Regulation of outside-in signaling and affinity

The adhesiveness of integrin L 2 is modulated by divalent cations. We mutated three metal ion-binding sites in the 2 I domain. The metal ion-dependent adhesion site (MIDAS) and the ligandinduced metal-binding site are required for ligand binding and sufficient for synergism between Ca2 and Mg2 . Adjacent to MIDAS (ADMIDAS) mutants are constitutively active but remain bent, with poor exposure of a 2 stalk region epitope. Fluorescence resonance energy transfer between fluorescent protein-fused L and 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, 2 I domain metal ionbinding sites regulate L I domain affinity, and the ADMIDAS is required for outside-in signaling.