Structural basis for angiopoietin-1-mediated signaling initiation.

Angiogenesis is a complex cellular process involving multiple regulatory growth factors and growth factor receptors. Among them, the ligands for the endothelial-specific tunica intima endothelial receptor tyrosine kinase 2 (Tie2) receptor kinase, angiopoietin-1 (Ang1) and Ang2, play essential roles in balancing vessel stability and regression during both developmental and tumor-induced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cell-signaling characteristics. Here, we present the crystal structures of Ang1 both unbound and in complex with the Tie2 ectodomain. Comparison of the Ang1-containing structures with their Ang2-containing counterparts provide insight into the mechanism of receptor activation and reveal molecular surfaces important for interactions with Tie2 coreceptors and associated signaling proteins. Using structure-based mutagenesis, we identify a loop within the angiopoietin P domain, adjacent to the receptor-binding interface, which confers the specific agonist/antagonist properties of the molecule. We demonstrate using cell-based assays that an Ang2 chimera containing the Ang1 loop sequence behaves functionally similarly to Ang1 as a constitutive Tie2 agonist, able to efficiently dissociate the inhibitory Tie1/Tie2 complex and elicit Tie2 clustering and downstream signaling.

The molecular balance between receptor tyrosine kinases Tie1 and Tie2 is dynamically controlled by VEGF and TNFα and regulates angiopoietin signalling.

Angiopoietin-1 (Ang1) signals via the receptor tyrosine kinase Tie2 which exists in complex with the related protein Tie1 at the endothelial cell surface. Tie1 undergoes regulated ectodomain cleavage in response to phorbol esters, vascular endothelial growth factor (VEGF) and tumour necrosis factor-α (TNFα). Recently phorbol esters and VEGF were found also to stimulate ectodomain cleavage of Tie2. Here we investigate for the first time the effects of factors activating ectodomain cleavage on both Tie1 and Tie2 within the same population of cells, and their impact on angiopoietin signalling. We find that phorbol ester and VEGF activated Tie1 cleavage within minutes followed by restoration to control levels by 24 h. However, several hours of PMA and VEGF treatment were needed to elicit a detectable decrease in cellular Tie2, with complete loss seen at 24 h of PMA treatment. TNFα stimulated Tie1 cleavage, and induced a sustained decrease in cellular Tie1 over 24 h whilst increasing cellular Tie2. These differential effects of agonists on Tie1 and Tie2 result in dynamic modulation of the cellular Tie2∶Tie1 ratio. To assess the impact of this on Ang1 signalling cells were stimulated with VEGF and TNFα for differing times and Ang1-induced Tie2 phosphorylation examined. Elevated Tie2∶Tie1, in response to acute VEGF treatment or chronic TNFα, was associated with increased Ang1-activated Tie2 in cells. These data demonstrate cellular levels of Tie1 and Tie2 are differentially regulated by pathophysiologically relevant agonists resulting in dynamic control of the cellular Tie2∶Tie1 balance and modulation of Ang1 signalling. These findings highlight the importance of regulation of signalling at the level of the receptor. Such control may be an important adaptation to allow modulation of cellular signalling responses in systems in which the activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.

Tie1-Tie2 interactions mediate functional differences between angiopoietin ligands.

The Tie family of endothelial-specific receptor tyrosine kinases is essential for cell proliferation, migration, and survival during angiogenesis. Despite considerable similarity, experiments with Tie1- or Tie2-deficient mice highlight distinct functions for these receptors in vivo. The Tie2 receptor is further unique with respect to its structurally homologous ligands. Angiopoietin-2 and -3 can function as agonists or antagonists; angiopoietin-1 and -4 are constitutive agonists. To address the role of Tie1 in angiopoietin-mediated Tie2 signaling and determine the basis for the behavior of the individual angiopoietins, we used an in vivo FRET-based proximity assay to monitor Tie1 and -2 localization and association. We provide evidence for Tie1-Tie2 complex formation on the cell surface and identify molecular surface areas essential for receptor-receptor recognition. We further demonstrate that the Tie1-Tie2 interactions are dynamic, inhibitory, and differentially modulated by angiopoietin-1 and -2. Based on the available data, we propose a unified model for angiopoietin-induced Tie2 signaling.

