Investigating the regulation of the endocytosis of Tyrosine Kinase Receptor Tie2

Tie2 receptor is a cell surface tyrosine kinase receptor expressed almost exclusively in endothelial cells, where it is mainly studied for its role in angiogenesis. Indeed, Tie2 has been related to various pathologies with vascular implication such as pulmonary hypertension, diabetes retinopathy and tumour growth. The regulation of Tie2 activation and function is complex, involving multiple factors that are still being investigated. For instance, after activation by the agonistic ligand Angiopoietin-1 (Ang1), Tie2 is internalized in cells by an endocytic mechanism that has yet to be fully characterised. As it has been shown that endocytosis of molecules can play a regulatory role in intracellular signalling in various ways I believe that the endocytosis of Tie2 may also be important in the regulation of its activity and cellular output. Therefore, I decided to characterise the endocytic mechanisms involved in the internalization of Tie2 to determine whether endocytosis can be a regulator of Tie2 signalling.
To facilitate the study of Tie2 I created a HeLa cell line with inducible expression of a Tie2FLAG receptor that emulates both expression levels and characteristics of endogenous Tie2 in Human Umbilical Vein Endothelial Cells. To study the endocytosis of Tie2 receptor I developed an immunofluorescence-based assay to quantify the amount of internalized agonistic ligand Ang1 in a high throughput Screening format. I also performed complementary immunofluorescence and western blot analysis to investigate the characteristics of Tie2 internalization.