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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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Inhibition by anti-integrin mAbs and synthetic peptides  of cell spreading mediated by recombinant FNs. HT1080 cells  were seeded on microtiter plates precoated with 5 μg/ml of  rFN(C) (open bars) or rFN(AC) (closed bars) in the presence or  absence of 10 μg/ml of the following anti-integrin mAbs or 1 mg/ ml of synthetic peptides (GRGDSP and GRGESP), and incubated for 30 min at 37°C: None, no mAb added; 8F1, anti-α5  mAb; 4G2, anti-β1 mAb; ST/73, anti-α4 mAb; LM609, anti-αvβ3  mAb. The cell spreading was quantified as described in Materials  and Methods. Each bar represents the mean ± SD (n = 6).
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Figure 6: Inhibition by anti-integrin mAbs and synthetic peptides of cell spreading mediated by recombinant FNs. HT1080 cells were seeded on microtiter plates precoated with 5 μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars) in the presence or absence of 10 μg/ml of the following anti-integrin mAbs or 1 mg/ ml of synthetic peptides (GRGDSP and GRGESP), and incubated for 30 min at 37°C: None, no mAb added; 8F1, anti-α5 mAb; 4G2, anti-β1 mAb; ST/73, anti-α4 mAb; LM609, anti-αvβ3 mAb. The cell spreading was quantified as described in Materials and Methods. Each bar represents the mean ± SD (n = 6).

Mentions: In support of this conclusion, function-blocking mAbs directed against the integrin α5 or β1 subunits inhibited spreading of HT1080 cells onto the rFN(AC)-coated substrates almost completely, whereas the mAbs directed against other types of FN-binding integrins, i.e., anti-α4 and anti-αvβ3 mAbs, were barely inhibitory (Fig. 6). These results indicated that spreading of HT1080 cells onto rFN(AC)-coated substrates was predominantly mediated by interaction of integrin α5β1 with CCBD, as was the case with spreading onto plasma FN-coated substrates (Aota et al., 1991). This conclusion was further supported by the observation that GRGDSP peptide, but not GRGESP, inhibited almost completely rFN(AC)-mediated spreading of HT1080 cells (Fig. 6). These results, together with the failure of EDA antagonists to inhibit rFN(AC)-mediated cell spreading, indicated that the EDA segment augments the cell-adhesive activity of FNs by promoting the interaction of integrin α5β1 with the RGD-containing CCBD and not by providing an additional cell-interactive site.


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)

Inhibition by anti-integrin mAbs and synthetic peptides  of cell spreading mediated by recombinant FNs. HT1080 cells  were seeded on microtiter plates precoated with 5 μg/ml of  rFN(C) (open bars) or rFN(AC) (closed bars) in the presence or  absence of 10 μg/ml of the following anti-integrin mAbs or 1 mg/ ml of synthetic peptides (GRGDSP and GRGESP), and incubated for 30 min at 37°C: None, no mAb added; 8F1, anti-α5  mAb; 4G2, anti-β1 mAb; ST/73, anti-α4 mAb; LM609, anti-αvβ3  mAb. The cell spreading was quantified as described in Materials  and Methods. Each bar represents the mean ± SD (n = 6).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139828&req=5

Figure 6: Inhibition by anti-integrin mAbs and synthetic peptides of cell spreading mediated by recombinant FNs. HT1080 cells were seeded on microtiter plates precoated with 5 μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars) in the presence or absence of 10 μg/ml of the following anti-integrin mAbs or 1 mg/ ml of synthetic peptides (GRGDSP and GRGESP), and incubated for 30 min at 37°C: None, no mAb added; 8F1, anti-α5 mAb; 4G2, anti-β1 mAb; ST/73, anti-α4 mAb; LM609, anti-αvβ3 mAb. The cell spreading was quantified as described in Materials and Methods. Each bar represents the mean ± SD (n = 6).
Mentions: In support of this conclusion, function-blocking mAbs directed against the integrin α5 or β1 subunits inhibited spreading of HT1080 cells onto the rFN(AC)-coated substrates almost completely, whereas the mAbs directed against other types of FN-binding integrins, i.e., anti-α4 and anti-αvβ3 mAbs, were barely inhibitory (Fig. 6). These results indicated that spreading of HT1080 cells onto rFN(AC)-coated substrates was predominantly mediated by interaction of integrin α5β1 with CCBD, as was the case with spreading onto plasma FN-coated substrates (Aota et al., 1991). This conclusion was further supported by the observation that GRGDSP peptide, but not GRGESP, inhibited almost completely rFN(AC)-mediated spreading of HT1080 cells (Fig. 6). These results, together with the failure of EDA antagonists to inhibit rFN(AC)-mediated cell spreading, indicated that the EDA segment augments the cell-adhesive activity of FNs by promoting the interaction of integrin α5β1 with the RGD-containing CCBD and not by providing an additional cell-interactive site.

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