Structural basis of Tie2 activation and Tie2/Tie1 heterodimerization Veli-Matti Leppänena,1, Pipsa Saharinena,b, and Kari Alitaloa,b,1 aWihuri Research Institute, Biomedicum Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland; and bTranslational Cancer Biology Program, Research Programs Unit, University of Helsinki, 00014 Helsinki, Finland Contributed by Kari Alitalo, March 8, 2017 (sent for review September 28, 2016; reviewed by Joseph Schlessinger and Michel O. Steinmetz) The endothelial cell (EC)-specific receptor tyrosine kinases mice lacking Tie1 develop severe edema around E13.5 because Tie1 and Tie2 are necessary for the remodeling and maturation of compromised microvessel integrity and defects in lymphatic of blood and lymphatic vessels. Angiopoietin-1 (Ang1) growth vasculature and die subsequently (15, 16). Furthermore, Tie1 has factor is a Tie2 agonist, whereas Ang2 functions as a context- critical functions in vascular pathologies, e.g., in tumor angio- dependent agonist/antagonist. The orphan receptor Tie1 modu- genesis and atherosclerosis progression (12, 17). lates Tie2 activation, which is induced by association of angio- In EC monolayers, angiopoietins stimulate Tie receptor trans- cis – trans location to cell–cell junctions for Tie2 trans-association, whereas poietins with Tie2 in and across EC EC junctions in . – Except for the binding of the C-terminal angiopoietin domains in the absence of cell cell adhesion the Tie receptors are an- to the Tie2 ligand-binding domain, the mechanisms for Tie2 chored to the extracellular matrix (ECM) by Ang1-induced Tie2 cis-association (10, 18). Integrins also have been implicated in activation are poorly understood. We report here the structural α β – basis of Ang1-induced Tie2 dimerization in cis and provide Tie2 signaling, and the 5 1 integrin heterodimer enhances Ang1-induced EC adhesion and Tie2 activation (13, 19, 20). The mechanistic insights on Ang2 antagonism, Tie1/Tie2 heterodi- Tie1 and Tie2 heteromeric complexes are promoted by angio- merization, and Tie2 clustering. We find that Ang1-induced poietin stimulation, resulting in Ang1-induced activation of both Tie2 dimerization and activation occurs via the formation of an – β Tie1 and Tie2 (13, 21 23). Several studies have indicated Tie1 as intermolecular -sheet between the membrane-proximal (third) a Tie2 inhibitor (22, 24, 25), whereas recent experiments show Fibronectin type III domains (Fn3) of Tie2. The structures of that Tie1 association with Tie2 is required for Tie2 activation by Tie2 and Tie1 Fn3 domains are similar and compatible with Tie2/ Ang1 and Ang2 in mice (13, 26). Tie1 expression inhibits Tie1 heterodimerization by the same mechanism. Mutagenesis Tie2 presentation at the cell surface in sprouting endothelial tip of the key interaction residues of Tie2 and Tie1 Fn3 domains cells, (23), but Tie1 sustains Tie2 signaling in contacting cells decreased Ang1-induced Tie2 phosphorylation and increased (13, 23). Thus, Tie1 exerts its context-dependent effects by the basal phosphorylation of Tie1, respectively. Furthermore, modulating Tie2 activity (13, 22, 23, 26). the Tie2 structures revealed additional interactions between Ligand-induced dimerization is regarded as a common, but the Fn 2 (Fn2) domains that coincide with a mutation of not the only, mechanism for activation of RTK signaling (27). Tie2 in primary congenital glaucoma that leads to defective Angiopoietin monomers form heterogeneous multimeric Tie2 Tie2 clustering and junctional localization. Mutagenesis of the ligands, and it has been suggested that Tie2 activation requires Fn2–Fn2 interface increased the basal phosphorylation of Tie2, receptor clustering (28–30). Also, Tie2 clustering without ligand suggesting that the Fn2 interactions are essential in preformed Tie2 oligomerization. The interactions of the membrane-proximal Significance domains could provide new targets for modulation of Tie recep- tor activity. Tie1 and Tie2 receptor tyrosine kinases are key regulators of blood and lymphatic vessel development and of pathological tyrosine kinase | angiopoietin | dimerization | homotypic interactions | processes including tumor angiogenesis, atherosclerosis, and crystallography vascular leakage, e.g., in sepsis. Tie1 is essential for the Tie2 agonist activity of angiopoietins, and the activated re- eceptor tyrosine kinases (RTKs) expressed in the endothelial ceptors form heteromeric complexes in endothelial cell–cell Rcells (ECs) of blood and lymphatic vessels control the de- junctions. However, little is known about the activation velopment and function of the cardiovascular and lymphatic mechanisms of the Tie receptors. Here we demonstrate that systems. The VEGFs and their endothelial receptors (VEGFRs) the membrane-proximal domains of Tie2 mediate homotypic are key regulators of angiogenesis and vascular integrity (1, 2). interactions, which occur via intermolecular β-sheet formation The angiopoietin ligand/Tie receptor pathway is necessary for and are necessary for Tie2 activation. The structural analysis blood and lymphatic vessel remodeling during embryonic and suggests that Tie2/Tie1 heterodimerization occurs by the same postnatal development and for homeostasis of the mature vas- mechanism. The crystal structures provide a model for culature (3, 4). Recently significant interest has focused on tar- angiopoietin-stimulated Tie2 ectodomain dimerization, clus- geting the VEGFR and Tie receptor pathways in antiangiogenic tering, and activation and insights into therapeutic targeting. and antilymphangiogenic therapies (5). Ang1 activation of Tie2 is indispensable for embryonic cardiac Author contributions: V.-M.L. and K.A. designed research; V.-M.L. performed research; development and angiogenesis, and both Ang1 and Ang2 are V.-M.L. analyzed data; and V.-M.L., P.S., and K.A. wrote the paper. necessary for the development of lymphatic and ocular vascula- Reviewers: J.S., Yale University School of Medicine; and M.O.S., Paul Scherrer Institut. ture. In adult tissues, Ang1 is required for vessel stabilization The authors declare no conflict of interest. – after angiogenesis (6 8). Ang2, which is produced by ECs and Freely available online through the PNAS open access option. – stored in their Weibel Palade bodies for rapid release, can Data deposition: The atomic coordinates, and structure factors reported in this paper function as a weak Tie2 agonist or as a context-dependent an- have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 5MYA, tagonist that inhibits Ang1-induced Tie2 activation and vascular 5MYB, and 5N06). – 1 stability (9 11). Tie2 is the major signal-transducing receptor of To whom correspondence may be addressed. Email: [email protected] or veli-matti. the angiopoietin/Tie signaling axis, and the homologous Tie1 [email protected]. receptor modulates Tie2 signaling (12, 13). Although Tie1, first This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. identified in human leukemia cells (14), is an orphan receptor, 1073/pnas.1616166114/-/DCSupplemental. 4376–4381 | PNAS | April 25, 2017 | vol. 114 | no. 17 www.pnas.org/cgi/doi/10.1073/pnas.1616166114 Downloaded by guest on September 28, 2021 binding has been reported (31). The Tie receptors have a unique The dimerization is based on symmetrical interfaces between the extracellular domain (ECD) for ligand binding, a single-pass membrane-proximal Fn3 domains, placing the C termini close to transmembrane domain, a two-partite cytoplasmic protein tyro- each other at a distance of about 25 Å. The C termini point in sine kinase domain, and a C-terminal tail. The ECDs consist of the same direction, whereas the N-terminal Fn-like domains are Ig, epidermal growth factor-like, and three fibronectin type III directed away from each other. Dimerization is mediated mainly (Fn) domains (Fn1, Fn2, Fn3) (30, 32). The angiopoietins have by hydrogen bonds between the main-chain atoms of antiparallel an N-terminal region responsible for their multimerization, a β-strands in residues Asp682 to Lys690. Other interactions occur coiled-coil domain for dimerization, and a C-terminal fibrinogen- between symmetry-related Lys700 and Gly701, and the inter- like domain (FLD) that contains the Tie2-binding region (28, 29). acting residues are largely conserved (Fig. 1C). According to the Crystal structures of the angiopoietin/Tie2 ligand-binding domain Pisa server analysis, the interface buries a surface area of about 2 (LBD) complexes demonstrate that Ang1 and Ang2 FLDs bind 580 Å per chain in space group C2; the solvation free energy Tie2 in a similar manner (30, 33). Ang1 is a strong Tie2 agonist, gains of −1.5 and −1.9 kcal/mol for each chain and the related P and Ang2 a weak Tie2 agonist, suggesting that the Tie2 agonism value of 0.54 suggest considerable specificity (Tables S2 and S3). of the native angiopoietins resides in sequences outside the FLDs. In space group P21, the interactions between the antiparallel β-strands are almost identical, but, because of additional inter- Because multimerization of Ang1 and Ang2 is critical for Tie2 2 phosphorylation, it has been suggested that the difference be- actions, the buried surface area is larger, about 700 Å per chain tween the agonistic activities of Ang1 and Ang2 is caused by (Tables S2 and S3). different oligomeric states (34, 35). Indeed, multiple studies in- The Tie2 homodimers in the two crystal structures share the dicate that the strongly activating Ang1 has higher tendency to