Urokinase Receptors Are Required for α5β1 Integrin-mediated Signaling in Tumor Cells

Abstract

-regulation of urokinase receptors is common during tumor progression and thought to promote invasion and metastasis. Urokinase receptors bind urokinase and a set of 1 integrins, but it remains unclear to what degree urokinase receptor/ integrin binding is important to 1 integrin signaling. Using site-directed mutagenesis, single amino acid mutants of the urokinase receptor were identified that fail to associate with either 31 (D262A) or 51 (H249A) but associate normally with urokinase. To study the effects of these mutations on 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 51-dependent ERK activation and metalloproteinase MMP-9 expression. Re-expression of wild type or D262A mutant receptors but not the 51 binding-deficient H249A mutant reconstituted fibronectin responses. Because urokinase receptor51 complexes bind in the fibronectin heparin-binding domain (Type III 12-14) whereas 51 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 12-14initiated signals to activate ERK and up-regulate MMP-9. Thus urokinase receptor binding to 51 is required for maximal responses to fibronectin and tumor cell invasion, and this operates through an enhanced Src/Rac/ERK signaling pathway.