Kinase insert domain receptor (KDR) extracellular immunoglobulin-like domains 4-7 contain structural features that block receptor dimerization and vascular endothelial growth factor-induced signaling

The vascular endothelial growth factor (VEGF) receptor tyrosine kinase subt ype kinase insert domain receptor (KDR) contains seven extracellular Ig-lik e domains, of which the three most amino-terminal contain the necessary str uctural features required for VEGF binding. To clarify the functional role of I(DR Ig-like domains 4-7, we compared VEGF-induced signaling in human em bryonic kidney and porcine aortic endothelial cells expressing native versu s mutant receptor proteins in which Ig-like domains 4-7, 4-6, or 7 had been deleted. Western blotting using an anti-receptor antibody indicated equiva lent expression levels for each of the recombinant proteins. As expected, V EGF treatment robustly augmented native receptor autophosphorylation, In co ntrast, receptor autophosphorylation, as well as downstream signaling event s, were VEGF-independent for cells expressing mutant receptors, I-125-VEGF( 165) bound with equal or better affinity to mutant versus native receptor, although the number of radioligand binding sites was significantly reduced because a significant percentage of mutant, but not native, receptors were localized to the cell interior. As was the case for native KDR,I-125-VEGF(1 65) binding to the mutant receptors was dependent upon cell surface heparan sulfate proteoglycans, and I-125-VEGF(121) bound with an affinity equal to that of I-125-VEGF(165) to the native and mutant receptors. It is conclude d that KDR Ig-like domains 4-7 contain structural features that inhibit rec eptor signaling by a mechanism that is independent of neuropilin-1 and hepa ran sulfate proteoglycans. We speculate that this provides a cellular mecha nism for blocking unwanted signaling events in the absence of VEGF.