MODULATION OF CELL-ADHESIVE ACTIVITY OF FIBRONECTIN BY THE ALTERNATIVELY SPLICED EDA SEGMENT

Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is promine ntly expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purifie d proteins. EDA(+) FN was significantly more potent than EDA(-) FN in promoting cell spreading and cell migration, irrespective of the prese nce or absence of a second alternatively spliced segment, EDB. The cel l spreading activity of EDA(+) FN was not affected by antibodies recog nizing the EDA segment but was abolished by antibodies against integri n alpha 5 and beta 1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin alpha 5 beta 1 . In support of this conclusion, purified integrin alpha 5 beta 1 boun d more avidly to EDA(+) FN than to EDA(-) FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding a ctivity between EDA(+) FN and EDA(-) FN was abolished after limited pr oteolysis with thermolysin. Consistent with this observation, binding of integrin alpha 5 beta 1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding doma in Hep2, was not affected by insertion of the EDA segment. Since the i nsertion of an extra type III module such as EDA into an array of repe ated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN m olecule may be globally altered upon insertion of the EDA segment, res ulting in an increased exposure of the RGD motif in III10 module and/o r local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integr in-binding affinity of FN operating when enhanced migration and prolif eration of cells are required.