Thrombospondin-1 (TSP1) and its receptors have long been thought to have important roles in regulating vascular cells, both circulating and mural. During the preceding grant period we have discovered that TSP1 and CD47 (integrin-associated protein) regulate the dynamic range of nitric oxide (NO) signaling in vascular cells. Thus TSP1-CD47 interactions are important not only in angiogenic regulation but in rapid regulation of tissue perfusion and many other roles where NO maintains the health of the cardiovascular system. We have identified the binding surface on the C-terminal domain of TSP1 that interacts with CD47. We have also discovered a new extracellular mechanism for integrin activation via CD47. This proposal focuses on the molecular interactions among TSP1, CD47 and 23 integrins. The molecular details of these interactions will be deduced by mutagenesis of all three interacting partners. Mutant proteins will be tested in vitro in binding assays and assays of CD47 and integrin function. The extracellular clasp mechanism for integrin activation that we identified within the 1v23 structure will be tested in cultured cells and in 23-null mice repopulated with bone marrow expressing activated 23 integrin constructs. A similar approach will test the ability of CD47 to activate 23 integrins via interaction with the clasp in cultured cells and in mice expressing mutant CD47 transgenes. Conditions for formation of TSP1-CD47 and CD47-integrin complexes will be evaluated using physical methods and several EM approaches. These methods will then be used to evaluate the interactions of mutant proteins in order to identify structural elements of each that are important for their molecular interactions. Based on these results, crystallization and X-ray diffraction studies will be initiated of CD47 in complexes with the TSP1 C-terminal domain and with 1v23 integrin. The sum of these studies will provide for the first time a physical model of TSP1-CD47-integrin interactions. Given our new paradigm for TSP1-CD47 regulation of NO signaling in the vascular system, this information will be vital in designing new strategies to modulate endogenous NO signaling, a new approach to ameliorating cardiovascular disease. ? ? ? ?
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