39A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor

The urokinase receptor, uPAR, is a GPI-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear if the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements, or if lateral interactions between uPAR and other membrane proteins can induce the same response. In this paper, we show that both of these triggering mechanisms can be operative and that uPAR dependent modulation of cell morphology can indeed occur independently of vitronectin binding. Expression of wildtype uPAR on HEK-293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements whereas a mutant uPAR, uPAR_W32A with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR_W32A with the protease ligand, pro-uPA or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored even though this did not rescue the vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPAR_Y57A, in the presence of a synthetic uPAR-binding peptide, as well as with wildtype uPAR which underwent cytoskeletal rearrangements even when cultivated in uPA deficient serum.