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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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A schematic model  for EDA-induced conformational change of FN. The FN  molecule is folded into a  compact conformation due  to intra- and/or inter-chain  interactions. Insertion of the  EDA segment (black) between CCBD (gray) and the  Hep2 domain rotates the  NH2-terminal region encompassing the NH2 terminus  through the III11 module up  to 180°C relative to the region COOH-terminal to the  inserted EDA segment, leading to a change in the global conformation of the FN molecule. Such a conformational change may increase  the accessibility of the RGD motif within CCBD to integrin α5β1 and/or alter the local conformation of the III10 module so as to optimize the binding of integrin α5β1 to the RGD motif. Arrowheads point to the position of the EDA insertion.
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Figure 12: A schematic model for EDA-induced conformational change of FN. The FN molecule is folded into a compact conformation due to intra- and/or inter-chain interactions. Insertion of the EDA segment (black) between CCBD (gray) and the Hep2 domain rotates the NH2-terminal region encompassing the NH2 terminus through the III11 module up to 180°C relative to the region COOH-terminal to the inserted EDA segment, leading to a change in the global conformation of the FN molecule. Such a conformational change may increase the accessibility of the RGD motif within CCBD to integrin α5β1 and/or alter the local conformation of the III10 module so as to optimize the binding of integrin α5β1 to the RGD motif. Arrowheads point to the position of the EDA insertion.

Mentions: There are several possible mechanisms that may explain enhanced integrin-binding affinity of EDA+ FNs. First, the EDA segment might directly interact with integrins α5β1 and αvβ3, thereby synergizing with the binding of the RGD motif to these integrins (ffrench-Constant, 1995). This possibility seems unlikely, however, since binding of integrin α5β1 to the GST-fusion protein consisting of CCBD and the Hep2 domain was not affected by the presence or absence of the EDA segment. A second possibility is that insertion of the EDA segment alters the conformation of the neighboring type III modules including III10, thereby enhancing the integrin-binding affinity of CCBD (ffrench-Constant, 1995). Analyses of the three- dimensional structure of a recombinant FN fragment consisting of III7–III10 modules revealed that two adjacent type III modules are interconnected with tilts and rotations along the long axis (Leahy et al., 1996). Insertion of an extra type III module (i.e., the EDA module) could alter the conformation of the neighboring modules (i.e., III11 and III12) by readjusting the intermodular rotations and tilts, which could in turn alter the conformation of their neighboring modules including III10 so as to optimize the conformation of the RGD-containing loop. The third possibility is that insertion of the EDA segment alters the global conformation of the FN molecule by rotating the NH2-terminal portion of the FN polypeptide relative to the COOH terminus (Fig. 12). Given a pseudo-twofold relationship between adjacent type III modules (Huber et al., 1994; Leahy et al., 1996), the insertion of the EDA segment is expected to rotate the NH2-terminal two-thirds (the NH2 terminus through III11) up to 180° relative to the COOH-terminal one-third (III12 through the COOH terminus). Such a change in global conformation may not only increase the accessibility of the RGD motif in CCBD to the integrins α5β1 and αvβ3 but also induce partial unfolding of the III10 module by altering the tension and/or torsion applied to CCBD. Our results obtained with thermolysin-cleaved recombinant FNs and GST fusion proteins consisting of CCBD and the Hep2 domain clearly showed that enhanced integrin-binding of EDA+ FN was only observable in the context of the intact FN molecule, consistent with the third possibility. The significant increase in the integrin-binding affinity of EDA− FN after limited proteolysis suggests that the integrin binding site of EDA− FN is either partially cryptic in the intact molecule or folded into a conformation with suboptimal affinity for integrin α5β1. In support of this notion, the binding of plasma FN to hamster kidney cells has been reported to increase up to twofold after tryptic digestion (Hayashi and Yamada, 1983; Akiyama et al., 1985). It should also be noted that the integrin-binding activity of EDA+ FN was slightly decreased after limited proteolysis, suggesting that conformational change induced by the inserted EDA segment not only increases the exposure of the integrin-binding site on the surface of the FN molecule but also perturbs the local conformation of CCBD, particularly the RGD-containing III10, to optimize the affinity for integrin α5β1. In support of this possibility, the FN type III modules have been proposed to undergo reversible unfolding with a relatively weak force that is comparable to that required to dissociate a noncovalent protein–protein interaction (Erickson, 1994).