Activation of aortic endothelial cells by oxidized phospholipids: a phosphoproteomic analysis.

Previous studies have shown that oxidized products of the phospholipid PAPC (Ox-PAPC) are strong activators of aortic endothelial cells and play an important role in atherosclerosis and other inflammatory diseases. We and others have demonstrated that Ox-PAPC activates specific signaling pathways and regulates a large number of genes. Using a phosphoproteomic approach based on phosphopeptide enrichment and mass spectrometry analysis, we identified candidate changes in Ox-PAPC-induced protein phosphorylation of 228 proteins. Functional annotation of these proteins showed an enrichment of the regulation of cytoskeleton, junctional components, and tyrosine kinases, all of which may contribute to the phenotypic and molecular changes observed in endothelial cells treated with Ox-PAPC. Many changes in protein phosphorylation induced by Ox-PAPC are reported here for the first time and provide new insights into the mechanism of activation by oxidized lipids, including phosphorylation-based signal transduction.

Structure of the extracellular domain of Tie receptor tyrosine kinases and localization of the angiopoietin-binding epitope.

Angiogenesis is essential for tissue repair and regeneration during wound healing but also plays important roles in many pathological processes including tumor growth and metastasis. The receptor protein tyrosine kinase Tie2 and its ligands, the angiopoietins, have important functions in the regulation of angiogenesis. Here, we report a detailed structural and functional characterization of the extracellular region of Tie2. Sequence analysis of the extracellular domain revealed an additional immunoglobulin-like domain resulting in a tandem repeat of immunoglobulin-like domains at the N terminus of the protein. The same domain organization was also found for the Tie1 receptor that shares a high degree of homology with Tie2. Based on structural similarities to other receptor tyrosine kinases and cell adhesion molecules, we demonstrate that the N-terminal two immunoglobulin-like domains of Tie2 harbor the angiopoietin-binding site. Using transmission electron microscopy we furthermore show that the extracellular domain of Tie receptors consists of a globular head domain and a short rod-like stalk that probably forms a spacer between the cell surface and the angiopoietin-binding site. Mutational analysis demonstrated that the head domain consists of the three immunoglobulin-like domains and the three epidermal growth factor-like modules and that the stalk is formed by the three fibronectin type III repeats. These findings might be of particular interest for drug development because Tie receptors are potential targets for treatment of angiogenesis-associated diseases.

The new Tie-1 monoclonal antibodies detect angiogenesis in metastatic malignancies.

Tie-1 is a transmembrane receptor expressed in vascular endothelium during angiogenic events and during embryonal growth in most mammalian species. The monoclonal antibodies (mAbs) used here are generated against the extracellular part of the Tie-1 receptor protein. We analyzed the specific binding of (125)I-labeled Tie-1 mAbs in the metastatic tumor model in mouse and in human serum samples to determine the possible use of the Tie-1 mAbs in detecting malignant growth. The in vivo biodistribution of (125)I-Tie-1 mAbs (IgG1) was evaluated in the mouse model. The same Tie-1 mAbs were used to analyze Tie-1 in patient serum samples. A high accumulation of two (125)I-Tie-1 mAbs, clones 10f11g6 and 3c4c7, was detected in the lung metastases of mouse tumor model. The uptake in the metastases is 12% injected dose/gram (ID/g; 10f11g6) and 7.8% ID/g (3c4c7) at 96 h after the injections of (125)I-Tie-1 mAbs, and the accumulation to the blood is 7% and 5% ID/g with the two mAbs, respectively. The finding directed us to analyze serum samples from lung, ovarian, and breast cancer patients. High levels of Tie-1 were detected in samples related to new pelvic or thoracic metastases in patients. Here we connect the Tie-1 levels in serum to tumor progression. The results suggest that Tie-1 mAbs can be used as a surrogate marker of progressive disease.