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...
Chronic inflammation is essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration/accumulation of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages in damaged tissue. The mechanism of macrophage migration and subsequent accumulation is still not fully understood. Currently, the amoeboid adhesion-independent motility is considered essential for leukocyte migration in the three-dimensional environment. We challenge this hypothesis by studying the contribution of leukocyte adhesive receptors, integrins αMβ2, and αDβ2, to three-dimensional migration of M1-polarized, M2-polarized, and resident macrophages. Both integrins have a moderate expression on M2 macrophages, while αDβ2 is upregulated on M1 and αMβ2 demonstrates high expression on resident macrophages. The level of integrin expression determines its contribution to macrophage migration. Namely, intermediate expression supports macrophage migration, while a high integrin density inhibits it. Using in vitro three-dimensional migration and in vivo tracking of adoptively-transferred fluorescently-labeled macrophages during the resolution of inflammation, we found that strong adhesion of M1-activated macrophages translates to weak 3D migration, while moderate adhesion of M2-activated macrophages generates dynamic motility. Reduced migration of M1 macrophages depends on the high expression of αDβ2, since αD-deficiency decreased M1 macrophage adhesion and improved migration in fibrin matrix and peritoneal tissue. Similarly, the high expression of αMβ2 on resident macrophages prevents their amoeboid migration, which is markedly increased in αM-deficient macrophages. In contrast, αD- and αM-knockouts decrease the migration of M2 macrophages, demonstrating that moderate integrin expression supports cell motility. The results were confirmed in a diet-induced diabetes model. αD deficiency prevents the retention of inflammatory macrophages in adipose tissue and improves metabolic parameters, while αM deficiency does not affect macrophage accumulation. Summarizing, β2 integrin-mediated adhesion may inhibit amoeboid and mesenchymal macrophage migration or support mesenchymal migration in tissue, and, therefore, represents an important target to control inflammation.
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.
The interaction between the leukocyte integrin alpha(M)beta(2) (CD11b/CD18, Mac-1, CR3) and fibrinogen mediates the recruitment of phagocytes during the inflammatory response. Previous studies demonstrated that peptides P2 and P1, duplicating gamma 377-395 and gamma 190-202 sequences in the gamma C domain of fibrinogen, respectively, blocked the fibrinogen-binding function of alpha(M)beta(2), implicating these sequences as possible binding sites for alpha(M)beta(2). To determine the role of these sequences in integrin binding, recombinant wild-type and mutant gamma C domains were prepared, and their interactions with the alpha(M)I-domain, a ligand recognition domain within alpha(M)beta(2), were tested. Deletion of gamma 383-411 (P2-C) and gamma 377-411 produced gamma C mutants which were defective in binding to the alpha(M)I-domain. In contrast, alanine mutations of several residues in P1 did not affect alpha(M)I-domain binding, and simultaneous mutations in P1 and deletion of P2 did not decrease the binding function of gamma C further. Verifying the significance of P2, inserting P2-C and the entire P2 into the homologous position of the beta C-domain of fibrinogen imparted the higher alpha(M)I-domain binding ability to the chimeric proteins. To further define the molecular requirements for the P2-C activity, synthetic peptides derived from P2-C and a peptide array covering P2-C have been analyzed, and a minimal recognition motif was localized to gamma(390)NRLTIG(395). Confirming a critical role of this sequence, the cyclic peptide NRLTIG retained full activity inherent to P2-C, with Arg and Leu being important residues. Thus, these data demonstrate the essential role of the P2, but not P1, sequence for binding of gamma C by the alpha(M)I-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.
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