Thrombospondin-1 (TSP-1) binding to its membrane receptor CD47 results in an inhibtion of the nitric oxide (NO) receptor soluble guanylate cyclase (sGC) and a decrease in intracellular cGMP levels. This causes physiologic effects such as vasoconstriction and a rise in blood pressure. The mechanism by which TSP-1 binds to CD47 at the membrane to decrease sGC activity is largely unknown. CD47 can physically associate with a number of binding partners, including α(v)β₃ and vascular endothelial growth factor receptor 2 (VEGFR2). Binding of a C-terminal fragment of TSP-1 called E3CaG1 to CD47 leads to a rise in intracellular calcium ([Ca²⁺](i)), which decreases sGC activity via a phosphorylation event. Binding of E3CaG1 is also known to disrupt the interaction between CD47 and VEGFR2, leading to a decrease in endothelial nitric-oxide synthase (eNOS) activity and cGMP levels through an Akt signaling pathway. However, it is not known whether other membrane proteins associated with CD47 are required for E3CaG1 binding and a subsequent [Ca²⁺](i) increase. Plasmon-waveguide resonance (PWR) spectroscopy was employed to elucidate the mechanism of TSP-1 inhibition of sGC activity through membrane complexes involving CD47. Using PWR, I found E3CaG1 can bind specifically to CD47 within native Jurkat membranes with picomolar and nanomolar dissociation constants (K(d)), suggesting multiple CD47 complexes are present. Among these complexes, CD47/VEGFR2 was found to bind E3CaG1 with a picomolar K(d)and CD47/α(v)β₃ was found to bind E3CaG1 with a nanomolar K(d). In addition, the presence of an anti-VEGFR2 antibody inhibited the E3CaG1-induced calcium response, which suggested CD47 in complex with VEGFR2 was responsible for TSP-1 reduction of sGC activity. I show that when both CD47 and VEGFR2 are returned to a HEK 293T cell line that does not contain these receptors, an increase in [Ca²⁺](i) upon E3CaG1 binding is restored. Interestingly, E3CaG1 was also found to bind to VEGFR2 in complex with the integrin α(v)β₃ on CD47-null cell lines and their derivations, causing a decrease in [Ca²⁺](i) levels. Therefore, the third type 2 repeat and C-terminal domains of TSP-1 can cause both increases and decreases in calcium based upon the availability of protein complexes to which it binds.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/293333 |
| Date | January 2013 |
| Creators | Green, Toni |
| Contributors | Montfort, William R., McEvoy, Megan, Tsao, Tsu-Shuen, Cordes, Matthew, Saavedra, Steven S., Montfort, William R. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | English |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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