N-glycosylation of the β-propeller domain of the integrin α5 subunit is essential for α5β1 heterodimerization, expression on the cell surface, and its biological function

Abstract

The N-glycosylation of integrin α5β1 is thought to play crucial roles in cell spreading, cell migration, ligand binding, and dimer formation, but the underlying mechanism remains unclear. To investigate the importance of the N-glycans of this integrin in detail, sequential site-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on the α5 integrin. Removal of the putative N-glycosylation sites on the β-propeller, Thigh, Calf-1, or Calf-2 domains of the α5 subunit resulted in a decrease in molecular weight compared with the wild type, suggesting that all of these domains contain attached N-glycans. Importantly, the absence of N-glycosylation sites (sites 1-5) on the β-propeller resulted in the persistent association of integrin subunit with calnexin in the endoplasmic reticulum, which subsequently blocked heterodimerization and its expression on the cell surface. Interestingly, the activities for cell spreading and migration for the α5 subunit carrying only three potential N-glycosylation sites (3-5 sites) on the β-propeller were comparable with those of the wild type. In contrast, mutation of these three sites resulted in a significant decrease in cell spreading as well as functional expression, although the total expression level of the Δ3-5 mutant on the cell surface was comparable with that of wild type. Furthermore, wefound that site 5 is a most important site for its expression on the cell surface, whereas the S5 mutant did not show any biological functions. Taken together, this study reveals for the first time that the N-glycosylation on the β-propeller domain of the α5 subunit is essential for heterodimerization and biological functions of α5β1 integrin and might also be useful for studies of the molecular structure. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.