Dual function for a unique site within the β2I domain of integrin αMβ2

Integrin activation has been postulated to occur in part via conformational changes in the I domain of the beta subunit ( the beta I domain), especially near the F-alpha(7) loop, in response to " inside- out" signaling. However, direct evidence for a role of the F-alpha(7) loop in ligand binding and activity modulation is still lacking. Here, we report our finding that the F- alpha(7) loop ( residues 344 - 358) within the beta(2)I domain has dual functions in ligand binding by alpha(M)beta(2). On the one hand, it supports intercellular adhesion molecule 1 (ICAM- 1) binding to alpha(M)beta(2) directly as part of a recognition interface formed by five noncontiguous segments ( Pro(192) - Glu(197), Asn(213) - Glu(220), Leu(225) - Leu(230), Ser(324) - Thr(329), and Glu(344) - Asp(348)) on the apex of the beta(2)I domain. On the other hand, it controls the open and closed conformation of the alpha(M)beta(2) receptor, thereby indirectly affecting alpha(M)beta(2) binding to other ligands. Switching the five constituent sequences of the ICAM- 1- binding site within the beta(2)I domain to their beta 1 counterparts destroyed ICAM- 1 binding but had no effect on the gross conformations of the receptor. Of the five ICAM- 1 bindingdefective mutants, four had normal or even stronger interaction with Fg and C3bi, as reported in our previous study. Synthetic peptides derived from the identified site inhibited alpha(M)beta(2)- ICAM- 1 interaction and supported direct binding to ICAM- 1. Most importantly, perturbation of the F- alpha7 loop caused conformational changes within the beta(2)I domain, which was further propagated to other regions of alpha(M)beta(2). Altogether, our data demonstrate that inside- out signaling could modulate ligand binding directly by changing the ligand- binding pocket per se and/ or indirectly by inducing multiple conformational changes within the receptor.