Multiple binding sites in fibrinogen for integrin alphaMbeta2 (Mac-1).
The leukocyte integrin alphaMbeta2 (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 alphaMbeta2 binds fibrinogen remains unknown. Previous studies indicated that a model in which two fibrinogen gammaC domain sequences, P1 (gamma190-202) and P2 (gamma377-395), serve as the alphaMbeta2 binding sites cannot fully account for recognition of fibrinogen by integrin. Here, using surface plasmon resonance, we examined the interaction of the ligand binding alphaMI-domain of alphaMbeta2 with the D fragment of fibrinogen and showed that this ligand is capable of associating with several alphaMI-domain molecules. To localize the alternative alphaMI-domain binding sites, we screened peptide libraries covering the complete sequences of the gammaC and betaC domains, comprising the majority of the D fragment structure, for alphaMI-domain binding. In addition to the P2 and P1 peptides, the alphaMI-domain bound to many other sequences in the gammaC and betaC scans. Similar to P1 and P2, synthetic peptides derived from gammaC and betaC were efficient inhibitors of alphaMbeta2-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 alphaMI-domain binding depends on basic and hydrophobic residues. These findings establish a new model of alphaMbeta2 binding in which the alphaMI-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 alphaMbeta2.
Lishko
Valeryi K
VK
J. J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Lerner Research Institute, Cleveland, Ohio 44195, USA.
Podolnikova
Nataly P
NP
Yakubenko
Valentin P
VP
Yakovlev
Sergiy
S
Medved
Leonid
L
Yadav
Satya P
SP
Ugarova
Tatiana P
TP
D000465:Algorithms; D000595:Amino Acid Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D004252:DNA Mutational Analysis; D005340:Fibrinogen / Q000737:chemistry*; D015723:Gene Library; D006801:Humans; D020128:Inhibitory Concentration 50; D008024:Ligands; D016177:Macrophage-1 Antigen / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors
D008024:Ligands; D016177:Macrophage-1 Antigen; D010455:Peptides; D005340:Fibrinogen