Tie2 is a receptor tyrosine kinase expressed predominantly in endothelial cells and is essential for blood vessel formation and maintenance. The receptor has potent antiinflammatory effects on endothelial cells, suppressing vascular endothelial growth factor- and tumor necrosis factor-induced expression of leukocyte adhesion molecules and procoagulant tissue factor and inhibiting vascular leakage. To delineate the signaling pathways utilized by Tie2, we performed yeast two-hybrid screening of a human endothelial cell cDNA library and identified a novel protein interacting with the intracellular domain of the receptor. This protein was found to be human A20 binding inhibitor of NF-kappaB activation-2, ABIN-2, an inhibitor of NF-kappaB-mediated inflammatory gene expression. Coexpression of Tie2 and ABIN-2 in CHO cells confirmed the interaction occurs in mammalian cells. In contrast, Tie1 did not interact with ABIN-2 in the yeast two-hybrid system or mammalian cells. Deletion analysis identified the Tie2 binding motif to be encompassed between residues 171 and 272 in ABIN-2. Interaction was dependent on Tie2 autophosphorylation but ABIN-2 was not tyrosine phosphorylated by Tie2. Furthermore, in endothelial cells the interaction was stimulated by the Tie2 ligand angiopoietin-1. Expression of ABIN-2 deletion mutants in endothelial cells suppressed the ability of angiopoietin-1 to inhibit phorbol ester-stimulated NF-kappaB-dependent reporter gene activity. These findings provide the first direct link between Tie2 and a key regulator of inflammatory responses in endothelial cells. Interaction between Tie2 and ABIN-2 may be important in the vascular protective antiinflammatory actions of Tie2.

The orphan receptor tyrosine kinase Tie-1 is expressed in endothelial cells and is essential for vascular development. Nothing is known about the signaling pathways utilized by this receptor. In this study we have used chimeric receptors composed of the TrkA ectodomain fused to the transmembrane and intracellular domains of Tie-1, or the related receptor Tie-2, to examine Tie-1 signaling capacity. In contrast to TrkA/Tie-2, the Tie-1 chimera was unable to phosphorylate cellular proteins or undergo autophosphorylation. Consistent with this Tie-1 exhibited negligible kinase activity. Co-immunoprecipitation analysis revealed Tie-1 was present in endothelial cells bound to Tie-2. Full-length Tie-1 and truncated receptor, formed by regulated endoproteolytic cleavage, were found to complex with Tie-2. Association was mediated by the intracellular domains of the receptors and did not require Tie-1 to be membranelocalized. Tie-1 bound to Tie-2 was not tyrosine-phosphorylated under basal conditions or following Tie-2 stimulation. This study provides the first evidence for the existence of a pre-formed complex of Tie-1 and Tie-2 in endothelial cells. The data suggest Tie-1 does not signal via ligand-induced kinase activation involving homo-oligomerization. The physical association between Tie-1 and Tie-2 is consistent with Tie-1 having a role in modulating Tie-2 signaling.

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