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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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The structure of recombinant FNs and bacterially expressed fusion fragments. Modular structures of recombinant  FNs are shown schematically on the basis of internally homologous modules termed types I, II, and III. The EDA and EDB segments are shown with filled rectangles, while the IIICS segment is  shown by a hatched oval. All recombinant FNs contain the complete IIICS sequence of 120 amino acids. Functional domains that  interact with heparin (Hep1, Hep2), fibrin (Fib1, Fib2), bacteria,  collagen, and cell surface integrins (CCBD) are indicated above  the schemes. Recombinant FN fragments encompassing different  intervals of type III modules were produced as fusion proteins  with either GST or MBP.
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Figure 1: The structure of recombinant FNs and bacterially expressed fusion fragments. Modular structures of recombinant FNs are shown schematically on the basis of internally homologous modules termed types I, II, and III. The EDA and EDB segments are shown with filled rectangles, while the IIICS segment is shown by a hatched oval. All recombinant FNs contain the complete IIICS sequence of 120 amino acids. Functional domains that interact with heparin (Hep1, Hep2), fibrin (Fib1, Fib2), bacteria, collagen, and cell surface integrins (CCBD) are indicated above the schemes. Recombinant FN fragments encompassing different intervals of type III modules were produced as fusion proteins with either GST or MBP.

Mentions: Two cDNAs encoding the III11 and III12 modules of human FN with or without the inserted EDA segment were amplified by reverse transcription PCR from mRNA extracted from WI-38 human fibroblasts using forward and reverse primers tagged with BamHI and SalI sites, respectively. The primers used were: 5′-AAAGTCGGATCCGAAATTGACAAACCATCC-3′ (forward) and 5′-AAAGTCGACCTACTCCAGAGTGGTGACAAC-3′ (reverse), where restriction sites and the stop codon are indicated with bold and italic characters, respectively. PCR-amplified fragments were cloned between the BamHI and SalI sites of pMAL-cRI (New England Biolabs, Beverly, MA) to engineer expression in Escherichia coli as fusion proteins with maltose-binding protein (MBP). The resulting fusion proteins, designated MBP11-12 and MBP11-A-12 (see Fig. 1), were purified from the bacterial lysate using amylose resin columns.


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)

The structure of recombinant FNs and bacterially expressed fusion fragments. Modular structures of recombinant  FNs are shown schematically on the basis of internally homologous modules termed types I, II, and III. The EDA and EDB segments are shown with filled rectangles, while the IIICS segment is  shown by a hatched oval. All recombinant FNs contain the complete IIICS sequence of 120 amino acids. Functional domains that  interact with heparin (Hep1, Hep2), fibrin (Fib1, Fib2), bacteria,  collagen, and cell surface integrins (CCBD) are indicated above  the schemes. Recombinant FN fragments encompassing different  intervals of type III modules were produced as fusion proteins  with either GST or MBP.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: The structure of recombinant FNs and bacterially expressed fusion fragments. Modular structures of recombinant FNs are shown schematically on the basis of internally homologous modules termed types I, II, and III. The EDA and EDB segments are shown with filled rectangles, while the IIICS segment is shown by a hatched oval. All recombinant FNs contain the complete IIICS sequence of 120 amino acids. Functional domains that interact with heparin (Hep1, Hep2), fibrin (Fib1, Fib2), bacteria, collagen, and cell surface integrins (CCBD) are indicated above the schemes. Recombinant FN fragments encompassing different intervals of type III modules were produced as fusion proteins with either GST or MBP.
Mentions: Two cDNAs encoding the III11 and III12 modules of human FN with or without the inserted EDA segment were amplified by reverse transcription PCR from mRNA extracted from WI-38 human fibroblasts using forward and reverse primers tagged with BamHI and SalI sites, respectively. The primers used were: 5′-AAAGTCGGATCCGAAATTGACAAACCATCC-3′ (forward) and 5′-AAAGTCGACCTACTCCAGAGTGGTGACAAC-3′ (reverse), where restriction sites and the stop codon are indicated with bold and italic characters, respectively. PCR-amplified fragments were cloned between the BamHI and SalI sites of pMAL-cRI (New England Biolabs, Beverly, MA) to engineer expression in Escherichia coli as fusion proteins with maltose-binding protein (MBP). The resulting fusion proteins, designated MBP11-12 and MBP11-A-12 (see Fig. 1), were purified from the bacterial lysate using amylose resin columns.

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