Mechanism of integrin activation by CD47 association. We have discovered a novel mechanism by which β3 integrins are restrained in the “off” state by a “clasp” interface between the αv/αIIb and β3 subunits. When the integrins are activated, this clasp is broken allowing the integrin to open up into a extended, ligand-binding conformation. We have mutagenized potential α-β contact residues in the clasp and generated constitutively activated integrins. The CD47 extracellular domain contains a homolog of the αv and αIIb clasp sequences. Expression, co-crystallization and mutagenesis of the CD47 extracellular (IgV) domain and the αvβ3 integrin extracellular domains will determine if CD47 activates integrins by associating with the “off” state and breaking the clasp.
Defining the TSP1 G domain interaction with CD47. Using synthetic peptides, we have mapped a site on the TSP1 C-terminal G domain that likely represents part of the CD47 binding site. The G domain and the peptide activate CD47 to inhibit NO signaling. Using mutants of the G domain and the CD47 extracellular IgV domain, we will map contact and activation residues of this important ligand-receptor pair. Expression of the soluble binding partners and the co-crystallization/structural approaches will be used as well.
The mechanism of CD47 inhibition of NO signaling. Stimulation of CD47 with the TSP1 G domain or certain peptides from it result in suppression of NO-stimulated cyclic-GMP productioin in vascular cells and may also block signaling downstream of guanylyl cyclase. The point(s) at which CD47 intervenes in NO signaling will be determined. Possible mechanisms include down-regulation of cAMP levels (via Gi), dephosphorylation of eNOS, activation of enzymes generating superoxide, an efficient NO scavenger, and alteratioins in localization and/or scaffolding of eNOS, G-cyclase, PKG and its substrates.
Regulation of platelet function by CD36/CD47. Platelets secrete large amounts of TSP1 upon activation. Until now, the function of TSP1 in platelets was unknown. We have shown that TSP1-CD47 (and likely -CD36) interaction down-regulates platelet cGMP levels, thus promoting platelet activation. We will use human platelets and platelets from mice lacking TSP1, CD36 and CD47 to determine how CD36 and CD47 interact to suppress NO signaling. Expression of mutant receptors in mammalian cell lines will be used to define interacting domains and residues. In vivo models of platelet adhesion, activation, aggregation and clot stability will be applied to knockout mice to understand the physiological role of TSP1, CD36 and CD47.