Potential roles of metalloprotease mediated ectodomain cleavage in signaling by the endothelial receptor tyrosine kinase Tie-1

The orphan receptor tyrosine kinase Tie-1 is expressed predominantly in end othelial cells. Expression of this receptor is increased in physiologic ang iogenesis and pathologic situations including tumor growth and arteriovenou s malformations. Tie-1 is essential for Vascular development where it acts in later stages of angiogenesis to suppress endothelial activation and stab ilize the newly formed vessel. Stimulation of protein kinase C in endotheli al cells results in endoproteolytic cleavage of Tie-1, releasing the extrac ellular ligand-binding domain of the receptor. We show that this is mediate d by a metalloprotease. Immunoprecipitation and immunoblotting of lysates p repared from human placentas confirm that Tie-1 truncation occurs in vivo. We propose cleavage of this receptor may be a mechanism for inducing vessel destabilization by preventing ligand-activated signaling through Tie-1. Us ing an antibody that recognizes the carboxy terminus of the intracellular d omain, we show that the Tie-1 endodomain formed on cleavage persists as a c ell-associated fragment for several hours. Subcellular fractionation reveal s this tyrosine kinase containing receptor fragment to be localized in the membrane fraction of the cell. Immunoprecipitation with antibodies recogniz ing phosphotyrosine demonstrates that cleavage of Tie-1 stimulates associat ion of newly generated endodomain with cellular phosphoproteins. Furthermor e, there was a marked induction of tyrosine phosphorylation of several prot eins after PMA-induced endodomain generation. These data indicate that ecto domain cleavage may be a mechanism for down-regulating ligand-induced signa ling through Tie-1 while activating an alternative ligand-independent signa ling pathway in endothelial cells. Ectodomain cleavage occurs in some other receptor tyrosine kinases. We suggest that rather than solely being a mean s of down-regulating receptor activity, ectodomain cleavage may be a novel way for a receptor to switch between two alternative signaling pathways.