Sequence γ377-395(P2), but not γ190-202(P1), is the binding site for the αMI-domain of integrin αMβ2 in the αC-domain of fibrinogen

Tatiana Ugarova, Valeryi K. Lishko, Nataly Podolnikova, Nobuo Okumura, Sergei M. Merkulov, Valentin P. Yakubenko, Vivien C. Yee, Susan T. Lord, Thomas A. Haas

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

The interaction between the leukocyte integrin αMβ2 (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 αMβ2, implicating these sequences as possible binding sites for αMβ2. To determine the role of these sequences in integrin binding, recombinant wild-type and mutant γC domains were prepared, and their interactions with the αMI-domain, a ligand recognition domain within αMβ2, were tested. Deletion of γ383-411 (P2-C) and γ377-411 produced γC mutants which were defective in binding to the αMI-domain. In contrast, alanine mutations of several residues in P1 did not affect αMI-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 βC-domain of fibrinogen imparted the higher αMI-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 γ390NRLTIG395. 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 αMI-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.

Original languageEnglish (US)
Pages (from-to)9365-9373
Number of pages9
JournalBiochemistry
Volume42
Issue number31
DOIs
StatePublished - Aug 12 2003
Externally publishedYes

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Integrins
Fibrinogen
Binding Sites
Cyclic Peptides
Peptides
Mutation
Phagocytes
Alanine
Adhesives
Leukocytes
Ligands
Proteins
P2 peptide

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sequence γ377-395(P2), but not γ190-202(P1), is the binding site for the αMI-domain of integrin αMβ2 in the αC-domain of fibrinogen. / Ugarova, Tatiana; Lishko, Valeryi K.; Podolnikova, Nataly; Okumura, Nobuo; Merkulov, Sergei M.; Yakubenko, Valentin P.; Yee, Vivien C.; Lord, Susan T.; Haas, Thomas A.

In: Biochemistry, Vol. 42, No. 31, 12.08.2003, p. 9365-9373.

Research output: Contribution to journalArticle

Ugarova, Tatiana ; Lishko, Valeryi K. ; Podolnikova, Nataly ; Okumura, Nobuo ; Merkulov, Sergei M. ; Yakubenko, Valentin P. ; Yee, Vivien C. ; Lord, Susan T. ; Haas, Thomas A. / Sequence γ377-395(P2), but not γ190-202(P1), is the binding site for the αMI-domain of integrin αMβ2 in the αC-domain of fibrinogen. In: Biochemistry. 2003 ; Vol. 42, No. 31. pp. 9365-9373.
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abstract = "The interaction between the leukocyte integrin αMβ2 (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 αMβ2, implicating these sequences as possible binding sites for αMβ2. To determine the role of these sequences in integrin binding, recombinant wild-type and mutant γC domains were prepared, and their interactions with the αMI-domain, a ligand recognition domain within αMβ2, were tested. Deletion of γ383-411 (P2-C) and γ377-411 produced γC mutants which were defective in binding to the αMI-domain. In contrast, alanine mutations of several residues in P1 did not affect αMI-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 βC-domain of fibrinogen imparted the higher αMI-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 γ390NRLTIG395. 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 αMI-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.",
author = "Tatiana Ugarova and Lishko, {Valeryi K.} and Nataly Podolnikova and Nobuo Okumura and Merkulov, {Sergei M.} and Yakubenko, {Valentin P.} and Yee, {Vivien C.} and Lord, {Susan T.} and Haas, {Thomas A.}",
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T1 - Sequence γ377-395(P2), but not γ190-202(P1), is the binding site for the αMI-domain of integrin αMβ2 in the αC-domain of fibrinogen

AU - Ugarova, Tatiana

AU - Lishko, Valeryi K.

AU - Podolnikova, Nataly

AU - Okumura, Nobuo

AU - Merkulov, Sergei M.

AU - Yakubenko, Valentin P.

AU - Yee, Vivien C.

AU - Lord, Susan T.

AU - Haas, Thomas A.

PY - 2003/8/12

Y1 - 2003/8/12

N2 - The interaction between the leukocyte integrin αMβ2 (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 αMβ2, implicating these sequences as possible binding sites for αMβ2. To determine the role of these sequences in integrin binding, recombinant wild-type and mutant γC domains were prepared, and their interactions with the αMI-domain, a ligand recognition domain within αMβ2, were tested. Deletion of γ383-411 (P2-C) and γ377-411 produced γC mutants which were defective in binding to the αMI-domain. In contrast, alanine mutations of several residues in P1 did not affect αMI-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 βC-domain of fibrinogen imparted the higher αMI-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 γ390NRLTIG395. 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 αMI-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.

AB - The interaction between the leukocyte integrin αMβ2 (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 αMβ2, implicating these sequences as possible binding sites for αMβ2. To determine the role of these sequences in integrin binding, recombinant wild-type and mutant γC domains were prepared, and their interactions with the αMI-domain, a ligand recognition domain within αMβ2, were tested. Deletion of γ383-411 (P2-C) and γ377-411 produced γC mutants which were defective in binding to the αMI-domain. In contrast, alanine mutations of several residues in P1 did not affect αMI-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 βC-domain of fibrinogen imparted the higher αMI-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 γ390NRLTIG395. 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 αMI-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.

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