The eighth fIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth fIII domain

Harri Altroff, Christopher F. Van Der Walle, Judith Asselin, Richard Fairless, Iain D. Campbell, Helen J. Mardon

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Binding of the extracellular matrix molecule fibronectin to the integrin receptor α5β1 elicits downstream signaling pathways that modulate cell function. Fibronectin-α5β1 interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin α5β1 to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin α5β1-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.
LanguageEnglish
Pages38885-38892
Number of pages8
JournalJournal of Biological Chemistry
Volume276
DOIs
Publication statusPublished - 2001

Fingerprint

Integrin alpha5beta1
Fibronectins
Integrins
Stabilization
Cell adhesion
Cell Adhesion
Denaturation
Assays
Protein Denaturation
Conserved Sequence
Biological Assay
Extracellular Matrix
Conformations
Chemical activation
Ligands
Scanning
Mutation
Molecules

Keywords

  • human fibronectin
  • signaling pathways
  • cell adhesion

Cite this

Altroff, Harri ; Van Der Walle, Christopher F. ; Asselin, Judith ; Fairless, Richard ; Campbell, Iain D. ; Mardon, Helen J. / The eighth fIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth fIII domain. In: Journal of Biological Chemistry. 2001 ; Vol. 276. pp. 38885-38892.
@article{a593c2c2131f4584a4176497f67c6bd4,
title = "The eighth fIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth fIII domain",
abstract = "Binding of the extracellular matrix molecule fibronectin to the integrin receptor α5β1 elicits downstream signaling pathways that modulate cell function. Fibronectin-α5β1 interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin α5β1 to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin α5β1-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.",
keywords = "human fibronectin, signaling pathways, cell adhesion",
author = "Harri Altroff and {Van Der Walle}, {Christopher F.} and Judith Asselin and Richard Fairless and Campbell, {Iain D.} and Mardon, {Helen J.}",
year = "2001",
doi = "10.1074/jbc.M105868200",
language = "English",
volume = "276",
pages = "38885--38892",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",

}

The eighth fIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth fIII domain. / Altroff, Harri; Van Der Walle, Christopher F.; Asselin, Judith; Fairless, Richard; Campbell, Iain D.; Mardon, Helen J.

In: Journal of Biological Chemistry, Vol. 276, 2001, p. 38885-38892.

Research output: Contribution to journalArticle

TY - JOUR

T1 - The eighth fIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth fIII domain

AU - Altroff, Harri

AU - Van Der Walle, Christopher F.

AU - Asselin, Judith

AU - Fairless, Richard

AU - Campbell, Iain D.

AU - Mardon, Helen J.

PY - 2001

Y1 - 2001

N2 - Binding of the extracellular matrix molecule fibronectin to the integrin receptor α5β1 elicits downstream signaling pathways that modulate cell function. Fibronectin-α5β1 interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin α5β1 to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin α5β1-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.

AB - Binding of the extracellular matrix molecule fibronectin to the integrin receptor α5β1 elicits downstream signaling pathways that modulate cell function. Fibronectin-α5β1 interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin α5β1 to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin α5β1-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.

KW - human fibronectin

KW - signaling pathways

KW - cell adhesion

U2 - 10.1074/jbc.M105868200

DO - 10.1074/jbc.M105868200

M3 - Article

VL - 276

SP - 38885

EP - 38892

JO - Journal of Biological Chemistry

T2 - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

ER -