This project involves the study of a rapidly emerging group of immune receptors. Many families of inhibitory immune receptors have recently been identified in both mice and humans. Interestingly, within each of these inhibitory families of receptors, there are proteins that have lost the inhibitory domains. Instead these receptors have gained a positively charged acid within their transmembrane domain, suggesting that they may interact with signal transduction chains and transmit positive signals. In this project, we study the signal transduction and biochemistry of both the positive and negative regulators of immune cell function. Through the study of the positive receptors, we and others demonstrated the association of some of these receptors with the novel signal transduction chain DAP12. Since then we have been characterizing the biochemistry of the DAP12 signal transduction pathway.Our studies of paired receptor systems have now largely involve the study of the Ly49s of NK cells and the Triggering Receptors Expressed on Myeloid cells (TREM). In NK cells we are focused on dissection of the role of a variety of adaptors in the transmission of the DAP12 signal. We have accumulated multiple knockout mice for these adaptors and are assaying Ly49, NKG2D and Fc receptor function in these mice. Crossing the various strains will generate multiple knockouts for further study. In contrast to NK cells, the TREM are a rapidly emerging immune receptor family. TREM-1 has recently been shown to be involved in the amplification of signals leading to septic shock and TREM-2 has been reported to be involved in the maturation of dendritic cells. Despite the established role of TREMs in both innate and adaptive immunity the signal transduction of the family is still relatively unknown. Each TREM family member, with the exception of TLT-1 (see below), is physically coupled to the DAP12 signaling chain. However, because of the expression of Fc receptors, studies using antibodies to cross link TREM are difficult to interpret. Therefore, we have recently developed an antibody-free, chimeric receptor approach to study DAP12 signaling in the myeloid compartment. The system uses the fusion of the ligand binding domain of GPVI to the cytoplasmic domain of DAP12. Stimulation of the cells with the soluble ligand, convulxin, results is phosphorylation of the chimera, activation of Syk, and downstream signaling including Erk1/2 activation. We are now using this system to dissect DAP12 signaling in RAW264 macrophages.In addition to the activating members of the TREM family, we recently described TREM Like Transcript-1, a putative inhibitory receptor within the TREM cluster. Immunohistochemical and immunofluorescence studies show that bone marrow TLT-1 is derived exclusively from megakaryocytes. The subcellular localization and regulation of TLT-1 in megakaryocytes and platelets suggests that TLT-1 is prepackaged into the platelet alpha granules by megakaryocytes for rapid surface expression after platelet degranulation. These findings suggest TLT-1 may be involved in the production and release of alpha granules and/or the thrombotic response that takes place on the platelet surface after activation. To fully elucidate the role of TLT-1 we have recently employed bacterial recombineering. This technology allows for the rapid modification of large pieces of DNA using in vivo recombination in bacteria. We have now used this technique to generate both traditional and BAC-derived gene targeting vectors for TLT-1. The traditional construct has 6Kb of flanking homology where as the BAC-based vector has nearly 250 Kb of homology. Both constructs were introduced into C57BL/6 ES cells and, for the first time, directly compared the effects of homologous arm length on rates of recombination. Our results showed that the long homologous arms of the BAC-derived construct yielded a five-fold increase in homologous recombination. The ES cells of both constructs are now being injected for the production of TLT-1-/- mice. These mice will be rigorously tested for the effects of TLT-1 on thrombosis.As we develop the mouse model we are dissecting the intracellular binding partners of TLT-1. A combination of GST pulldowns and protein sequencing has identified 2 partners so far and we are investigating these further. One of the TLT-1 binding proteins is Nedd4, a WW domain containing E3 ubiquitin ligase. Our current model suggests that Nedd4 may participate in the packaging and/or trafficking of TLT-1 to the alpha granules. These possibilities are being addressed using a variety of models.