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)

A schematic model  for EDA-induced conformational change of FN. The FN  molecule is folded into a  compact conformation due  to intra- and/or inter-chain  interactions. Insertion of the  EDA segment (black) between CCBD (gray) and the  Hep2 domain rotates the  NH2-terminal region encompassing the NH2 terminus  through the III11 module up  to 180°C relative to the region COOH-terminal to the  inserted EDA segment, leading to a change in the global conformation of the FN molecule. Such a conformational change may increase  the accessibility of the RGD motif within CCBD to integrin α5β1 and/or alter the local conformation of the III10 module so as to optimize the binding of integrin α5β1 to the RGD motif. Arrowheads point to the position of the EDA insertion.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139828&req=5

Figure 12: A schematic model for EDA-induced conformational change of FN. The FN molecule is folded into a compact conformation due to intra- and/or inter-chain interactions. Insertion of the EDA segment (black) between CCBD (gray) and the Hep2 domain rotates the NH2-terminal region encompassing the NH2 terminus through the III11 module up to 180°C relative to the region COOH-terminal to the inserted EDA segment, leading to a change in the global conformation of the FN molecule. Such a conformational change may increase the accessibility of the RGD motif within CCBD to integrin α5β1 and/or alter the local conformation of the III10 module so as to optimize the binding of integrin α5β1 to the RGD motif. Arrowheads point to the position of the EDA insertion.
Mentions: There are several possible mechanisms that may explain enhanced integrin-binding affinity of EDA+ FNs. First, the EDA segment might directly interact with integrins α5β1 and αvβ3, thereby synergizing with the binding of the RGD motif to these integrins (ffrench-Constant, 1995). This possibility seems unlikely, however, since binding of integrin α5β1 to the GST-fusion protein consisting of CCBD and the Hep2 domain was not affected by the presence or absence of the EDA segment. A second possibility is that insertion of the EDA segment alters the conformation of the neighboring type III modules including III10, thereby enhancing the integrin-binding affinity of CCBD (ffrench-Constant, 1995). Analyses of the three- dimensional structure of a recombinant FN fragment consisting of III7–III10 modules revealed that two adjacent type III modules are interconnected with tilts and rotations along the long axis (Leahy et al., 1996). Insertion of an extra type III module (i.e., the EDA module) could alter the conformation of the neighboring modules (i.e., III11 and III12) by readjusting the intermodular rotations and tilts, which could in turn alter the conformation of their neighboring modules including III10 so as to optimize the conformation of the RGD-containing loop. The third possibility is that insertion of the EDA segment alters the global conformation of the FN molecule by rotating the NH2-terminal portion of the FN polypeptide relative to the COOH terminus (Fig. 12). Given a pseudo-twofold relationship between adjacent type III modules (Huber et al., 1994; Leahy et al., 1996), the insertion of the EDA segment is expected to rotate the NH2-terminal two-thirds (the NH2 terminus through III11) up to 180° relative to the COOH-terminal one-third (III12 through the COOH terminus). Such a change in global conformation may not only increase the accessibility of the RGD motif in CCBD to the integrins α5β1 and αvβ3 but also induce partial unfolding of the III10 module by altering the tension and/or torsion applied to CCBD. Our results obtained with thermolysin-cleaved recombinant FNs and GST fusion proteins consisting of CCBD and the Hep2 domain clearly showed that enhanced integrin-binding of EDA+ FN was only observable in the context of the intact FN molecule, consistent with the third possibility. The significant increase in the integrin-binding affinity of EDA− FN after limited proteolysis suggests that the integrin binding site of EDA− FN is either partially cryptic in the intact molecule or folded into a conformation with suboptimal affinity for integrin α5β1. In support of this notion, the binding of plasma FN to hamster kidney cells has been reported to increase up to twofold after tryptic digestion (Hayashi and Yamada, 1983; Akiyama et al., 1985). It should also be noted that the integrin-binding activity of EDA+ FN was slightly decreased after limited proteolysis, suggesting that conformational change induced by the inserted EDA segment not only increases the exposure of the integrin-binding site on the surface of the FN molecule but also perturbs the local conformation of CCBD, particularly the RGD-containing III10, to optimize the affinity for integrin α5β1. In support of this possibility, the FN type III modules have been proposed to undergo reversible unfolding with a relatively weak force that is comparable to that required to dissociate a noncovalent protein–protein interaction (Erickson, 1994).

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