IDENTIFYING THE PUTATIVE METAL ION-DEPENDENT ADHESION SITE IN THE BETA(2) (CD18) SUBUNIT REQUIRED FOR ALPHA(L)BETA(2) AND ALPHA(M)BETA(2) LIGAND INTERACTIONS

We have previously demonstrated that Asp(134) and Ser(136) of the beta (2) subunit are essential for alpha(L) beta(2) and alpha(M) beta(2) li gand recognition. It has been proposed that these residues may be part of a metal ion-dependent adhesion site (MIDAS) within the beta subuni t homologous to the alpha(M) I domain MIDAS structure (Lee, J.-0., Rie u, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638). In the present study, we evaluated the role of additional candidate meta l ion-coordinating residues in the beta(2) subunit in ligand interacti ons. Cells bearing the recombinant alpha(L) beta(2) or alpha(M) beta(2 ) mutant(s) were tested for the ability to bind to immobilized ligands , Alanine substitution at Asp(232) in beta(2) produced a complete loss in the capacity of both alpha(L) beta(2) and alpha(M) beta(2) to supp ort cell adhesion and suppressed the expression of a divalent cation-d ependent conformation recognized by mAb 24. Alanine substitution at Gl u(235) differentially affected receptor function dependent upon the co -transfected alpha subunit. Cells expressing alpha(L) beta(2) with a s ubstitution at Glu(235) failed to adhere to intercellular adhesion mol ecule 1 (ICAM-1) but did retain the capacity to bind mAb 24. Moreover, cells expressing alpha(M) beta(2) with a substitution at Glu(235) fai led 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 anti body-induced activation, These results implicate Asp(232) and Glu(235) , along with Asp(134) and Ser(136), in ligand binding function of alph a(L) beta(2) and alpha(M) beta(2).. These findings provide evidence in support of the existence of a MIDAS structure in beta(2) analogous to that seen in the alpha(M) I domain.