Multiple Binding Sites in Fibrinogen for Integrin αMβ2 (Mac-1) (original) (raw)
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Multiple Binding Sites in Fibrinogen for Integrin M 2 (Mac-1)
Journal of Biological Chemistry, 2004
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 .
A Molecular Basis for Integrin αMβ2 Ligand Binding Promiscuity
Journal of Biological Chemistry, 2002
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 Lys 245-Arg 261 from the ␣ M I domain of ␣ M  2 was inserted into the framework of ␣ L  2. The construct was expressed in HEK 293 cells, and the ability of generated cells to adhere to fibrinogen and its derivatives was characterized first. Grafting the ␣ M (Lys 245-Arg 261) sequence converted ␣ L  2 into a fibrinogen-binding protein capable of mediating efficient and specific adhesion similar to that of wild-type ␣ M  2. Verifying a switch in the binding specificity of ␣ L  2 , the chimeric receptor became competent to support cell migration to fibrinogen. Mutations at positions Phe 246 , Asp 254 , and Pro 257 within Lys 245-Arg 261 of ␣ M  2 produced significant decreases in cell adhesion, illustrating the critical role of these residues in ligand binding. The insertion of ␣ M (Lys 245-Arg 261) imparted to the chimeric integrin the ability to recognize many typical ␣ M  2 protein ligands. Furthermore, cells expressing the chimeric receptor, but not ␣ L  2 , were able to stick to uncoated plastic, which represents the hallmark of wild-type ␣ M  2. These results suggest that ␣ M (Lys 245-Arg 261) serves as a consensus binding site for interaction with a variety of distinct molecules and, thus, may define the degenerate recognition properties inherent to ␣ M  2 .
Biochemistry, 2003
The interaction between the leukocyte integrin R M 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 γ377-395 and γ190-202 sequences in the γC domain of fibrinogen, respectively, blocked the fibrinogen-binding function of R M 2 , implicating these sequences as possible binding sites for R M 2. To determine the role of these sequences in integrin binding, recombinant wildtype and mutant γC domains were prepared, and their interactions with the R M I-domain, a ligand recognition domain within R M 2 , were tested. Deletion of γ383-411 (P2-C) and γ377-411 produced γC mutants which were defective in binding to the R M I-domain. In contrast, alanine mutations of several residues in P1 did not affect R M I-domain binding, and simultaneous mutations in P1 and deletion of P2 did not decrease the binding function of γC further. Verifying the significance of P2, inserting P2-C and the entire P2 into the homologous position of the C-domain of fibrinogen imparted the higher R 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 γ 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 γC by the R M I-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.
Binding of a fibrinogen mimetic stabilizes integrin αIIbβ3's open conformation
Protein Science, 2001
The platelet integrin ␣IIb3 is representative of a class of heterodimeric receptors that upon activation bind extracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of ␣IIb3's fibrinogen binding site affected the receptor's solution structure and stability. Eptifibatide, an integrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent model ligand with fibrinogen-like selectivity for ␣IIb3. Eptifibatide binding promptly and reversibly perturbed the conformation of the ␣IIb3 complex. Ligand-specific decreases in its diffusion and sedimentation coefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptorperturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatide promoted ␣IIb3 dimerization 10-fold more effectively than less selective RGD ligands, as determined by sedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation and enhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmission electron microscopy. Ligation with eptifibatide protected ␣IIb3 from SDS-induced subunit dissociation, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the open conformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the first demonstration that binding a monovalent ligand to ␣IIb3's extracellular fibrinogen-recognition site stabilizes the receptor's open conformation and enhances self-association through its distant transmembrane and/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct binding sites, each affects ␣IIb3's conformation, our findings provide new mechanistic insights into ligand-linked integrin activation, clustering and signaling.
