Binding Studies of Membrane Receptor CD47 with the C-terminal Domain of Thrombospondin-1

Secreted glycoprotein Thrombospondin-1 (TSP1), among its many functions, binds to membrane receptor CD47 in order to downregulate the nitric oxide pathway via inhibition of soluble guanylate cyclase (sGC). Physiologically, this process is anti-angiogenic and also affects vasodilation, platelet aggregation and tissue ischemia. Aside from the key players TSP1, CD47 and sGC, little is known about the mechanism of this pathway. This project seeks to understand the binding interaction between trimeric TSP1 and transmembrane receptor CD47. It is hypothesized that the C-terminal binding domain of TSP1 (E3CaG1) and the N-terminal extracellular domain of CD47 (CD47-NT) are sufficient to mediate binding. E3CaG1 was expressed and purified from Sf9 insect cells while CD47-NT was expressed and refolded from inclusion bodies in E. coli. Binding was tested via co-immunoprecipitation experiments utilizing both nickel and GST affinity resins. It was found that the GST- and His-tagged constructs of CD47-NT did not show any interaction with E3CaG1. While E3CaG1 has been shown to be fully active, further work is underway to determine whether CD47-NT is also properly folded and active