A study of the receptor tyrosine kinase Tie2 and its interaction with novel signalling intermediates

The receptor tyrosine kinase Tie2 is expressed predominantly in endothelial cells and has a role in blood vessel formation, remodelling and maturation. This receptor also has anti-apoptotic and anti-inflammatory effects on endothelial cells. To delineate the signalling pathways utilised by Tie2 two approaches were used. The first approach involved the use of Tie2 chimeric receptors in order to gain control over Tie2 activation. When transiently expressed in endothelial cells Tie2 chimeric receptors were autophosphorylated and insensitive to further increases in tyrosine phosphorylation following ligand treatment. In stable cell lines Tie2 chimeras showed reduced levels of autophosphorylation but once again were insensitive to further increases in tyrosine phosphorylation following ligand treatment. Furthermore stably expressed Tie2 chimeras could not be readily immunoprecipitated and so this approach was deemed unsuitable for determining Tie2 interacting proteins. The second approach was to use the yeast 2-hybrid system to screen a human endothelial cell cDNA library. This screen identified a novel protein, termed FL9.1 that interacted with the intracellular domain of Tie2. FL9.1 was found to represent the human homologue of the recently described murine A20 binding inhibitor of NFkB activation-2 (ABIN-2). FL9.1 was determined not be endothelial cell specific, to bind Tie2 in a phosphotyrosine-dependent manner but not become tyrosine phosphorylated by Tie2. Furthermore FL9.1 was shown to bind specifically to Tie2 but not Tiel. Deletion analysis showed that within FL9.1 amino acid residues 171-272 are required for Tie2 binding. NFkB regulates genes participating in immune and inflammatory responses. Expression of FL9.1 deletion mutants in human endothelial cells suppressed the ability of the Tie2 activating ligand angiopoietin-1 to inhibit phorbol ester-induced NFkB-dependent reporter gene activity. These findings represent a potentially novel Tie2 signalling pathway and may be the pathway through which Tie2 exerts its anti-inflammatory actions in endothelial cells.