title: Sequence gamma 377-395(P2), but not gamma 190-202(P1), is the binding site for the alpha MI-domain of integrin alpha M beta 2 in the gamma C-domain of fibrinogen.
abstract: 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.
authors.affiliation: Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Lerner Research Institute, Cleveland, Ohio 44195, USA. ugarovt@ccf.org
authors.forename: Tatiana P
authors.initials: TP
authors.lastname: Ugarova
authors.forename: Valeryi K
authors.initials: VK
authors.lastname: Lishko
authors.forename: Nataly P
authors.initials: NP
authors.lastname: Podolnikova
authors.forename: Nobuo
authors.initials: N
authors.lastname: Okumura
authors.forename: Sergei M
authors.initials: SM
authors.lastname: Merkulov
authors.forename: Valentin P
authors.initials: VP
authors.lastname: Yakubenko
authors.forename: Vivien C
authors.initials: VC
authors.lastname: Yee
authors.forename: Susan T
authors.initials: ST
authors.lastname: Lord
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Haas
mesh_terms: D000409:Alanine / Q000235:genetics; D001665:Binding Sites; D002448:Cell Adhesion; D002460:Cell Line; D005340:Fibrinogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008024:Ligands; D016177:Macrophage-1 Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D008024:Ligands; D016177:Macrophage-1 Antigen; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen; D000409:Alanine