Overlapping, but not identical, sites are involved in the recognition of C3bi, neutrophil inhibitory factor, and adhesive ligands by the alpha(M)beta(2) integrin

The alpha(M) beta(2) (CD11b/CD18, Mac-1) integrin receptor binds numerous ligands, including neutrophil inhibitory factor (NIF), C3bi, and certain immobilized protein substrates, represented by denatured ovalbumin. These ligands share no obvious structural similarities, yet their interactions with receptor are inhibited by NIF and involve the I domain, a stretch of similar to 200 amino acids in the alpha(M) subunit. Recombinant wild-type and mutant forms of alpha(M) beta(2) have been used to compare the recognition requirements of these ligands. The various constructs were expressed efficiently on the surface of human embryonic kidney 293 cells and formed alpha .beta heterodimeric complexes. The wild-type transfectants bound the three ligands in a similar fashion to naturally occurring alpha(M) beta(2). NIF inhibited these interactions, and deletion of the D(248)PLGY from within the I domain abolished binding of all three ligands, suggesting an overlapping recognition specificity. A single point mutation of Ser(138) to Ala in the beta(2) subunit abolished C3bi binding and cell adhesion but did not affect NIF binding. A switch of the R(281)QELNTI sequence in helix 6 of the alpha(M) I domain to the corresponding sequence in the I domain of the alpha(L) (QETLHKF) subunit completely abrogated adhesion while not affecting C3bi and NIF binding. The two mutant receptors also did not support activation-dependent adhesion to fibrinogen. Thus, the contact sites for NIF, C3bi, and adhesive proteins, represented by denatured ovalbumin and fibrinogen, in alpha(M) beta(2) are overlapping but not identical.