A Key Regulator of Cell Adhesion: Identification and Characterization of Important N -Glycosylation Sites on Integrin α 5 for Cell Migration

Abstract

The N -glycosylation of integrin α5β1 is thought to control many fundamental aspects of cell behavior, including cell adhesion and migration. However, the mechanism of how N -glycans function remains largely obscure. Here, we used a loss-of-function approach. The wild-type (WT) and N -glycosylation mutant integrin α5, S3-5 (sites 3, 4 and 5), which contains fewer N -glycans, were stably reconstituted in α5-knock-out cancer cells. We found that the migration ability of the S3-5 cells was decreased by comparison with the WT. Interestingly, the levels of phosphorylated focal adhesion kinase and actin stress-fiber formation were greatly enhanced in the S3-5 mutant. In a mechanistic manner, the active, but not the total integrin α5β1 internalization was inhibited in the S3-5 cells, which is a process that is related to an enhanced expression of active integrin α5β1 on the cell surface. Importantly, restoration of N -glycosylation on the β-propeller domain of α5 reinstated the cell migration ability, active α5β1 expression and internalization. Moreover, these N -glycans are critical for α5-syndecan-4 complex formation. These findings indicate that N -glycosylation on the β-propeller domain functions as a molecular switch to control the dynamics of α5β1 on the cell surface that in turn is required for an optimum adhesion for cell migration.