid rafts mediates Fas-associated apoptotic calcium release. In Specific Aim 2: Determine if rapid stimulus-dependent palmitoylation/depalmitoylation of Lck by DHHC21 mediates Fas-associated apoptotic calcium release. We will use a variety of molecular biological, biochemical, and novel live cell imaging techniques to determine the mechanisms by which Fas receptor engages the TCR complex to mediate T lymphocyte calcium release and cell death. These studies may uncover novel therapeutic targets for diseases associated with altered T cell homeostasis.