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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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Binding of integrin α5β1 to recombinant FNs. Integrin  α5β1 was purified from human placenta and reconstituted in  phosphatidylcholine liposomes as described in Materials and  Methods. The integrin α5β1-liposomes were added to microtiter  plates precoated either with 20 μg/ml of rFN(C) (open bars) or  rFN(AC) (closed bars), or with 5 μg/ml of vitronectin (hatched  bars) in the presence or absence of the anti-integrin α5 mAb 8F1  (10 μg/ml) and incubated for 6 h at room temperature. Quantities  of bound integrin α5β1 liposomes are expressed as percentage of  the total input radioactivity after subtraction of the radioactivity  bound to plates coated only with BSA. Each bar represents the  mean ± SD (n = 6).
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Figure 8: Binding of integrin α5β1 to recombinant FNs. Integrin α5β1 was purified from human placenta and reconstituted in phosphatidylcholine liposomes as described in Materials and Methods. The integrin α5β1-liposomes were added to microtiter plates precoated either with 20 μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars), or with 5 μg/ml of vitronectin (hatched bars) in the presence or absence of the anti-integrin α5 mAb 8F1 (10 μg/ml) and incubated for 6 h at room temperature. Quantities of bound integrin α5β1 liposomes are expressed as percentage of the total input radioactivity after subtraction of the radioactivity bound to plates coated only with BSA. Each bar represents the mean ± SD (n = 6).

Mentions: The results described above left us with the possibility that the binding affinity of integrin α5β1 to CCBD could be enhanced by inclusion of the EDA segment. To explore this possibility further, integrin α5β1 purified from human placenta and reconstituted into phosphatidylcholine liposomes containing [3H]dipalmitoyl phosphatidylcholine was tested for its binding avidity to recombinant FNs with or without the EDA segment. As depicted in Fig. 8, integrin α5β1 liposomes bound to rFN(AC) significantly more avidly than to rFN(C). Binding of integrin α5β1 liposomes to rFN(AC) was blocked by the anti-integrin α5 mAb 8F1, as was the case with the binding to rFN(C). No significant binding was observed with vitronectin, confirming that the purified α5β1 used in this study was devoid of other integrins capable of binding to both FN and vitronectin (i.e., αvβ3, αvβ5, αvβ6, and αIIbβ3).


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)

Binding of integrin α5β1 to recombinant FNs. Integrin  α5β1 was purified from human placenta and reconstituted in  phosphatidylcholine liposomes as described in Materials and  Methods. The integrin α5β1-liposomes were added to microtiter  plates precoated either with 20 μg/ml of rFN(C) (open bars) or  rFN(AC) (closed bars), or with 5 μg/ml of vitronectin (hatched  bars) in the presence or absence of the anti-integrin α5 mAb 8F1  (10 μg/ml) and incubated for 6 h at room temperature. Quantities  of bound integrin α5β1 liposomes are expressed as percentage of  the total input radioactivity after subtraction of the radioactivity  bound to plates coated only with BSA. 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 8: Binding of integrin α5β1 to recombinant FNs. Integrin α5β1 was purified from human placenta and reconstituted in phosphatidylcholine liposomes as described in Materials and Methods. The integrin α5β1-liposomes were added to microtiter plates precoated either with 20 μg/ml of rFN(C) (open bars) or rFN(AC) (closed bars), or with 5 μg/ml of vitronectin (hatched bars) in the presence or absence of the anti-integrin α5 mAb 8F1 (10 μg/ml) and incubated for 6 h at room temperature. Quantities of bound integrin α5β1 liposomes are expressed as percentage of the total input radioactivity after subtraction of the radioactivity bound to plates coated only with BSA. Each bar represents the mean ± SD (n = 6).
Mentions: The results described above left us with the possibility that the binding affinity of integrin α5β1 to CCBD could be enhanced by inclusion of the EDA segment. To explore this possibility further, integrin α5β1 purified from human placenta and reconstituted into phosphatidylcholine liposomes containing [3H]dipalmitoyl phosphatidylcholine was tested for its binding avidity to recombinant FNs with or without the EDA segment. As depicted in Fig. 8, integrin α5β1 liposomes bound to rFN(AC) significantly more avidly than to rFN(C). Binding of integrin α5β1 liposomes to rFN(AC) was blocked by the anti-integrin α5 mAb 8F1, as was the case with the binding to rFN(C). No significant binding was observed with vitronectin, confirming that the purified α5β1 used in this study was devoid of other integrins capable of binding to both FN and vitronectin (i.e., αvβ3, αvβ5, αvβ6, and αIIbβ3).

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