Distinct roles of talin and kindlin in regulating integrin α5β1 function and trafficking.
BACKGROUND
Integrins are heterodimeric αβ 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 β1 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 α5β1 function and trafficking, we introduced Y > A mutations in either motif. Disruption of the membrane-proximal NPxY completely prevented α5β1-induced morphological changes, cell scattering and migration, and fibronectin fibrillogenesis. In addition, it reduced α5β1 internalization but not its recycling. In contrast, disruption of the membrane-distal NPxY promoted degradation of α5β1 in late endosomes/lysosomes but did not prevent α5β1-dependent cell scattering, migration, or fibronectin fibrillogenesis. Whereas depletion of either talin-1 or kindlin-2 reduced α5β1 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 α5β1 accumulation in lysosomes and degradation.


CONCLUSIONS
The two NPxY motifs of β1 play distinct and separable roles in controlling the function and trafficking of α5β1. Whereas talin binding to the membrane-proximal NPxY is crucial for connecting α5β1 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 α5β1 surface expression and degradation.
Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Coert
C
Margadant
Maaike
M
Kreft
Dirk-Jan
DJ
de Groot
Jim C
JC
Norman
Arnoud
A
Sonnenberg
D000595:Amino Acid Sequence; D000818:Animals; D002465:Cell Movement; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D039081:Integrin alpha5beta1 / Q000378:metabolism / Q000502:physiology*; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D021382:Protein Sorting Signals; D021381:Protein Transport; D016415:Sequence Alignment; D016608:Talin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D003598:Cytoskeletal Proteins; D039081:Integrin alpha5beta1; D009124:Muscle Proteins; D021382:Protein Sorting Signals; D016608:Talin; C527802:kindlin-2 protein, mouse; C496077:Tln1 protein, mouse