Urokinase-Type Plasminogen Activator Receptor (uPAR, CD87) Regulates Integrin Redistribution in Endothelial Cells Via Interaction with Low Density Lipoprotein Receptor- (LDLR-) Like Proteins

Abstract 5315

angiogenesis by degradation of extracellular matrix proteins as well as induction of intracellular signal transduction. We recently could demonstrate that in VEGF-stimulated endothelial cells pro-uPA becomes activated, which leads to uPAR-complex formation, it's internalization and redistribution of uPAR to newly formed focal adhesions ad the leading edge of migrating endothelial cells. Thereby, uPAR surface expression is tightly transcriptional regulated via the Density Enhanced Phosphatase-1 (DEP-1), but also via the LDLR-family members, which regulate subcellular uPAR distribution.

Here, we describe a mechanisms by which uPAR-internalization regulates integrin redistribution. We have characterized a novel binding motif on uPAR domain 3 for LDLR-protein interaction by using affinity chromatography as well as co-immunoprecipitation experiments. To proof a functional relevance of a direct uPAR/LDLR protein interaction, we reconstituted either uPAR mutants (mutL3/uPAR), lacking the binding site for LDLR-proteins, or wild type uPAR into endothelial cells derived from uPAR−/− mice. Reconstitution of mutL3/uPAR was incapable to redistribute uPAR as well as integrins during VEGF-induced endothelial cell migration when compared to wild type uPAR reconstitutes. The functional importance of uPAR / LDLR interaction was further reflected by the use of an inhibitory peptide (P1) interfering with uPAR/LDLR-protein interaction, which functionally reverted full length uPAR reconstitution, or the chaperon Receptor Associated Protein (RAP), a high affinity ligand for LDLR-proteins, which prevents uPAR/LDLR interactions. Thus, interfering with uPAR/LDLR-protein interaction at different levels led to an impaired endothelial cell spreading behavior on integrin-adhesive matrix proteins as well as a reduced pY576 FAK phosphorylation upon endothelial cell adhesion, leading to an reduced migratory response towards VEGF.

These data suggest a central role of uPAR/LDLR-protein interaction in VEGF-induced endothelial cell migration via induction of integrin redistribution. Thus, uPAR/LDLR interaction might represent a novel therapeutic target in angiogenesis-related diseases.