Urokinase receptors are required for α5β1 integrin-mediated signaling in tumor cells

Up-regulation of urokinase receptors is common during tumor progression and thought to promote invasion and metastasis. Urokinase receptors bind urokinase and a set of beta 1 integrins, but it remains unclear to what degree urokinase receptor/integrin binding is important to beta 1 integrin signaling. Using site-directed mutagenesis, single amino acid mutants of the urokinase receptor were identified that fail to associate with either alpha 3 beta 1 (D262A) or alpha 5 beta 1 (H249A) but associate normally with urokinase. To study the effects of these mutations on beta 1 integrin function, endogenous urokinase receptors were first stably silenced in tumor cell lines HT1080 and H1299, and then wild type or mutant receptors were expressed. Knockdown of urokinase receptors resulted in markedly reduced fibronectin and alpha 5 beta 1-dependent ERK activation and metalloproteinase MMP-9 expression. Re-expression of wild type or D262A mutant receptors but not the alpha 5 beta 1 binding-deficient H249A mutant reconstituted fibronectin responses. Because urokinase receptor-alpha 5 beta 1 complexes bind in the fibronectin heparin-binding domain (Type III 12-14) whereas alpha 5 beta 1 primarily binds in the RGD-containing domain (Type III 7-10), signaling pathways leading to ERK and MMP-9 responses were dissected. Binding to III 7-10 led to Src/focal adhesion kinase activation, whereas binding to III 7-14 caused Rac 1 activation. Tumor cells engaging fibronectin required both Type III 7-10- and 12-14-initiated signals to activate ERK and up-regulate MMP-9. Thus urokinase receptor binding to alpha 5 beta 1 is required for maximal responses to fibronectin and tumor cell invasion, and this operates through an enhanced Src/Rac/ERK signaling pathway.