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Research Institute, Osaka Medical Center for Maternal and Child Health, Izumi, Osaka 590-02, Japan
Fibronectin (FN) has a complex pattern of
alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently 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 purified proteins. EDA+ FN
was significantly more potent than EDA
FN in promoting cell spreading and cell migration, irrespective of
the presence or absence of a second alternatively spliced
segment, EDB. The cell spreading activity of EDA+
FN was not affected by antibodies recognizing the
EDA segment but was abolished by antibodies against
integrin 
5 and 
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 51. In support of
this conclusion, purified integrin 51 bound 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 activity between
EDA+ FN and EDA FN was abolished after limited
proteolysis with thermolysin. Consistent with this observation, binding of integrin 51 to a recombinant
FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2,
was not affected by insertion of the EDA segment.
Since the insertion of an extra type III module such as
EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180° at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA
segment, resulting in an increased exposure of the RGD
motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the
EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced
migration and proliferation of cells are required.
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