The leukocyte integrin ␣ M ␤ 2 is a highly promiscuous leukocyte receptor capable of binding a multitude of unrelated ligands. To understand the molecular basis for the broad ligand recognition of ␣ M ␤ 2 , the inter-integrin chimera was created. In the chimeric integrin, the ␤D-␣5 loop-␣5 helix segment comprised of residues

Fibrinogen is a ligand for leukocyte integrin alpha(M)beta2 (CD11b/CD18, Mac-1) and mediates adhesion and migration of leukocytes during the immune-inflammatory responses. The binding site for alpha(M)beta2 resides in gammaC, a constituent subdomain in the D-domain of fibrinogen. The sequence gamma383-395 (P2-C) in gammaC was implicated as the major binding site for alpha(M)beta2. It is unknown why alpha(M)beta2 on leukocytes can bind to immobilized fibrinogen in the presence of high concentrations of soluble fibrinogen in plasma. In this study, we have investigated the accessibility of the binding site in fibrinogen for alpha(M)beta2. We found that the alpha(M)beta2-binding site in gammaC is cryptic and identified the mechanism that regulates its unmasking. Proteolytic removal of the small COOH-terminal segment(s) of gammaC, gamma397/405-411, converted the D100 fragment of fibrinogen, which contains intact gammaC and is not able to inhibit adhesion of the alpha(M)beta2-expressing cells, into the fragment D98, which effectively inhibited cell adhesion. D98, but not D100, bound to the recombinant alpha(M)I-domain, and the alpha(M)I-domain recognition peptide, alpha(M)(Glu253-Arg261). Exposure of the P2-C sequence in fibrinogen, D100, and D98 was probed with a site-specific mAb. P2-C is not accessible in soluble fibrinogen and D100 but becomes exposed in D98. P2-C is also unmasked by immobilization of fibrinogen onto a plastic and by deposition of fibrinogen in the extracellular matrix. Thus, exposure of P2-C by immobilization and by proteolysis correlates with unmasking of the alpha(M)beta2-binding site in the D-domain. These results demonstrate that conformational alterations regulate the alpha(M)beta2-binding site in gammaC and suggest that processes relevant to tissue injury and inflammation are likely to be involved in the activation of the alpha(M)beta2-binding site in fibrinogen.

The leukocyte integrin ␣ M ␤ 2 (Mac-1) is a multiligand receptor that mediates a range of adhesive reactions of leukocytes during the inflammatory response. This integrin binds the coagulation protein fibrinogen providing a key link between thrombosis and inflammation. However, the mechanism by which ␣ M ␤ 2 binds fibrinogen remains unknown. Previous studies indicated that a model in which two fibrinogen ␥C domain sequences, P1 (␥190 -202) and P2 (␥377-395), serve as the ␣ M ␤ 2 binding sites cannot fully account for recognition of fibrinogen by integrin. Here, using surface plasmon resonance, we examined the interaction of the ligand binding ␣ M I-domain of ␣ M ␤ 2 with the D fragment of fibrinogen and showed that this ligand is capable of associating with several ␣ M I-domain molecules. To localize the alternative ␣ M I-domain binding sites, we screened peptide libraries covering the complete sequences of the ␥C and ␤C domains, comprising the majority of the D fragment structure, for ␣ M I-domain binding. In addition to the P2 and P1 peptides, the ␣ M I-domain bound to many other sequences in the ␥C and ␤C scans. Similar to P1 and P2, synthetic peptides derived from ␥C and ␤C were efficient inhibitors of ␣ M ␤ 2 -mediated cell adhesion and were able to directly support adhesion suggesting that they contain identical recognition information. Analyses of recognition specificity using substitutional peptide libraries demonstrated that the ␣ M I-domain binding depends on basic and hydrophobic residues. These findings establish a new model of ␣ M ␤ 2 binding in which the ␣ M I-domain interacts with multiple sites in fibrinogen and has the potential to recognize numerous sequences. This paradigm may have implications for mechanisms of promiscuity in ligand binding exhibited by integrin ␣ M ␤ 2 .Integrins are noncovalently associated cell surface ␣␤ heterodimer receptors that mediate adhesive interactions with the extracellular matrix and with other cells. By providing a link between the cell cytoplasm and the surrounding matrix integrins regulate a diverse range of processes, including cellular differentiation, cell migration, the immune response, and the maintenance of tissue architecture. Integrins also play key roles in a variety of pathological conditions. Many integrins exhibit a very broad binding specificity and are able to recognize diverse ligands representing several protein families.Integrin ␣ M ␤ 2 (Mac-1, CD11b/CD18, and CR3) is the most promiscuous member of the entire family with more than 30 proteins being reported as its ligands. ␣ M ␤ 2 is abundantly expressed on activated leukocytes, primarily neutrophils and monocytes, and mediates critical adhesive reactions during the inflammatory responses. Specifically, it contributes to firm adhesion of neutrophils to endothelial cells, promotes their subsequent diapedesis, and participates in neutrophil migration through the interstitial matrix (reviewed in Ref. 1). Many other neutrophil responses, including phagocytosis, homotypic aggregation, degranulatio...

The broad recognition specificity exhibited by integrin αMβ2 (Mac-1, CD11b/CD18) has allowed this adhesion receptor to play innumerable roles in leukocyte biology, yet we know little about how and why αMβ2 binds its multiple ligands. Within αMβ2, the αMI-domain is responsible for integrin’s multiligand binding properties. To identify its recognition motif, we screened peptide libraries spanning sequences of many known protein ligands for αMI-domain binding and also selected the αMI-domain recognition sequences by phage display. Analyses of >1400 binding and nonbinding peptides derived from peptide libraries showed that a key feature of the αMI-domain recognition motif is a small core consisting of basic amino acids flanked by hydrophobic residues. Furthermore, the peptides selected by phage display conformed to a similar pattern. Identification of the recognition motif allowed the construction of an algorithm that reliably predicts the αMI-domain binding sites in the αMβ2 ligands. The recognition specificity of the αMI-domain resembles that of some chaperones, which allows it to bind segments exposed in unfolded proteins. The disclosure of the αMβ2 binding preferences allowed the prediction that cationic host defense peptides, which are strikingly enriched in the αMI-domain recognition motifs, represent a new class of αMβ2 ligands. This prediction has been tested by examining the interaction of αMβ2 with the human cathelicidin peptide LL-37. LL-37 induced a potent αMβ2-dependent cell migratory response and caused activation of αMβ2 on neutrophils. The newly revealed recognition specificity of αMβ2 toward unfolded protein segments and cationic proteins and peptides suggests that αMβ2 may serve as a previously proposed “alarmin” receptor with important roles in innate host defense.