Distinct Roles of Talin and Kindlin in Regulating Integrin α5β1 Function and Trafficking

Background: Integrins are heterodimeric alpha beta 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 beta 1 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 alpha 5 beta 1 function and trafficking, we introduced Y > A mutations in either motif. Disruption of the membrane-proximal NPxY completely prevented alpha 5 beta 1-induced morphological changes, cell scattering and migration, and fibronectin fibrillogenesis. In addition, it reduced alpha 5 beta 1 internalization but not its recycling. In contrast, disruption of the membrane-distal NPxY promoted degradation of alpha 5 beta 1 in late endosomes/lysosomes but did not prevent alpha 5 beta 1-dependent cell scattering, migration, or fibronectin fibrillogenesis. Whereas depletion of either talin-1 or kindlin-2 reduced alpha 5 beta 1 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 alpha 5 beta 1 accumulation in lysosomes and degradation. Conclusions: The two NPxY motifs of beta 1 play distinct and separable roles in controlling the function and trafficking of alpha 5 beta 1. Whereas talin binding to the membrane-proximal NPxY is crucial for connecting alpha 5 beta 1 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 alpha 5 beta 1 surface expression and degradation.