RESUMO
BACKGROUND: Integrins are heterodimeric αß transmembrane receptors that play key roles in cellular physiology and pathology. Accumulating data indicate that the two NPxY motifs in the cytoplasmic domain of the ß1 subunit synergistically promote integrin activation through the binding of talin and kindlin. However, it is unclear how the individual motifs regulate integrin function and trafficking. RESULTS: To investigate how the two NPxY motifs individually control integrin α5ß1 function and trafficking, we introduced Y > A mutations in either motif. Disruption of the membrane-proximal NPxY completely prevented α5ß1-induced morphological changes, cell scattering and migration, and fibronectin fibrillogenesis. In addition, it reduced α5ß1 internalization but not its recycling. In contrast, disruption of the membrane-distal NPxY promoted degradation of α5ß1 in late endosomes/lysosomes but did not prevent α5ß1-dependent cell scattering, migration, or fibronectin fibrillogenesis. Whereas depletion of either talin-1 or kindlin-2 reduced α5ß1 binding to fibronectin and cell adhesion, talin-1 depletion recapitulated the loss-of-function phenotype of the membrane-proximal NPxY mutation, whereas kindlin-2 depletion induced α5ß1 accumulation in lysosomes and degradation. CONCLUSIONS: The two NPxY motifs of ß1 play distinct and separable roles in controlling the function and trafficking of α5ß1. Whereas talin binding to the membrane-proximal NPxY is crucial for connecting α5ß1 to the actin cytoskeleton and thus permit the tension required for fibronectin fibrillogenesis and cell migration, kindlin binding to the membrane-distal NPxY is dispensable for these events but regulates α5ß1 surface expression and degradation.