OVERALL PROJECT SUMMARY/ABSTRACT This application is a revision of a renewal of an ongoing Program Project in which five investigators with different but complementary expertise have worked together for 10 years to understand poorly characterized but fundamentally important aspects of TCR signaling. Synergy resulting from interactions and collaborations has yielded impressive progress. The Program benefitted enormously from the complementary expertise, skills and approaches by the 5 investigators from structural biology, proteomics, immunology, biophysics, imaging and computational biology. In addition to the synergy created by the distinct expertise of each of the investigators and the progress we have made over the previous 10 years, we also take advantage of a Proteomics Core that has extended our strategies and analytic skills in proteomics. Our collaborative studies have resulted in considerable progress and productivity. In this revision, a major change that we have made is an increase in the number of projects from 2 to 4 in order to allow for more fully developed aims and approaches to answer the following unanswered questions: 1) What are the unique features of the kinases employed by TCR that have been optimized for regulating critical downstream tyrosine phosphorylation and for unique characteristics involved in ligand discrimination? 2) What are the properties of the circuitry that allow for discrimination of self-pMHC from agonist-pMHC recognition to establish a stable homeostatic state versus a robust cellular response? 3) How is the adaptor LAT condensation regulated and serve to coordinate and amplify the diverse signaling events associated with a robust cellular response? 4) How does the TCR signaling network regulate Ras guanine nucleotide exchange factors (GEFs) with downstream, canonical and non-canonical effector kinase signals in a properly regulated and faithful manner to incoming TCR signals? To answer these questions, a new Project is now incorporated into this proposal to address each of these issues. Our overall goal then is to capitalize on our current progress and leverage our distinct knowledge and skills related to TCR signaling to more completely understand the distinct features of the molecules, the circuitry and the regulation of TCR signaling that enables and ensures appropriate biological responses.