Mutagenesis studies of the β I domain metal ion binding sites on integrin αVβ3 ligand binding affinity

Three divalent cation binding sites in the integrin beta I domain have been shown to regulate ligand binding and adhesion. However, the degree of ligand binding and adhesion varies among integrins. The aL beta 2 and a4 beta 7 integrins show an increase in ligand binding affinity and adhesion when one of their ADMIDAS (adjacent to MIDAS, or the metal ion-dependent adhesion site) residues is mutated. By contrast, the a2 beta 1, a5 beta 1, and aIIb beta 3 integrins show a decrease in binding affinity and adhesion when their ADMIDAS is mutated. Our study here indicated that integrin aV beta 3 had lower affinity when the ADMIDAS was mutated. By comparing the primary sequences of these integrin subunits, we propose that one residue associated with the MIDAS (beta 3 Ala252) may account for these differences. In the beta 1 integrin subunit, the corresponding residue is also Ala, whereas in both beta 2 and beta 7 integrin subunits, it is Asp. We mutated the beta 3 residue Ala252 to Asp and combined this mutant with mutations of one or two ADMIDAS residues. The mutant A252D showed reduced ligand binding affinity and adhesion. The ligand binding affinity and adhesion were increased when this A252D mutant was paired with mutations of one ADMIDAS residue. But when paired with mutations of two ADMIDAS residues the mutant nearly abolished ligand-binding ability, which was restored by the activating glycosylation mutation. Our study suggests that the variation of this residue contributes to the different ligand binding affinities and adhesion abilities among different integrin families. J. Cell. Biochem. 113: 11901197, 2012. (c) 2011 Wiley Periodicals, Inc.