Structural basis for angiopoietin-1–mediated signaling initiation

Abstract

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 tumorinduced angiogenesis. Despite possessing a high degree of sequence identity, Ang1 and Ang2 have distinct functional roles and cellsignaling 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. cellular signaling | Tie receptor tyrosine kinase | X-ray crystallography P roper development of the cardiovascular system involves two highly integrated and dynamic processes: vasculogenesis and angiogenesis (1). Vasculogenesis involves the in situ differentiation of endothelial cells from precursor angioblasts and results in their subsequent migration and proliferation, leading to the formation of the endocardium of the heart, as well as the major primitive blood vessels (2-6). Angiogenesis occurs following vasculogenesis and results in the remodeling and extension of the endothelial tubes into the adult microvasculature, as well as in the growth and expansion of vessels into organs including the kidney and brain. Endothelial cells lining these newly formed vessels are surrounded by support cells, such as smooth muscle cells and pericytes, which serve to regulate blood flow and provide survival signals (7). Angiogenesis predominantly takes place during embryonic development, although it also recurs during wound repair in adulthood. Its role is essential for tumor development, as well as metastasis and, therefore, represents a viable target for therapeutic intervention (8).The angiopoietins are a small set of growth factor ligands for the tunica intima endothelial receptor tyrosine kinase 2 (Tie2) endothelial-specific receptor tyrosine kinase and are critical for both development and pathological angiogenesis (9-11). The prototypic family member, angiopoietin-1 (Ang1), is a Tie2 agonist, whereas the highly homologous Ang2 is a context-dependent agonist/antagonist. All angiopoietin family members, Ang1 to -4, contain an amino-terminal "superclustering" and...

Angiopoietin receptor TEK mutations underlie primary congenital glaucoma with variable expressivity

“…Furthermore, our cell-based assays have identified new functional roles for various domains. The Ig2 domain, which functions as a ligand-binding domain (37,38), is also critical for stable protein expression, as loss of this Ig domain in p.T19_R210del and IgII del leads to reduced protein levels. The mutation in the EGF1 domain highlights the critical role of cysteine residues, which are needed to disulfide cross-link the protein for stability (38,55).…”

“…To As portions of the ectodomain are responsible for ligand binding (37,38), subsequent multimerization (38), and subcellular localization (39-41), we predicted that the mutations in these regions could weaken or abolish ligand-mediated activation and signal transduction through the intracellular catalytic domain. Anticipating that TEK ectodomain mutations may affect ligandmediated signaling, we expressed TEK variants in HUVECs and examined their subcellular localization after ligand stimulation.…”

The progress of angiogenic factors in the development of leukemias

“…Although Ang-1 doesn't take part in initiation of angiogenesis, it can maintain stability of the vessels and promote maturation of the newly formed vessels. Ang1 and Ang-2 are widely different in their biological roles although they are homologous (91). As the antagonist of Ang-1, on the one hand, during vasculogenesis and inflammation Ang-2 can be strongly released.…”

Harnessing developmental processes for vascular engineering and regeneration

“…TGFβ inhibits EC proliferation and stimulates mural cell differentiation into SMCs or perivascular cells, as well as affecting matrix deposition (Heimark et al, 1986;D'Amore and Smith, 1993). ANG1, which activates ligands for TIE2 (endothelial-specific receptor tyrosine kinase; TEK), is expressed by mural cells and stimulates mural coverage and basement membrane deposition, thereby promoting vessel tightness, whereas angiopoietin 2 (ANG2 or ANGPT2), which is an antagonist to ANG1 and TIE2 signaling, promotes mural cell detachment and EC sprouting, resulting in angiogenesis (Fagiani et al, 2011;Yu et al, 2013). Accumulating evidence demonstrates that stromal cell-derived factor 1 (SDF1) and insulin-like GF (IGF) stimulate angiogenesis through recruitment of EPCs from bone marrow and enhanced EPC mobilization (Maeng et al, 2009;Jujo et al, 2010).…”