Regulated Unmasking of the Cryptic Binding Site for Integrin αMβ2 in the γC-Domain of Fibrinogen
Biochemistry, 2002
Fibrinogen is a ligand for leukocyte integrin R M 2 (CD11b/CD18, Mac-1) and mediates adhesion and migration of leukocytes during the immune-inflammatory responses. The binding site for R M 2 resides in γC, a constituent subdomain in the D-domain of fibrinogen. The sequence γ383-395 (P2-C) in γC was implicated as the major binding site for R M 2. It is unknown why R M 2 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 R M 2. We found that the R M 2-binding site in γC is cryptic and identified the mechanism that regulates its unmasking. Proteolytic removal of the small COOH-terminal segment(s) of γC, γ397/405-411, converted the D 100 fragment of fibrinogen, which contains intact γC and is not able to inhibit adhesion of the R M 2-expressing cells, into the fragment D 98 , which effectively inhibited cell adhesion. D 98 , but not D 100 , bound to the recombinant R M I-domain, and the R M I-domain recognition peptide, R M (Glu 253-Arg 261). Exposure of the P2-C sequence in fibrinogen, D 100 , and D 98 was probed with a site-specific mAb. P2-C is not accessible in soluble fibrinogen and D 100 but becomes exposed in D 98. 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 R M 2-binding site in the D-domain. These results demonstrate that conformational alterations regulate the R M 2-binding site in γC and suggest that processes relevant to tissue injury and inflammation are likely to be involved in the activation of the R M 2-binding site in fibrinogen.
Structural basis for distinctive recognition of fibrinogen C peptide by the platelet integrin IIb 3
The Journal of Cell Biology, 2008
Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin αIIbβ3 on platelets, resulting in platelet aggregation. αvβ3 binds fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen's α subunit. αIIbβ3 also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the γ subunit (γC peptide). These distinct modes of fibrinogen binding enable αIIbβ3 and αvβ3 to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin αIIbβ3–γC peptide interface, and, for comparison, integrin αIIbβ3 bound to a lamprey γC primordial RGD motif. Compared with RGD, the GAKQAGDV motif in γC adopts a different backbone configuration and binds over a more extended region. The integrin metal ion–dependent adhesion site (MIDAS) Mg2+ ion binds the γC Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca2+ ion binds the γC C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this nati...
Journal of Biological Chemistry, 1998
The type III connecting segment of fibronectin contains two cell binding sites, represented by the peptides CS1 and CS5, that are recognized by the integrin receptor oAl1. Using assays measuring the spreading of A375-SM human melanoma cells, we now report that the adhesion promoting activity of a 29 kDa protease fragment of fibronectin containing the COOH-terminal heparinbinding domain (Hepll), but lacking CS1 and CS5, is completely sensitive to anti-a4 and anti-/l antibodies, suggesting that Hepll contains a third A04f1-binding sequence. Examination of the primary structure of HepIl revealed a sequence with homology to CS1. A i9mer peptide spanning this region (designated H1) was found to support cell spreading to the same level as the 29 kDa fragment. Hl-dependent adhesion was completely sensitive to anti-A4 and anti-Il antibodies. When soluble peptides were tested for their ability to block cell spreading on the 29 kDa fragment, a 13mer peptide comprising the central core of HI was found to be completely inhibitory. The active region of Hi was localized to the pentapeptide IDAPS, which is homologous to LDVPS from the active site of CSI. Taken together, these results identify a novel peptide sequence in the HepIl region of fibronectin that supports A4f1-dependent cell adhesion.
Journal of Cell Biology - J CELL BIOL, 2009
Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin α{sub IIb}βâ on platelets, resulting in platelet aggregation. α{sub v}βâ binding fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen's α subunit. α{sub IIb}βâ also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the γ subunit (γC peptide). These distinct modes of fibrinogen binding enable α{sub IIb}βâ and α{sub v}βâ to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin α{sub IIb}βâ-γC peptide interface, and, for comparison, integrin α{sub IIb}βâ bound to a lamprey γC primordial RGD motif. Compared with RGD, the GAKQAGDV motif in γC adopts a different backbone configuration and binds over a more extended region. The integrin metal ion-dependent adhesion site (MIDAS) Mg{sup 2+} ion binds the γC Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca{sup 2+} ion binds the γC C terminus, reveali...