Regulation of outside-in signaling and affinity by the β2I domain of integrin αLβ2

Abstract

The adhesiveness of integrin alpha(L)beta(2) is modulated by divalent cations. We mutated three metal ion-binding sites in the beta(2) I domain. The metal ion-dependent adhesion site (MIDAS) and the ligand-induced metal-binding site are required for ligand binding and sufficient for synergism between Ca(2+) and Mg(2+). Adjacent to MIDAS (ADMIDAS) mutants are constitutively active but remain bent, with poor exposure of a beta(2) stalk region epitope. Fluorescence resonance energy transfer between fluorescent protein-fused alpha(L) and beta(2) cytoplasmic domains showed that ADMIDAS mutation abrogated ligand binding-induced spatial separation of cytoplasmic domains. Furthermore, ADMIDAS mutation abolished spreading on ligand-bearing substrates. Thus, beta(2) I domain metal ion-binding sites regulate alpha(L) I domain affinity, and the ADMIDAS is required for outside-in signaling.