DISTINCT LIGAND-BINDING SITES IN THE I-DOMAIN OF INTEGRIN ALPHA(M)BETA(2) THAT DIFFERENTIALLY AFFECT A DIVALENT CATION-DEPENDENT CONFORMATION

The I domains of the leukocyte beta(2) integrins have been shown to be essential for Ligand recognition, Amino acid substitutions of Asp(140 ) and Ser(142), which reside in a conserved cluster of oxygenated resi dues, abrogate divalent cation ligand binding function of alpha(M) bet a(2). Presently, we evaluated the role of two I domain regions in alph a(M) beta(2) ligand recognition: 1) the conserved cluster of oxygenate d residues (Asp(134), Asp(140), Ser(142), and Ser(144)) and 2) a 7-ami no acid region (Phe(246)-Tyr(252)), conserved in alpha(M), and alpha(X ) but absent in alpha(L) of the beta(2) integrins. Recombinant alpha(M ) beta(2) was expressed on COS-7 cells, and function was assessed by i C3b recognition. Alanine substitution at position Asp(140), Asp(140)/S er(142), Ser(142), or Ser(144) produced a complete loss in the capacit y of alpha(M) beta(2) to recognize iC3b and attenuated the binding of a divalent cation-dependent epitope recognized by monoclonal antibody 24. Moreover, alanine substitution at Asp(248) or Tyr(252) or deletion of Phe(246)-Tyr(252) abolished iCSb ligand recognition as web as the binding of a blocking antibody. In contrast, these mutations did not a ffect the binding of the cation-dependent epitope. These data implicat e a second region within the I domain important for alpha(M) beta(2) l igand binding function and suggest that this region does not affect a divalent cation-dependent conformation of alpha(M) beta(2).