Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with β1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the β-propeller of α3β1 empowers vitronectin adhesion by this integrin. How uPAR modifies other β1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds α5β1 and rather than blocking, renders fibronectin (Fn) binding by α5β1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of α5β1–uPAR to Fn type III repeats 12–15 in addition to type III repeats 9–11 bound by α5β1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall α5β1 conformation. A β1 peptide (res 224NLDSPEGGF232) that models near the known α-chain uPAR-binding region, or a β1-chain Ser227Ala point mutation, abrogated effects of uPAR on α5β1. Direct binding and regulation of α5β1 by uPAR implies a modified “bent” integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.

Urokinase-type plasminogen activator (uPA) induces cell adhesion and chemotactic movement. uPA signaling requires its binding to uPA receptor (uPAR/CD87), but how glycosylphosphatidylinositol-anchored uPAR mediates signaling is unclear. uPAR is a ligand for several integrins (e.g. ␣ 5 ␤ 1 ) and supports cell-cell interaction by binding to integrins on apposing cells (in trans). We studied whether binding of uPAR to ␣ 5 ␤ 1 in cis is involved in adhesion and migration of Chinese hamster ovary cells in response to immobilized uPA. This process was temperature-sensitive and required mitogen-activated protein kinase activation. Anti-uPAR antibody or depletion of uPAR blocked, whereas overexpression of uPAR enhanced, cell adhesion to uPA. Adhesion to uPA was also blocked by deletion of the growth factor domain (GFD) of uPA and by anti-GFD antibody, whereas neither the isolated uPA kringle nor serine protease domain supported adhesion directly. Interestingly, anti-␣ 5 antibody, RGD peptide, and function-blocking mutations in ␣ 5 ␤ 1 blocked adhesion to uPA. uPA-induced cell migration also required GFD, uPAR, and ␣ 5 ␤ 1 , but ␣ 5 ␤ 1 alone did not support uPA-induced adhesion and migration. Thus, binding of uPA causes uPAR to act as a ligand for ␣ 5 ␤ 1 to induce cell adhesion, intracellular signaling, and cell migration. We demonstrated that uPA induced RGD-dependent binding of uPAR to ␣ 5 ␤ 1 in solution. These results suggest that uPA-induced adhesion and migration of Chinese hamster ovary cells occurs as a consequence of (a) uPA binding to uPAR through GFD, (b) the subsequent binding of a uPA⅐uPAR complex to ␣ 5 ␤ 1 via uPAR, and (c) signal transduction through ␣ 5 ␤ 1 .