) The lymphotoxin (LT)-beta receptor (LTbR) is essential for the development of secondary lymphoid tissue, micro architecture of the spleen, and differentiation of NK and dendritic cells. This receptor is activated by LTa1b2, and LIGHT, a new member of the TNF family that also binds to Herpesvirus entry mediator (HVEM). At the molecular level, LTbR signal transduction is initiated via recruitment of TRAFs, however LTbR, a non-death domain receptor, can induce apoptosis. TRAF3 is essential for LTbR induced death of HT29 adenocarcinoma as defined by dominant negative mutants of TRAF3. LTbR signaling also activates NFkB and AP1 independently of cell death and is mediated via TRAF 2 and 5, yet TRAFs may not account for all the signaling by the LTbR. We have identified a signaling domain in the LTbR that is essential for apoptosis and NFkB activation. This region contains a distinct TRAF binding motif, subcellular compartmentalization motif, and region that regulates death signaling through receptor aggregation. The goal of this project is to understand how the molecular components of the LTbR and functionally related TNFR signaling pathways are integrated into the physiological processes observed in vivo. We have created a variety of reagents, cell lines, and screening methods to identify proteins involved in signal regulation by the LTbR. Candidate regulators, such as SODD will be tested for interactions with LTbR by coimmunoprecipitation, two-hybrid complementation and colocalization using retrovirus gene transfer system in HT29 adenocarcinoma cells. A tyrosine motif is proprosed to control localization to subcellular compartments via caveolin. Viral immune evasion proteins that interfere with signal transduction will be used to probe the nexus connecting death domain and TRAF binding receptors to caspase pathway and tested by use of point mutation in the tyrosine motif, which will create a dominant negative mutant. A putative TRAF-independent pathway for NFkB activation exists which will be defined using dominant negative mutants of NIK and IkB. Together these specific aims will provide new knowledge concerning the molecular steps in signal transduction mediated by the LTbR and functionally related receptors.
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