Structural basis of Tie2 activation and Tie2/Tie1 heterodimerization

Abstract

The endothelial cell (EC)-specific receptor tyrosine kinases Tie1 and Tie2 are necessary for the remodeling and maturation of blood and lymphatic vessels. Angiopoietin-1 (Ang1) growth factor is a Tie2 agonist, whereas Ang2 functions as a contextdependent agonist/antagonist. The orphan receptor Tie1 modulates Tie2 activation, which is induced by association of angiopoietins with Tie2 in cis and across EC-EC junctions in trans. Except for the binding of the C-terminal angiopoietin domains to the Tie2 ligand-binding domain, the mechanisms for Tie2 activation are poorly understood. We report here the structural basis of Ang1-induced Tie2 dimerization in cis and provide mechanistic insights on Ang2 antagonism, Tie1/Tie2 heterodimerization, and Tie2 clustering. We find that Ang1-induced Tie2 dimerization and activation occurs via the formation of an intermolecular β-sheet between the membrane-proximal (third) Fibronectin type III domains (Fn3) of Tie2. The structures of Tie2 and Tie1 Fn3 domains are similar and compatible with Tie2/ Tie1 heterodimerization by the same mechanism. Mutagenesis of the key interaction residues of Tie2 and Tie1 Fn3 domains decreased Ang1-induced Tie2 phosphorylation and increased the basal phosphorylation of Tie1, respectively. Furthermore, the Tie2 structures revealed additional interactions between the Fn 2 (Fn2) domains that coincide with a mutation of Tie2 in primary congenital glaucoma that leads to defective Tie2 clustering and junctional localization. Mutagenesis of the Fn2-Fn2 interface increased the basal phosphorylation of Tie2, suggesting that the Fn2 interactions are essential in preformed Tie2 oligomerization. The interactions of the membrane-proximal domains could provide new targets for modulation of Tie receptor activity.eceptor tyrosine kinases (RTKs) expressed in the endothelial cells (ECs) of blood and lymphatic vessels control the development and function of the cardiovascular and lymphatic systems. The VEGFs and their endothelial receptors (VEGFRs) are key regulators of angiogenesis and vascular integrity (1, 2). The angiopoietin ligand/Tie receptor pathway is necessary for blood and lymphatic vessel remodeling during embryonic and postnatal development and for homeostasis of the mature vasculature (3, 4). Recently significant interest has focused on targeting the VEGFR and Tie receptor pathways in antiangiogenic and antilymphangiogenic therapies (5).Ang1 activation of Tie2 is indispensable for embryonic cardiac development and angiogenesis, and both Ang1 and Ang2 are necessary for the development of lymphatic and ocular vasculature. In adult tissues, Ang1 is required for vessel stabilization after angiogenesis (6-8). Ang2, which is produced by ECs and stored in their Weibel-Palade bodies for rapid release, can function as a weak Tie2 agonist or as a context-dependent antagonist that inhibits Ang1-induced Tie2 activation and vascular stability (9-11). Tie2 is the major signal-transducing receptor of the angiopoietin/Tie signaling axis, and the homologous Tie1 receptor m...

Angiopoietin-1 is required for Schlemm’s canal development in mice and humans

“…Interestingly, while both novel ANGPT1 nonsense mutations we identified have a clear impact on ANGPT1 function and its ability to interact with the TEK receptor, each has a different mechanism. p.Q236* completely lacks the C-terminal receptor binding domain, which is required for interaction with TEK (19,20). In addition, the truncated ANGPT1 Q236* protein can readily form oligomeric complexes through its intact N-terminal domain with WT ANGPT1, a process that is critical to the signaling function of ANGPT1 (18,19).…”

“…p.Q236* completely lacks the C-terminal receptor binding domain, which is required for interaction with TEK (19,20). In addition, the truncated ANGPT1 Q236* protein can readily form oligomeric complexes through its intact N-terminal domain with WT ANGPT1, a process that is critical to the signaling function of ANGPT1 (18,19). We hypothesize that in heterozygous PCG patients, any truncated protein that escapes NMD might have a dominant-negative function on ANGPT1 signaling.…”

“…However, K249 is evolutionarily well conserved ( Figure 5C), and this mutation has been observed only once (in a South Asian individual) out of more than 121,000 alleles in the ExAC database. Owing to the location of this modified amino acid, we predicted that ANGPT1 K249R might be unable to properly oligomerize, which is important for its ability to activate TEK (18)(19)(20).…”

Endothelial Tie1–mediated angiogenesis and vascular abnormalization promote tumor progression and metastasis

“…Angiopoietin-1 (Ang1) is paracrine acting and is the primary agonistic ligand of Tie2 (8). Its multimeric structure can bind the Tie2 extracellular domain homodimer for strong Tie2 activation (9).…”

“…Indeed, because of its dimeric conformation, Ang2 fails to bridge Tie2 extracellular domain homodimers, limiting receptor dimerization and activation (9). As an antagonist of constitutive Ang1/Tie2 signaling, Ang2 acts contextually, enabling angiogenesis in the presence of angiogenic growth factors or driving vessels into regression in the absence of angiogenic stimuli (12).…”