Distinct Ligand Binding Sites in the I Domain of Integrin That Differentially Affect a Divalent Cation-dependent Conformation (*)

Abstract

The I domains of the leukocyte β integrins have been shown to be essential for ligand recognition. Amino acid substitutions of Asp and Ser, which reside in a conserved cluster of oxygenated residues, abrogate divalent cation ligand binding function of αβ. Presently, we evaluated the role of two I domain regions in αβ ligand recognition: 1) the conserved cluster of oxygenated residues (Asp, Asp, Ser, and Ser) and 2) a 7-amino acid region (Phe-Tyr), conserved in α and α but absent in α of the β integrins. Recombinant αβ was expressed on COS-7 cells, and function was assessed by iC3b recognition. Alanine substitution at position Asp, Asp/Ser, Ser, or Ser produced a complete loss in the capacity of αβ to recognize iC3b and attenuated the binding of a divalent cation-dependent epitope recognized by monoclonal antibody 24. Moreover, alanine substitution at Asp or Tyr or deletion of Phe-Tyr abolished iC3b ligand recognition as well as the binding of a blocking antibody. In contrast, these mutations did not affect the binding of the cation-dependent epitope. These data implicate a second region within the I domain important for αβ ligand binding function and suggest that this region does not affect a divalent cation-dependent conformation of αβ.