Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Curr Biol. 2012 Sep 11;22(17):1554-63. doi: 10.1016/j.cub.2012.06.060. Epub 2012 Jul 12.
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.
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.
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.
整合素是异二聚体的 αβ 跨膜受体，在细胞生理学和病理学中发挥关键作用。越来越多的证据表明，β1 亚基胞质域中的两个 NPxY 基序通过与 talin 和 kindlin 的结合协同促进整合素的激活。然而，目前尚不清楚这两个基序如何分别调节整合素的功能和运输。
为了研究两个 NPxY 基序如何分别控制整合素 α5β1 的功能和运输，我们在每个基序中引入了 Y > A 突变。膜近端 NPxY 的破坏完全阻止了 α5β1 诱导的形态变化、细胞分散和迁移以及纤维连接蛋白纤维的形成。此外，它减少了 α5β1 的内化，但不影响其回收。相比之下，破坏膜远端 NPxY 促进了晚期内体/溶酶体中 α5β1 的降解，但不阻止 α5β1 依赖性细胞分散、迁移或纤维连接蛋白纤维的形成。尽管 talin-1 或 kindlin-2 的耗竭均降低了 α5β1 与纤维连接蛋白的结合和细胞黏附，但 talin-1 的耗竭再现了膜近端 NPxY 突变的功能丧失表型，而 kindlin-2 的耗竭则诱导 α5β1 在溶酶体中积累和降解。
β1 的两个 NPxY 基序在控制 α5β1 的功能和运输方面发挥着独特且可分离的作用。虽然 talin 与膜近端 NPxY 的结合对于将 α5β1 连接到肌动蛋白细胞骨架并因此允许纤维连接蛋白纤维形成和细胞迁移所需的张力至关重要，但 kindlin 与膜远端 NPxY 的结合对于这些事件是可有可无的，但调节 α5β1 的表面表达和降解。
