Studies on the endothelial cell specific receptor tyrosine kinase, tie-1.

Tie-1 is an endothelial cell specific tyrosine kinase receptor that is essential for the stabilisation of newly developed vessels and maintenance of endothelial cell integrity in the latter stages of angiogenesis. There is presently no known ligand for the tie-1 receptor, little is known of the factors that regulate its expression and information is lacking on downstream signalling events that occur following its activation. This study demonstrates that tie-1 protein expression in endothelial cells is increased at the level of transcription by VEGF and low oxygen tension, factors which are known to initiate angiogenesis. The cell surface expression of the receptor is also regulated by a metalloprotease enzyme which results in tie-1 ectodomain cleavage at a region close to the transmembrane region. This event generates a tie-1 endodomain fragment that persists in the cell membrane for several hours. This results in downstream signalling events, which lead to further secretion of the metalloprotease enzyme that can cleave tie-1 ectodomain in surrounding endothelial cells. The cleavage event may well be protein kinase C dependent and can be activated by VEGF. The loss of the extracellular domain of tie-1 inhibits tie-1 ligand binding at the cell surface and probably results in destabilising the endothelial cell allowing it to undergo angiogenesis or vessel remodelling. A potential tie-1 ligand, produced by a malignant melanoma cell line, results m activation of the tie-1 receptor by autophosphorylation of tyrosine kinase residues. Attempts at isolation of this protein suggest that it is a glycoprotein with a molecular weight of approximately 60-90kDa and this protein has been visualised on a nitrocellulose membrane and is currently awaiting N-terminal sequencing. Work presented in this thesis demonstrates for the first time factors that regulate expression of tie-1, novel downstream signalling events and the possible isolation of the tie-1 ligand.