Function and Receptor Specificity of a Minimal 20 Kilodalton Cell Adhesive Fragment of Fibronectin

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Abstract
Previous studies have reached conflicting conclusions about the minimal size and sequences of the fibronectin cell-adhesive domain necessary for retention of high cell adhesive activity. We have expressed a recombinant 20 kDa cell-binding fragment of human fibronectin consisting of the ninth and tenth type III modules, which includes the Arg-Gly-Asp (RGD) cell recognition site and a second cell adhesive domain that acts synergistically with the RGD site. This polypeptide retained a similar activity as a larger 110 kDa fibronectin fragment when used in soluble form in inhibition assays, but it displayed low cell adhesive activity if assayed after direct adsorption to a plastic substrate. However, adhesive function was restored if the fragment was bound to a non-inhibitory anti-fibronectin antibody pre-adsorbed to the plastic substrate. The antibody-bound fragment also promoted cell migration. Both cell spreading and migration were specifically mediated by the α5β1 integrin. Affinity columns containing immobilized 20 kDa cell-binding fragment effectively bound α5- aL3-, and αv-containing fibronectin-binding integrins. In contrast, an immobilized 11.5 kDa fragment that contained the RGD sequence but lacked the synergistic sequence was bound only poorly by α5-containing fibronectin receptor integrins, even though the α3- and αv- containing integrins bound readily. Our results indicate that the manner in which adhesion proteins are presented to cells is important and that most cell adhesive activity is retained in a minimal 20 kDa segment of fibronectin.