The eighth FIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth FIII domain.
Binding of the extracellular matrix molecule fibronectin to the integrin receptor alpha(5)beta(1) elicits downstream signaling pathways that modulate cell function. Fibronectin-alpha(5)beta(1) 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 alpha(5)beta(1) 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 alpha(5)beta(1)-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.
Altroff
H
H
Nuffield Department of Obstetrics and Gynecology, University of Oxford, Women's Centre, Level 3, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom.
van der Walle
C F
CF
Asselin
J
J
Fairless
R
R
Campbell
I D
ID
Mardon
H J
HJ
D001665:Binding Sites; D002448:Cell Adhesion; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017469:Receptors, Fibronectin / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013816:Thermodynamics
D005353:Fibronectins; D008024:Ligands; D017469:Receptors, Fibronectin; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D019791:Guanidine