uPAR Directly Interacts with LDLR-like Proteins to Induce Angiogenic Endothelial Cell Behavior

In this study we characterized a conserved motif of domain 3 of the urokinase-type plasminogen activator receptor (uPAR) to directly interact with low-density lipoprotein receptor (LDLR)-related protein (LRP) family proteins, thereby affecting endothelial cell motility and angiogenesis in vitro and in vivo.

There is increasing evidence that uPAR plays a central role in growth factor induced endothelial cell activation. Beside its proteolytic role, urokinase-type plasminogen activator (uPA) / uPAR-complex formation induces intracellular signal transduction, which leads to endothelial cell migration and invasion. Since uPAR is a GPI-anchored protein, an interaction with transmembrane proteins - such as members of the LDL-receptor family - is required, inducing signal transduction but also regulating distribution of uPAR via its internalization and recycling to the leading edge. Recently, a direct interaction between uPAR and LRP-family members has been suggested to be sufficient to mediate internalization of uPAR-complex. A crystal structure analysis revealed a small sequence of domain 3 (D3) of uPAR, to be highly exposed upon uPA binding to its receptor.

Applying affinity chromatography analysis as well as mutation expression studies, we identified the sequence as an LRP-binding motif, which affects endothelial cell spreading, migration and invasion upon VEGF in vivo as well as in vitro.

In detail, matrigel-filled angioreactors with embedded retroviral constructs, carrying wild-type or modified uPAR genes, were implanted subcutaneously into uPAR deficient C57BL/6 mice. After explantation, blood vessel in-growth analysis revealed that only angioreactors with reconstituted wild-type uPAR but not reactors with modified uPAR, being deficient in LDLR interaction, showed angiogenesis.

To test a therapeutic impact, peptides mimicking the binding motif and competitive for LDLR binding were used. We found that in a dose dependent manner the peptides did not only block uPAR/LDLR-like protein interaction, but were also capable of blocking VEGF-induced endothelial cell migration in vitro.

In summary, our data show that a conserved motif of uPAR domain 3 is capable to interact with LDLR-like proteins, which is required for efficient growth-factor induced endothelial cell behavior. Preliminary functional data suggest that this extracellular motif might be a potential therapeutic target in angiogenesis dependent diseases such as cancer.