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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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SDS-PAGE and immunoblot analyses of recombinant  FNs. (A) Recombinant FNs as well as plasma and cellular FNs  (abbreviated pFN and cFN, respectively), both purified by gelatin-affinity chromatography and subsequent ion exchange chromatography on a HiTrap Q column (see Materials and Methods),  were subjected to SDS-PAGE under reducing (top gel) or nonreducing (bottom gel) conditions and visualized by Coomassie  staining. 2 μg of protein was applied to each lane. Positions of the  dimeric and monomeric forms of FNs are indicated in the right  margin. Shown in the left are the positions of molecular size  markers. (B) Purified FNs (0.6 μg/lane) were subjected to SDS-PAGE under reducing conditions followed by immunoblotting  with the following anti-FN mAbs: FN8-12 recognizing Fib2 domain (top panel); IST-9 recognizing the EDA segment (middle  panel); BC-1 recognizing the EDB+ FNs (bottom panel).
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Figure 2: SDS-PAGE and immunoblot analyses of recombinant FNs. (A) Recombinant FNs as well as plasma and cellular FNs (abbreviated pFN and cFN, respectively), both purified by gelatin-affinity chromatography and subsequent ion exchange chromatography on a HiTrap Q column (see Materials and Methods), were subjected to SDS-PAGE under reducing (top gel) or nonreducing (bottom gel) conditions and visualized by Coomassie staining. 2 μg of protein was applied to each lane. Positions of the dimeric and monomeric forms of FNs are indicated in the right margin. Shown in the left are the positions of molecular size markers. (B) Purified FNs (0.6 μg/lane) were subjected to SDS-PAGE under reducing conditions followed by immunoblotting with the following anti-FN mAbs: FN8-12 recognizing Fib2 domain (top panel); IST-9 recognizing the EDA segment (middle panel); BC-1 recognizing the EDB+ FNs (bottom panel).

Mentions: Purified FNs gave single bands with apparent molecular masses of 220–250 kD upon SDS-PAGE under reducing conditions (Fig. 2 A). The relative molecular masses of the recombinant FNs were in the order of rFN(BAC) > rFN(AC) > rFN(C), consistent size differences expected due to the presence or absence of the EDA and/or EDB segments. The recombinant FNs gave sharper bands in SDS-PAGE compared to native FNs purified from plasma (plasma FN) and from conditioned medium of cultured fibroblasts (cellular FN), confirming the homogeneity of the recombinant FNs. SDS-PAGE under nonreducing conditions showed that almost all of the recombinant FNs exist as dimers, as observed for plasma and cellular FNs (Fig. 2 A). Presence or absence of the EDA and EDB segments were confirmed by immunoblot analysis (Fig. 2 B).


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)

SDS-PAGE and immunoblot analyses of recombinant  FNs. (A) Recombinant FNs as well as plasma and cellular FNs  (abbreviated pFN and cFN, respectively), both purified by gelatin-affinity chromatography and subsequent ion exchange chromatography on a HiTrap Q column (see Materials and Methods),  were subjected to SDS-PAGE under reducing (top gel) or nonreducing (bottom gel) conditions and visualized by Coomassie  staining. 2 μg of protein was applied to each lane. Positions of the  dimeric and monomeric forms of FNs are indicated in the right  margin. Shown in the left are the positions of molecular size  markers. (B) Purified FNs (0.6 μg/lane) were subjected to SDS-PAGE under reducing conditions followed by immunoblotting  with the following anti-FN mAbs: FN8-12 recognizing Fib2 domain (top panel); IST-9 recognizing the EDA segment (middle  panel); BC-1 recognizing the EDB+ FNs (bottom panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139828&req=5

Figure 2: SDS-PAGE and immunoblot analyses of recombinant FNs. (A) Recombinant FNs as well as plasma and cellular FNs (abbreviated pFN and cFN, respectively), both purified by gelatin-affinity chromatography and subsequent ion exchange chromatography on a HiTrap Q column (see Materials and Methods), were subjected to SDS-PAGE under reducing (top gel) or nonreducing (bottom gel) conditions and visualized by Coomassie staining. 2 μg of protein was applied to each lane. Positions of the dimeric and monomeric forms of FNs are indicated in the right margin. Shown in the left are the positions of molecular size markers. (B) Purified FNs (0.6 μg/lane) were subjected to SDS-PAGE under reducing conditions followed by immunoblotting with the following anti-FN mAbs: FN8-12 recognizing Fib2 domain (top panel); IST-9 recognizing the EDA segment (middle panel); BC-1 recognizing the EDB+ FNs (bottom panel).
Mentions: Purified FNs gave single bands with apparent molecular masses of 220–250 kD upon SDS-PAGE under reducing conditions (Fig. 2 A). The relative molecular masses of the recombinant FNs were in the order of rFN(BAC) > rFN(AC) > rFN(C), consistent size differences expected due to the presence or absence of the EDA and/or EDB segments. The recombinant FNs gave sharper bands in SDS-PAGE compared to native FNs purified from plasma (plasma FN) and from conditioned medium of cultured fibroblasts (cellular FN), confirming the homogeneity of the recombinant FNs. SDS-PAGE under nonreducing conditions showed that almost all of the recombinant FNs exist as dimers, as observed for plasma and cellular FNs (Fig. 2 A). Presence or absence of the EDA and EDB segments were confirmed by immunoblot analysis (Fig. 2 B).

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