Tie2 Receptor Dimerization Mediated by its Extracellular FNIII Domains

Abstract

The Tie family of receptor tyrosine kinases (RTKs) regulate a number of angiogenic processes that are critical in vascular development, as well as vascularization of tumor masses. The current signaling paradigm is that angiopoietin (Ang)1 binds to Tie2, promoting Tie2 homodimerization and autophosphorylation. This in turn results in cell proliferation, vessel branching, and sprouting. The extracellular regions of the Tie receptors contain three each of immunoglobulin-like (Ig) domains, EGF-like domains, and fibronectin type III (FNIII) domains. The structure of the Ig/EGF domain region of Tie2 in complex with an Ang protein has been described. However, this structure does not fully explain receptor activation or dimerization. Focusing on the three membrane-proximal FNIII domains - missing from the previously reported structure - we have found that this region can independently drive Tie2 dimerization, indicating that the FNIII domains play an important role in defining the activated dimeric configuration of this receptor. To determine the molecular basis for this observation we have solved a 2.5 A resolution crystal structure of the Tie2 FNIII domains, which reveals a domain architecture with intermolecular interactions between the second and third FNIII domains that are highly reminiscent of those seen in ligand-induced dimers of the hGHR receptor. Guided by this crystal structure we have generated mutations in the region that defines receptor dimerization. These mutations appear to reduce dimer formation in solution, and to reduce Ang1 stimulated phosphorylation of Tie2 in a cellular context.