Abstract
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 membrane-localized. 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.
Highlights
The receptor kinases Tie-1 and Tie-2 are expressed predominantly in endothelial cells and are essential for developmental vascularization where they appear to have roles in promoting microvessel maturation and stability [1]
Phosphotyrosine Signaling through Tie-1 Is Impaired—Despite the importance of Tie-1 in blood vessel formation, the signaling pathways utilized by this receptor and its cellular functions are not known
Endothelial cells were transfected with TrkA/Tie-1 chimeric receptors and 24 h later challenged with nerve growth factor (NGF) or control vehicle (Fig. 1)
Summary
Angiopoietin; BAEC, bovine aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; NGF, nerve growth factor; PMA, phorbol 12-myristate 13-actetate; PAGE, polyacrylamide gel electrophoresis; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; PBS, phosphate-buffered saline.
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