Structure of the Angiopoietin-2 Receptor Binding Domain and Identification of Surfaces Involved in Tie2 Recognition
ABSTRACT The angiopoietins comprise a small class of secreted glycoproteins that play crucial roles in the maturation and maintenance of the mammalian vascular and lymphatic systems. They exert their effects through a member of the tyrosine kinase receptor family, Tie2. Angiopoietin/Tie2 signaling is unique among tyrosine kinase receptor-ligand systems in that distinct angiopoietin ligands, although highly homologous, can function as agonists or antagonists in a context-dependent manner. In an effort to understand this molecular dichotomy, we have crystallized and determined the 2.4 A crystal structure of the Angiopoietin-2 (Ang2) receptor binding region. The structure reveals a fibrinogen fold with a unique C-terminal P domain. Conservation analysis and structure-based mutagenesis identify a groove on the Ang2 molecular surface that mediates receptor recognition.
- SourceAvailable from: Scott C Henderson
[Show abstract] [Hide abstract]
- "Interestingly, our crystal structure of Tie2 bound to Ang2(Barton et al., 2006) and to Ang1 (unpublished data) show that all angiopoietins bind Tie2 in a similar conformation, excluding the possibility of differential Tie2 activation resulting from alternate Tie2/angiopoietin structural arrangements. However, our structural analysis does suggest that the different angiopoietin ligands could present distinct molecular surfaces outside of the receptor-binding interface that could influence the Tie1-Tie2 receptor complexes(Barton et al., 2005). Based upon our findings we hypothesized that the observed direct Tie2/Tie1 interactions are inhibitory and the ability, or inability, of individual angiopoietins to effectively destabilize the Tie1/Tie2 complexes at the cell surface would define their respective agonistic or antagonistic roles. "
ABSTRACT: 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.Molecular cell 03/2010; 37(5):643-55. DOI:10.1016/j.molcel.2010.02.007 · 14.46 Impact Factor
[Show abstract] [Hide abstract]
- "All the angiopoietins are secreted glycoproteins with molecular masses of approximately 70 kDa and similar overall structures, comprising of a secretory leader sequence, coiled coil domain and carboxy-terminal fibrinogen related domain (Davis et al. 1996; Maisonpierre et al. 1997; Valenzuela et al. 1999). It is this C-terminal domain that is responsible for binding of the ligands to their primary receptor Tie2 (Barton et al. 2005; Procopio et al. 1999), a receptor tyrosine kinase expressed almost exclusively in vascular endothelial cells (Dumont et al. 1992; Runting et al. 1993). Interaction of Ang1 or Ang4 with Tie2 stimulates receptor phosphorylation and initiates downstream signalling and functional effects in endothelial cells (Davis et al. 1996; Valenzuela et al. 1999). "
ABSTRACT: Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) were originally identified as endothelial-specific ligands regulating key functions of the vasculature important in stroke. There is increasing evidence that these ligands also exert effects on neurons. Here we review the neuronal effects of VEGF and Ang1 and highlight their potential for therapeutic manipulation in stroke. VEGF stimulates angiogenesis whereas Ang1 suppresses leakage, inflammation and regression of microvessels. Expression of both ligands change dramatically in the brain in experimental stroke, correlating with increased vascular leakage and inflammation. In addition to vascular effects, VEGF can stimulate survival, migration and proliferation of neurons suggesting roles in neural protection and possible therapeutic applications, an idea supported by preclinical studies. Recent reports now demonstrate that Ang1 can also act directly on neurons and enhance neural repair. The realization that VEGF and Ang1 have effects on both neural and vascular compartments impacted by stroke provides new opportunities for therapeutic manipulation to promote neuroprotection and extend the thrombolytic window, as well as stimulating neurogenesis and revascularization.Current neurovascular research 12/2008; 5(4):236-45. DOI:10.2174/156720208786413433 · 2.74 Impact Factor
[Show abstract] [Hide abstract]
- "The extracellular regions of murine ephrin-B2 and EphB2, as well as of human ephrin-A5 and EphA3, were expressed as Fc-fusion proteins in HEK293 cells from a modified pcDNA3.1 vector (Invitrogen) containing a CD4 signal sequence (Barton et al. 2005). A thrombin cleavage site followed by a constant domain of IgG was placed at the C terminus of the Eph or ephrin genes. "
ABSTRACT: Eph receptors and ephrins play important roles in regulating cell migration and positioning during both normal and oncogenic tissue development. Using a surface plasma resonance (SPR) biosensor, we examined the binding kinetics of representative monomeric and dimeric ephrins to their corresponding Eph receptors and correlated the apparent binding affinity with their functional activity in a neuronal growth cone collapse assay. Our results indicate that the Eph receptor binding of dimeric ephrins, formed through fusion with disulfide-linked Fc fragments, is best described using a bivalent analyte model as a two-step process involving an initial monovalent 2:1 binding followed by a second bivalent 2:2 binding. The bivalent binding dramatically decreases the apparent dissociation rate constants with little effect on the initial association rate constants, resulting in a 30- to 6000-fold decrease in apparent equilibrium dissociation constants for the binding of dimeric ephrins to Eph receptors relative to their monomeric counterparts. Interestingly, the change was more prominent in the A-class ephrin/Eph interactions than in the B-class of ephrins to Eph receptors. The increase in apparent binding affinities correlated well with increased activation of Eph receptors and the resulting growth cone collapse. Our kinetic analysis and correlation of binding affinity with function helped us better understand the interactions between ephrins and Eph receptors and should be useful in the design of inhibitors that interfere with the interactions.Protein Science 04/2007; 16(3):355-61. DOI:10.1110/ps.062608807 · 2.85 Impact Factor