Abstract
Vascular endothelial growth factors (VEGFs) activate cellular receptor tyrosine kinases (RTKs) such as VEGFR-1, -2, and -3. These receptors are activated upon ligand binding to the extracellular receptor domain (ECD), resulting in receptor dimerization and activation of the intracellular kinase domain. Here we investigated the molecular mechanism of activation of the human VEGFR-2 expressed in human HEK293, monkey COS-1, and porcine aortic endothelial cells. To study the role of dimerization in receptor activation we created a series of dimerization-promoting transmembrane domain (TMD) mutants lacking the extracellular domain. The TMDs consisted of 23 valine and 2 glutamic acid residues spaced 7 aa apart in different positions of the transmembrane helix. All TMDs dimerized VEGFR-2, each in a specific orientation, giving rise to a series of either active or inactive receptor dimers. One particular TMD, V6/13E, gave rise to highly active kinase dimers, while all the other dimerizing TMD mutant receptors had 6- to 10-fold lower activity. When the V6/13E TMD was introduced into the full-length receptor in place of the native TMD, it promoted ligand-independent activation. Nonactivating TMDs, on the other hand, gave rise to inactive receptor mutants, both in the absence and in the presence of VEGF. These data demonstrate that dimerization is necessary, but not sufficient, for receptor activation, and that ligand-mediated receptor activation requires specific orientation of receptor monomers. Our study also shows that dimerization is mimicked by distinct dimerization-promoting TMDs that position the intracellular kinase domain either in an active or inactive conformation.