PMC:2575619 / 29188-31106 JSONTXT

{"project":"2_test","denotations":[{"id":"18593464-11121117-4200944","span":{"begin":212,"end":214},"obj":"11121117"}],"text":"Tie1-751 binds to membrane Tie1 and Tie2 on human umbilical vein endothelial cells\nSome soluble receptor splice variants have been shown to bind cognate cell surface receptors and to modulate response to ligand [48]. Tie1-751 comprises most of the extracellular domain of Tie1 plus 11 C-terminal intron-derived amino acids. To begin understanding the functionality of Tie1-751, we tested whether Tie1-751 binds to endothelial cells. Proliferating endothelial cells (HUVEC) were incubated with 125I-labeled Tie1-751. Our results showed that 125I-Tie1-751 specifically bound to HUVEC, with an estimated dissociation constant (Kd) of 121 nM (Figure 4a). Binding of 125I-Tie1-751 to HUVEC was competed by increasing amounts of unlabeled Tie1-751 (Figure 4b).\nFigure 4 Tie1-751 interacts with Tie1 and Tie2. (a) Specific binding of 125I-Tie1-751(6His) to human umbilical vein endothelial cells (HUVEC). Nonspecific binding was determined in the presence of 100-fold excess of unlabelled Tie1-751 and was subtracted from the total binding. CPM, counts per minute; Kd, dissociation constant. (b) Binding of 125I-Tie1-751(6His) to HUVEC was competed by increasing amounts of cold Tie1-751. Data are the mean ± standard error of the mean. (c) Binding of Tie1-751(6His) to HUVEC. At the end of binding, cells were treated with or without the cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), immunoprecipitated using a C-terminal-specific anti-Tie1 (top panel) or anti-Tie2 (middle panel) antibody, and were analyzed by western blotting using anti-His antibody. To confirm equal loading, cell lysates were blotted with anti-Tie1 antibody (bottom panel). IP, Immunprecipitation; WB, Western blot. Direct binding of Tie1-751(6His) to Tie1 and Tie2 on HUVEC was also examined. Our results demonstrated interaction of Tie1-751(6His) with the transmembrane Tie1, as well as with the transmembrane Tie2 (Figure 4c)."}