Motogenic sites in human fibronectin are masked by long range interactions.
Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains (4-5)FnI and (3)FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes (3)FnIII and disrupts the observed (4-5)FnI-(3)FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites.
Department of Biochemistry, University of Oxford, Oxford OX1 3QU , UK. ioannis.vakonakis@bioch.ox.ac.uk
Ioannis
I
Vakonakis
David
D
Staunton
Ian R
IR
Ellis
Peter
P
Sarkies
Aleksandra
A
Flanagan
Ana M
AM
Schor
Seth L
SL
Schor
Iain D
ID
Campbell
D017398:Alternative Splicing; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D003094:Collagen / Q000502:physiology; D016207:Cytokines / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D014644:Genetic Variation; D006801:Humans; D007668:Kidney; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000502:physiology; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013623:Tail; D013710:Tendons; D014945:Wound Healing
D016207:Cytokines; C067625:FN1 protein, human; D005353:Fibronectins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003094:Collagen