Function and receptor specificity of a minimal 20 kilodalton cell adhesive fragment of fibronectin.
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 alpha 5 beta 1 integrin. Affinity columns containing immobilized 20 kDa cell-binding fragment effectively bound alpha 5-, alpha 3-, and alpha v-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 alpha 5-containing fibronectin receptor integrins, even though the alpha 3- and alpha v-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.
Akiyama
S K
SK
Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA.
Aota
S
S
Yamada
K M
KM
D001483:Base Sequence; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D017469:Receptors, Fibronectin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D005353:Fibronectins; D016023:Integrins; D009842:Oligopeptides; D017469:Receptors, Fibronectin; D011993:Recombinant Fusion Proteins; C047981:arginyl-glycyl-aspartic acid