Identifying the putative metal ion-dependent adhesion site in the beta2 (CD18) subunit required for alphaLbeta2 and alphaMbeta2 ligand interactions.

We have previously demonstrated that Asp134 and Ser136 of the beta2 subunit are essential for alphaLbeta2 and alphaMbeta2 ligand recognition. It has been proposed that these residues may be part of a metal ion-dependent adhesion site (MIDAS) within the beta subunit homologous to the alphaM I domain MIDAS structure (Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638). In the present study, we evaluated the role of additional candidate metal ion-coordinating residues in the beta2 subunit in ligand interactions. Cells bearing the recombinant alphaLbeta2 or alphaMbeta2 mutant(s) were tested for the ability to bind to immobilized ligands. Alanine substitution at Asp232 in beta2 produced a complete loss in the capacity of both alphaLbeta2 and alphaMbeta2 to support cell adhesion and suppressed the expression of a divalent cation-dependent conformation recognized by mAb 24. Alanine substitution at Glu235 differentially affected receptor function dependent upon the co-transfected alpha subunit. Cells expressing alphaLbeta2 with a substitution at Glu235 failed to adhere to intercellular adhesion molecule 1 (ICAM-1) but did retain the capacity to bind mAb 24. Moreover, cells expressing alphaMbeta2 with a substitution at Glu235 failed to adhere to fibrinogen or ICAM-1 and did not bind mAb 24. However, these cells did retain the capacity to adhere to iC3b following antibody-induced activation. These results implicate Asp232 and Glu235, along with Asp134 and Ser136, in ligand binding function of alphaLbeta2 and alphaMbeta2. These findings provide evidence in support of the existence of a MIDAS structure in beta2 analogous to that seen in the alphaM I domain.