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Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment.

Manabe R, Ohe N, Maeda T, Fukuda T, Sekiguchi K - J. Cell Biol. (1997)

Bottom Line: 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.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 degrees 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.

View Article: PubMed Central - PubMed

Affiliation: Research Institute, Osaka Medical Center for Maternal and Child Health, Japan.

ABSTRACT
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 alpha5 and beta1 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 alpha5beta1. In support of this conclusion, purified integrin alpha5beta1 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 alpha5beta1 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 degrees 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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Dose dependence of the spreading of HT1080 cells on  recombinant FNs. HT1080 cells were seeded on plates coated  with various concentrations of rFN(C) (open circles), rFN(AC)  (closed circles), or rFN(BAC) (closed squares) and incubated for  30 min at 37°C. Spreading of HT1080 cells was quantified as described in Materials and Methods and expressed as the number of  cells adopting a well spread morphology per square millimeter.  Each bar represents the mean ± SD (n = 3).
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Figure 4: Dose dependence of the spreading of HT1080 cells on recombinant FNs. HT1080 cells were seeded on plates coated with various concentrations of rFN(C) (open circles), rFN(AC) (closed circles), or rFN(BAC) (closed squares) and incubated for 30 min at 37°C. Spreading of HT1080 cells was quantified as described in Materials and Methods and expressed as the number of cells adopting a well spread morphology per square millimeter. Each bar represents the mean ± SD (n = 3).

Mentions: Fig. 4 shows the dose dependence of the spreading of HT1080 cells on different FN isoforms. The number of spread cells reached a plateau at >10 μg/ml of recombinant FNs irrespective of the presence or absence of the alternatively spliced segments. rFN(AC) and rFN(BAC) were more potent than rFN(C) in promoting cell spreading throughout the range of FN concentrations examined. A maximal difference between EDA+ and EDA− isoforms was observed at 5 μg/ml recombinant protein.


Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment.

Manabe R, Ohe N, Maeda T, Fukuda T, Sekiguchi K - J. Cell Biol. (1997)

Dose dependence of the spreading of HT1080 cells on  recombinant FNs. HT1080 cells were seeded on plates coated  with various concentrations of rFN(C) (open circles), rFN(AC)  (closed circles), or rFN(BAC) (closed squares) and incubated for  30 min at 37°C. Spreading of HT1080 cells was quantified as described in Materials and Methods and expressed as the number of  cells adopting a well spread morphology per square millimeter.  Each bar represents the mean ± SD (n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139828&req=5

Figure 4: Dose dependence of the spreading of HT1080 cells on recombinant FNs. HT1080 cells were seeded on plates coated with various concentrations of rFN(C) (open circles), rFN(AC) (closed circles), or rFN(BAC) (closed squares) and incubated for 30 min at 37°C. Spreading of HT1080 cells was quantified as described in Materials and Methods and expressed as the number of cells adopting a well spread morphology per square millimeter. Each bar represents the mean ± SD (n = 3).
Mentions: Fig. 4 shows the dose dependence of the spreading of HT1080 cells on different FN isoforms. The number of spread cells reached a plateau at >10 μg/ml of recombinant FNs irrespective of the presence or absence of the alternatively spliced segments. rFN(AC) and rFN(BAC) were more potent than rFN(C) in promoting cell spreading throughout the range of FN concentrations examined. A maximal difference between EDA+ and EDA− isoforms was observed at 5 μg/ml recombinant protein.

Bottom Line: 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.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 degrees 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.

View Article: PubMed Central - PubMed

Affiliation: Research Institute, Osaka Medical Center for Maternal and Child Health, Japan.

ABSTRACT
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 alpha5 and beta1 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 alpha5beta1. In support of this conclusion, purified integrin alpha5beta1 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 alpha5beta1 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 degrees 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.

Show MeSH
Related in: MedlinePlus